Target Research Dec 2010 by Muscular Dystrophy UK

Muscular Dystrophy Campaign Research Magazine Issue 3 â&#x20AC;&#x201C; December 2010

Target Research

Gene therapy

Delivering new genes and repairing old ones

Translational research The winding road to treatment

New DNA technology Improving the diagnosis and understanding of neuromuscular conditions

Also inside...hydrotherapy, groundbreaking research we fund, clinical trial news, and more

Welcome

New research projects awarded funding by us this year

Gene therapy – delivering new genes and repairing old ones

Research news from around the world

Focus on hydrotherapy

Translational research – the winding road to treatment

Clinical trial update

The puzzle of research funding

Funding our research

Updates and highlights from research funded by the Muscular Dystrophy Campaign

Partnership project improves exon skipping and develops non-invasive muscle monitoring

The sequencing revolution

Ask a scientist

Spotlight on two of our long-term supporters Professor Alan Emery and Lord John Walton

Workshops bring international scientists together

Research projects currently funded by the Muscular Dystrophy Campaign

Feedback

Glossary

Words in italics can be found in the glossary at the end of the magazine.

 More than 70,000 babies, children and adults in the UK have a neuromuscular condition.  More than 350,000 people are indirectly affected as family, friends and carers.  The Muscular Dystrophy Campaign supports more than 60 different neuromuscular conditions.  The Muscular Dystrophy Campaign has pioneered the search for treatments and cures since 1959.  Each year we invest more than £1 million into high quality research in the UK and an equal amount is invested into the provision of care and information.  We currently fund 26 research projects with each project lasting between one and four years.  Many research advances for neuromuscular conditions in the past 50 years have been by Muscular Dystrophy Campaign-funded scientists including vital preclinical research for the Duchenne muscular dystrophy exon-skipping trial.

Dr Marita Pohlschmidt – Director of Research (right), Dr Kristina Elvidge – Research Communications Officer (centre) and Dr Julia Ambler – Grants Manager (left)

The Lay Research Panel discuss the research grant applications

Welcome to the third edition of Target Research, once again bringing news of exciting research advances direct to your home.

buzz word in the scientific world and the article on page 12 will bring you up to speed with what this entails.

It has been a challenging year for our commitment to support research into neuromuscular conditions. However, we are proud to announce that we were able to award funding to seven new cutting-edge research projects in this year’s grant round – you can read about them on page 2. A profound change in how we fund research was also implemented in 2010. A Lay Research Panel was established and our families and supporters are now playing an active part in deciding what research projects should be funded.

Last but not least, we asked artist Jacqueline Donachie whose family is affected by myotonic dystrophy to design the front cover. Her drawing emphasises the importance of the alliance between the scientific community and the families affected by neuromuscular conditions. Together we’re stronger to keep science moving forward to ensure that life-saving treatments will become available in the future.

Internationally the scientific community is advancing fast. A decade ago the completion of a £6 billion project was announced revealing the sequence of the human genome. Thanks to new technology the cost and length of time it takes to sequence a human genome are now a tiny fraction of this first attempt. This means that sequencing an individual’s DNA is set to become an integral part of research into genetic conditions and will revolutionise diagnosis. This definitely has advantages but there are also challenges which are discussed in detail on page 24. The search for treatments is tirelessly continuing. Great advances have been made in gene therapy and you can read about the new technologies on page 4. However, most neuromuscular conditions are rare and the speedy transfer of promising technology into treatments requires the establishment of an international infrastructure. This area of translational research is currently the

A big thank you to everybody who has supported our research programme and contributed to the magazine. We hope you enjoy reading it! The Research Team, Muscular Dystrophy Campaign

Target Research The research magazine for families and supporters of the Muscular Dystrophy Campaign The Muscular Dystrophy Group of Great Britain and Northern Ireland, 61 Southwark St, London SE1 0HL Registered Charity No. 205395 and Registered Scottish Charity No. SC039445

For all magazine enquiries: Kristina Elvidge research@muscular-dystrophy.org Disclaimer While every effort has been made to ensure the information contained within Target Research is accurate, the Muscular Dystrophy Campaign accepts no responsibility or liability where errors or omissions are made. The views expressed in this magazine are not necessarily those of the charity. ISSN 1663-4538

Edited and designed by the Muscular Dystrophy Campaign.

New research projects awarded funding by us this year

The Muscular Dystrophy Campaign has awarded seven new grants for the 2010/11 financial year; four PhD studentships and three “pump-priming” grants. Pump-priming grants give researchers the opportunity to investigate new and innovative ideas and technologies for one year to obtain proof of principle prior to applying for further funding.

Prof. Beeson and colleagues previously discovered that one form of myasthenia gravis is caused by antibodies attacking a protein called MuSK, which has a similar role to DOK7. Since the underlying disease mechanisms are similar, they will explore, using cells grown in the laboratory, whether these myasthenia gravis patients might also respond to treatment with ephedrine.

Pump-priming awards

is affecting the amount of calcium being released during muscle contraction and how this affects the mitochondria.

How does ephedrine improve the symptoms of congenital myasthenia and could it be used for myasthenia gravis? Prof. David Beeson, at the University of Oxford, previously found that some individuals with congenital myasthenic syndrome have a mistake in the gene that codes for a protein called DOK7. This protein has an important role in the normal functioning of a specialised structure where the nerve and muscle meet called the neuromuscular junction (NMJ). The research team also found that these patients respond very well to a drug called ephedrine. How ephedrine works to improve the symptoms in these patients is unknown and this project will investigate this.

Investigating the role of the mitochondria in the core myopathies At University College London Prof. Michael Duchen and his student aim to study how the genetic defect that causes core myopathies can affect the “batteries” of the cell – the mitochondria. Core myopathies are often caused by a defect in the ryanodine receptor 1 gene (RYR1). This impairs the flow of calcium in muscle cells which is needed for muscle contraction. Mitochondria also need calcium to function properly so Prof. Duchen will investigate whether they are being impaired in these conditions, which may be compounding the muscle weakness. The team will use a variety of cutting-edge imaging techniques to look at how the faulty RYR1

Searching for new genes that cause periodic paralysis and the myotonias The channelopathies are a group of conditions that includes periodic paralysis, myotonia congenita and paramyotonia congenita. Symptoms of these conditions include episodes of muscle stiffness and/or paralysis. They are all caused by defects in genes that contain the instructions for making ion channels – pores in the walls of muscle cells that allow the flow of molecules such as potassium into and out of muscle cells. About 30 percent of channelopathy patients have yet to be given a genetic diagnosis because no defect has been found in any of the genes known to cause their symptoms. This makes it hard to predict how their condition might progress and it is difficult to give these patients family planning advice. Prof. Michael Hanna and his student at University College London will examine the DNA from this group of individuals, initially looking at a set of genes suspected to be

Prof. David Beeson

Prof. Michael Duchen

Prof. Michael Hanna

New PhD Studentships

Dr Matthew Wood involved in the functioning of ion channels. If nothing arises they will employ whole genome sequencing (see p24) to identify new causative genes.

Improving molecular patch therapy for Duchenne muscular dystrophy Exon skipping is a gene therapy approach that is currently in clinical trial for Duchenne muscular dystrophy (see p6). It involves short strands of DNA, known as ‘antisense oligonucleotides’ (AOs), or ‘molecular patches’, that can restore production of the protein dystrophin (which is missing in Duchenne muscular dystrophy). Researchers are facing several major challenges in trying to get exon skipping technology from the bench to the bedside. One of these is finding ways to improve the delivery of the molecular patches to the heart. It is very important that the heart muscle, as well as the skeletal muscle, can be treated with the molecular patches to help prevent heart complications occurring. Dr Matthew Wood and his team at the University of Oxford have previously discovered that short protein fragments known as peptides can be attached to the patches to dramatically improve their delivery into cells. The aim of this project, therefore, is to further investigate the use of these peptides to enhance the delivery of the molecular patches. Dr Wood and his student will be using a mouse model to determine the best dose of peptide-patch, how often it should be administered and the best way to give the drug. See also www.muscular-dystrophy.org/currentgrants

How does the interaction of genes affect the progression of congenital myasthenic syndromes? This project led by Prof. Dame Kay Davies at the University of Oxford will be examining the way that different genes interact in the hopes of gaining new insight into the congenital myasthenic syndromes (CMS). The team will look at a specialised structure where the nerve and muscle meet called the neuromuscular junction (NMJ). It is here that signals are passed from the nerve to the muscle telling it to contract. Individuals with CMS have fault in a gene that affects the NMJ. This research will study the NMJ in mouse models of congenital myasthenic syndromes and compare them to healthy mice. They aim to scrutinise the DNA to determine how specific genes with a role at the NMJ are interacting with each other. It is thought that these interactions help to coordinate the normal development of the NMJ. If one of the genes is faulty, then it may not be able to interact with the other NMJ genes. So, as well as the original faulty gene which prevents the production of a functional protein, there might also be a knock-on effect on the activity of other NMJ genes making the problem more complex. If the genes of the NMJ have an effect on each other, it may be that certain types of gene therapy would be unsuitable for people with congenital myasthenic syndromes. This research will help scientists focus on pursuing the most practical therapeutic approaches.

Studying a gene involved in muscle growth Following an injury or a long period of inactivity (such as a long stay in hospital) muscles may waste away and need a period of rehabilitation in order to regain strength and be able to fully function. People with neuromuscular conditions also often experience muscle wasting as part of their condition and so in conjunction with the current efforts to

develop treatments to directly address the genetic defect, such as gene therapy, it would be very useful to develop ways to promote muscle growth. Such muscle building treatments could be used alone or alongside other therapies to maximise their effectiveness. Prof. Peter Rigby, at the Institute of Cancer Research in London, will be focusing his research on this topic and studying how a gene called Mrf4 is able to contribute to muscle growth. The research team hope to gain further knowledge about the function of Mrf4 in order to use that knowledge to develop therapies that could promote muscle growth.

Investigating a group of genes called microRNAs in Duchenne muscular dystrophy The aim of this project is to examine the role that a group of genes known as microRNAs (miRNAs) have in Duchenne muscular dystrophy. miRNA genes carry the instructions for small molecules that control the activities of other genes. They are an integral part of the normal functioning of the body, including normal muscle development and maintenance. Work carried out by Dr Matthew Wood in a mouse model of Duchenne muscular dystrophy has revealed that a number of important miRNA molecules are present in different amounts in diseased muscle compared with healthy muscle. Given the important role that miRNAs play in controlling the activities of other genes, altered miRNA activity might contribute to disease progression in Duchenne muscular dystrophy. Over the course of this one-year project based at the University of Oxford, Dr Wood and his colleagues will study in detail the miRNAs in the different muscles of the mdx mouse. They will then study any ‘abnormal’ miRNAs using computer prediction and cutting-edge experimental techniques to determine which genes they might be controlling and how this could affect the progression of Duchenne muscular dystrophy.

Gene therapy – delivering new genes and repairing old ones

Gene therapy time line

Dr Kristina Elvidge, Research Communications Officer, Muscular Dystrophy Campaign and Dr Alexandra Dedman, University College London It is now 20 years since the first gene therapy procedure was performed on a human being. The patient was a four-year-old girl with a genetic disorder called severe combined immunodeficiency (SCID) that made her vulnerable to even the mildest infections. Although billions of dollars have been pumped into gene therapy research, SCID remains one of the only diseases that can be successfully treated this way. Since the 1990s intense research efforts have been made to develop gene therapy technology for neuromuscular conditions and over the past five years these efforts have come to fruition with the commencement of clinical trials for Duchenne and limb girdle muscular dystrophies. Gene therapy approaches for other neuromuscular conditions are following in these footsteps, so it is a good time to put this technology under the spotlight.

What is gene therapy? DNA is a the ‘book of life’; containing all the instructions for building and operating our bodies. The book has about 25,000 ‘chapters’ or genes, each of which holds the instructions for creating a particular protein, such as collagen to hold your skin together or an enzyme to digest your food. With more than three billion characters in this book, mistakes affecting the production of an important protein sometimes happen, resulting in disease. These mistakes are known as mutations. Gene therapy aims to treat conditions arising from DNA mutations by introducing working versions of genes into cells. It is hoped that the introduced gene can compensate for the faulty gene and improve disease symptoms. The definition of gene therapy can be widened to include approaches that aim to repair mutations in genes using techniques known as exon skipping and gene silencing (see p6).

The challenges of gene therapy The development of a specific gene therapy approach bears a number of enormous challenges which might explain why it has not yet delivered what it originally promised. For example, the delivery vehicle for the new functioning gene has to be very efficient and transport its cargo into as many of the right type of cells as possible and it has to be safe. These key points are discussed in more detail below.

space for a human gene within their own DNA and at the same time these modifications prevent them from multiplying inside the cell and spreading uncontrollably throughout the body. Many different viruses have been tested for gene-therapy suitability but the adeno-associated virus (AAV) is currently the most attractive candidate for gene therapy because it is not known to cause any severe disease in humans and is capable of infecting many different cell types, including muscle cells and the heart. Unlike some other types of virus which insert their DNA randomly into the DNA of the cell, potentially disrupting a gene that prevents cancer, AAVs keep their DNA as a separate ‘package’ inside the cell. However, the disadvantage of using a virus that keeps its DNA separate, is that the DNA might be lost and the treatment not as long-lasting.

Targeting the right cells Another challenge is to get the therapeutic gene into the right cells. In neuromuscular conditions the cells that need to be targeted are the muscle cells (including the heart) or the nerves. Forty percent of our body is muscle, so distributing the gene to that muscle mass is a big task. To distribute the virus throughout the body it needs to be injected into the bloodstream. But AAV naturally infects a range of different cell types which will inevitably also deliver the virus and therapeutic gene to organs such as the liver and the kidneys. In order to avoid large amounts of a muscle protein being made in organs where it is not naturally produced, and potentially damaging, scientists have developed a trick. They attach a piece of DNA to the therapeutic gene which carries a signal that allows the gene to be produced only where it is needed – in the muscle cells.

Gene therapy using adeno-associated virus (AAV)

A computer generated image of an adeno-associated virus (AAV) The development of a gene therapy approach that targets the nerves is a more difficult matter. The nerves are relatively inaccessible and the safety of injecting viruses into the nervous system is still uncertain. However, in animal models it has been shown that certain types of AAV are able to reach the nerves.

Milestone

1966

The concept of gene therapy in humans is first discussed by scientists

1987

Discovery of the dystrophin gene

1990

Start of the first ever gene therapy clinical trial for a genetic immune disorder (SCID)

1990

The possibility of a mini-dystrophin gene therapy is proposed by scientists

1995

Idea of exon skipping for Duchenne muscular dystrophy first discussed by scientists

1998

Gene therapy successful in a mouse model of limb girdle muscular dystrophy 2D

1999

Exon skipping successful in a Duchenne muscular dystrophy mouse model

1999

Big set back for gene therapy as a participant dies in a clinical trial for a metabolic disorder called OTC

2000

Gene therapy successful in a mouse model of Duchenne muscular dystrophy using AAV delivery of a mini-dystrophin gene

2006

Start of the first clinical trial of mini-dystrophin delivered by AAV into the bicep muscle of boys with Duchenne

2006

Dutch pharmaceutical company Prosensa starts the first clinical trial of exon skipping for Duchenne muscular dystrophy; injecting into a muscle in the ankle

2008

Start of the phase I clinical trial of AAV gene therapy for limb girdle muscular dystrophy type 2D – it is injected in the foot

2008

Prosensa starts first body wide trial of exon skipping for Duchenne – injecting the AOs under the skin

2008

First UK exon skipping trial for Duchenne muscular dystrophy – the molecular patch is injected into a muscle in the foot

2009

Second UK exon skipping trial begins – the molecular patch is injected into the blood stream

2010

Prosensa starts phase III clinical trial of exon skipping for Duchenne which will last for 12 months

Immune reactions A major concern is that gene therapy can activate immune reactions in patients. Any foreign material that is not recognised by the body may cause an immune reaction as the body tries to reject it. Besides causing side effects, an immune response that quickly clears the introduced material from the body will render the gene therapy ineffective. The choice of virus is very important as some trigger more severe reactions than others. People are more likely to have an immune response primed to attack if they have had a prior infection with the same virus. A solution could be to prescreen patients to find those that are more likely to react to the virus and combine their treatment with immune suppressing drugs. There is also a risk that the immune system might regard the newly produced protein as foreign. This is a possibility if a person’s condition is caused by a genetic mutation that prevents any protein being made from that gene. It is less of a threat for mutations that reduce the amount of protein or cause the protein that is produced to not work correctly. In these cases the body will have seen the protein before and will probably accept it. There are several strategies under development to avoid triggering an immune response. Various regimes of drugs to suppress the immune system while the patient undergoes gene therapy are being tested. Secondly, the method of administration is very important in influencing immune response. Injection directly into the muscle is not ideal because high local concentrations of the virus in the muscle are more likely to trigger an immune response. It is hoped that administration into the bloodstream will dilute the introduced virus and cause milder immune reactions.

Gene delivery Viruses are often used to deliver the therapeutic gene as they are expert at invading human cells, and once inside, are adept at hijacking the cell machinery to read their own DNA sequence and make proteins (see diagram right). Viruses used for gene therapy are engineered to create

Year

Difficult genes Sometimes the therapeutic gene itself can represent a problem. Duchenne muscular dystrophy, for example, is caused by mutations in the gene responsible for producing dystrophin. Unfortunately

the dystrophin gene is too big to be inserted into a virus. However, researchers have taken inspiration from a man with Becker muscular dystrophy who despite having almost half of the dystrophin gene missing has very mild symptoms. Based on his gene smaller versions of the dystrophin gene, with the non-essential parts removed, have been designed. These are known as ‘mini-’ or ‘micro-dystrophin’. This is a good example of how understanding the underlying genetic defect is vital to develop therapeutic approaches.

How far have we come? Progress in gene therapy research for neuromuscular disease continues to gain momentum. Animal trials are producing promising results and several phase I clinical trials have been conducted. In a recent report researchers used AAV to deliver a therapeutic gene to the nerve cells of newborn mice with spinal muscular atrophy (see p8). The pups showed hugely improved neuromuscular function and life span, highlighting the enormous potential of gene therapy and setting the stage for trials in patients. Two phase I clinical trials of gene therapy for limb girdle muscular dystrophy (LGMD) have been conducted. The sarcoglycan genes that are responsible for causing LGMD types C, D, E and F are small, so they

are good candidates for this approach. The clinical trials in the US and France tested AAV gene therapy in patients with LGMD2D and LGMD2C respectively. These trials were designed to assess safety by injection into one muscle – either in the arm or foot. In both trials the gene therapy was deemed to be safe and a significant amount of the sarcoglycocan proteins was produced in the injected muscle. One patient out of the six in the LGMD2D trial had an immune response to AAV and as a result little alpha-sarcoglycan protein was produced. A safety study of AAV delivery of a mini-dystrophin gene has been carried out in six boys with Duchenne muscular dystrophy which involved injection into a muscle in the arm. The results of this trial were disappointing with only two of the boys producing a small amount of dystrophin protein. Some of the boys in the trial were found to have an immune response to the newly produced dystrophin protein, which may have resulted in its destruction. Researchers believe that the improvements in gene therapy technology currently being developed will be able to avoid this immune response in the future.

Repairing old genes Therapies that aim to repair or change existing genes have recently stepped into the limelight. This approach is most advanced for Duchenne muscular dystrophy with clinical trials underway, but variations of the technology are being explored for other conditions. These potential therapies rely on small pieces of DNA synthesised in the laboratory called ‘antisense oligonucleotides’ (AOs), otherwise known as ‘molecular patches’, which are designed specifically to bind to part of a gene.

Exon skipping Exons are the essential segments of genes. AOs can be used to mask an exon, which results in the processes inside the cell skipping over that piece of information. To understand exon skipping we need to look at how the DNA code is read inside a cell. The genetic code consists of a sequence of letters read in groups of three, like three-letter words. However, there is no punctuation to indicate where the words start or stop. Take this example: ‘themanranforthebus’. This gives the sentence ‘The man ran for the bus’. If a mutation caused one of the letters to be deleted, in our example the first ‘e’, it would read like this ‘Thm anr anf ort heb’, which no longer makes sense. So, mutations are particularly detrimental if they occur in anything other than multiples of three because they disrupt the reading of the rest of the code. Exon skipping is one way of attempting to overcome these errors in the code.

Exon skipping therapy for Duchenne muscular dystrophy currently shows great promise for restoring dystrophin protein expression, with very encouraging data reported by the MDEX consortium in the UK and also from Prosensa in the Netherlands. It will now be vital to extend the study length in Duchenne patients and to test this treatment in many more patients in order see to what extent this restored dystrophin can prevent or delay progression of the disease. These are exciting times for Duchenne muscular dystrophy gene therapy Dr Matthew Wood, University of Oxford

Exon skipping comes into play when a gene is ‘switched on’ and all of the exons of a gene are copied and assembled together into a molecule called RNA, which the cell then uses to make a protein. Exon skipping causes a segment to be excluded during the assembly of the RNA copy of the gene. So, in addition to the mutation which removes part of the gene, exon skipping removes an extra segment. But how can this be a good thing? If we return to our ‘The man ran for the bus’ analogy with the first ‘e’ deleted to give ‘Thm anr anf ort heb’. Exon skipping would aim to remove the ‘th’ before the deleted ‘e’ to give the sentence ‘Man ran for the bus’ which isn’t perfect but we still understand what it means. The dystrophin gene, which is faulty in boys with Duchenne muscular dystrophy, is particularly amenable to exon skipping. It is a very large gene with 79 exons which produces a very large protein with lots of repeated segments in the middle (see diagram below). It is known that the protein can still work, at least to some extent, if some of these repeated segments in the middle are missing, as long as the important ends are intact. This is the case for Becker muscular dystrophy which has more mild symptoms – affected individuals are often still able to walk into their 40s and 50s. So exon skipping aims to transform the severe symptoms of Duchenne into the more mild symptoms of Becker muscular dystrophy. As a starting point, AOs have been designed to skip exon number 51 of the dystrophin gene, which would be applicable to about 13 percent of boys with Duchenne muscular dystrophy. Preliminary results from the exon skipping clinical trials are encouraging and more clinical trials are

Dystrophin protein with repeats Dystrophin is a very large protein with a section in the middle consisting of many repeated segments (in dark pink) and it is known

that the protein can still work to some extent if some of these repeated segments are missing.

Quick facts about antisense oligonuleotides (AOs)

Gene therapy research funded by the Muscular Dystrophy Campaign 1) Improving AAV gene therapy for Duchenne muscular dystrophy Prof. George Dickson at the University of London is investigating the AAV delivery of dystrophin in a mouse model of Duchenne muscular dystrophy. His team will compare different minidystrophins to discover which is the most efficient at restoring muscle function. Innovative new techniques will also be explored to deliver the full dystrophin gene in three parts to be reassembled once inside the cell. Preventing an immune response during gene therapy is also being studied.

ongoing (see p15). Exon skipping enters the realm of personalised medicine as molecular patches will need to be tailor made to correct mutations that can occur anywhere along the dystrophin gene. Following on from this initial success, researchers and pharmaceutical companies are now developing patches to treat other regions of the dystrophin gene which will make this technology applicable to more boys with Duchenne muscular dystrophy.

2) Delivering molecular patches to the muscle using a virus Prof. Davies and her colleagues at the University of Oxford are aiming to improve exon skipping technology by using AAV to deliver AOs to the muscle. They are testing this approach in a mouse model of Duchenne muscular dystrophy. They hope that this will improve the efficiency of exon skipping throughout the body for boys with the condition.

Preventing exon skipping for spinal muscular atrophy

3) Improving the delivery of molecular patches using peptides. Dr Wood at the University of Oxford received a new grant this year to improve the delivery of molecular patches (AOs) by attaching small protein fragments called peptides to them. For more details of this project please see p3.

For the treatment of spinal muscular atrophy (SMA) the aim of the AOs is reversed – preventing unwanted exon skipping. SMA is caused by the lack of the SMN gene which is critical for motor neuron survival. However, scientists can take advantage of the fact that everybody has at least one copy of the closely related SMN2 gene. This gene mostly produces SMN protein that does not work because an essential segment is removed when the gene is copied into RNA. AOs can be used in this instance to mask the signal in the SMN2 DNA code that indicates that this essential segment should be removed. This can result in the production of functional SMN protein. This approach has been successfully tested in a mouse model of SMA.

Blocking interactions between genes and proteins in myotonic dystrophy DM1 – the most common form of myotonic dystrophy – is caused by the expansion of a repeated section of DNA in a gene called DMPK. A three-letter code in this gene is repeated many hundreds of times instead of the usual number – less than 30. The RNA copy of this gene gets stuck inside the nucleus of the cell and sticks to certain proteins causing them to form clumps. This prevents these proteins from performing their normal function within the cell and leads to the wide range of symptoms of myotonic dystrophy. Researchers have successfully tested AOs in mice to specifically bind to the repeated section of RNA and block it from sticking to proteins.

Gene silencing Another way of using AOs is to block the activity of genes, a technique called RNA interference. In this case a special type of AOs, different from those used for exon skipping, specifically binds to the RNA copy of a gene and causes it to degrade before it can produce a protein.

This approach is relevant when a mutation results in the over production of a protein or the mutation changes the function of a protein so that it is toxic. For example, recent research has revealed that facioscapulohumeral muscular dystrophy may be a potential candidate for gene silencing because it is caused by the activation of a gene called DUX4 which is thought to be toxic (see p9 and 27). Similarly the most common form of Charcot-Marie-Tooth disease, CMT1A, is caused by an extra copy of a gene called PMP22 which results in too much PMP22 protein being produced, which is toxic to the nerves.

Conclusions Using DNA as drug has tremendous potential – it targets the primary cause, can have a long-lasting effect, and it doesn’t necessarily require detailed knowledge of the disease process. However, over the past 20 years gene therapy has not delivered what it originally promised which might be partly due to unanticipated challenges that clinicians and researchers have had to face. Research efforts have remained strong though, and in the past few years gene therapy has come along in leaps and bounds. A number of clinical trials have started and if they are successful we will see this technology move quickly forward for a range of conditions. For more information go to: www.muscular-dystrophy.org/research/news and www.muscular-dystrophy.org/exonskipping

Research news from around the world

SMA gene therapy success Scientists in Ohio, USA led by Dr Brian Kaspar have successfully treated a mouse model of spinal muscular atrophy (SMA) using a gene therapy approach. Day-old mice injected with AAV9 virus (see p4) containing the gene missing in these mice (SMN1) survived for more than 250 days compared to the average 13-day lifespan of their untreated counterparts. They were also able to move around almost as well as normal mice. However, the treatment was only successful if administered within the first two days of life. Researchers in Sheffield led by Prof. Azzouz have also since reported similar results. The American researchers also showed in their study that this type of virus was capable of penetrating the nervous system of monkeys. The researchers hope that after further safety tests in the laboratory, the stage will be set for clinical trials to test the benefit of this gene therapy approach in people affected by SMA.

Promising new research on a drug to protect the heart muscle Researchers in the USA have published exciting results of a potential method to protect the heart muscle from damage caused by muscular dystrophy. Individuals with Duchenne, Becker and some other forms of muscular dystrophy are susceptible to tears in the fragile membrane surrounding heart muscle cells which eventually leads to cell death and a heart condition known as cardiomyopathy. The researchers in Minnesota led by Dr Joseph Metzger therefore investigated a man-made chemical called poloxamer-188 (P188) which is thought to be able to seal tears in cell membranes. The researchers repeatedly injected P188 into the blood stream of a dog model of Duchenne muscular dystrophy over an eight-week period. This large animal model is considered to be better than the mdx mouse model because it better mirrors the severity of the symptoms experienced by boys with Duchenne muscular dystrophy. They found that P188 helped to protect heart cells from dying and maintained the heart structure. Because of its ability to target cell membranes, P188 is already used as an

ingredient in some medicines and it is approved for short-term use for various heart conditions. After further experiments to determine the best dose of P188, the next step will be to conduct clinical trials assessing the effectiveness and safety of P188 treatment in humans with muscular dystrophy.

Critical breakthrough in understanding FSHD

of action – one which involves interacting with DNA and one which involves interacting with proteins. Research has shown that it is the interaction with DNA that causes side effects. Drugs are now being developed that are similar to glucocorticoids but do not interact with DNA. Testing these new drugs in a mouse model of Duchenne muscular dystrophy showed that they are at least as effective as prednisone and did not cause the usual side effects, even at doses forty times higher than usual. Due to the vast experience with steroids in humans, the planned clinical trials of such alternative steroid drugs are expected to be relatively short.

Collaboration between researchers from the Netherlands, USA, France and Spain has resulted in a greater understanding of the genetics of FSHD. This is a critical breakthrough because although the genetic mutation causing FSHD was identified nearly 20 years ago, scientists have struggled to understand how this mutation causes this complex condition. FSHD is caused by changes to a region of DNA on chromosome 4 called D4Z4 that has the same piece of DNA code repeated many times. In healthy individuals the number of repeats varies between 11 and 100. People with FSHD have fewer than 11 repeats. The scientific community has previously disagreed about the function of D4Z4; some believed that D4Z4 regulates the activity of neighbouring genes without being a gene itself. However, this new research has confirmed that the repeated pieces of DNA do indeed contain a gene – called DUX4 – and proposes a new model for how the condition develops. Firstly, scientists have known for a while that a reduction in the number of repeats on D4Z4 to less than 11 changes the way this piece of DNA is folded (this is known as the chromatin structure). This exposes the DNA code to be read by the cell, like opening a book. Secondly, this new research has shown that an activation signal also needs to be present next to the DUX4 gene. In other words, the book needs to be open and the light switched on before you can read it. This causes the DUX4 gene to be active and its product – the DUX4 protein – is then toxic to the cell. This new information will help scientists move closer to developing targeted treatments for the condition. We still don’t understand the function of the DUX4 gene though, and why it is toxic. This will be the next piece of the puzzle to put in place. Prof. Jane Hewitt, Nottingham and Dr Peter Zammit, London are currently funded by the Muscular Dystrophy Campaign to investigate the detailed function of the DUX4 gene. D4Z4 region contains 11 to 100 repeats – DNA is tightly coiled and can’t be read

D4z4 region contains less than 11 repeats – DNA is more relaxed and can be read

New steroids for Duchenne muscular dystrophy Our Research Communications Officer, Dr Kristina Elvidge, attended the International Congress on Neuromuscular Diseases in July and heard Prof. Eric Hoffman from the US talk about how recent research may lead to the development of new steroids that work better and have fewer side effects. Steroids are currently the only treatment for Duchenne muscular dystrophy proven to slow down the progression of symptoms, but they can cause side effects such as bone fragility, mood changes and weight gain. New research into how glucocorticoid steroids such as prednisone work has shown that they appear to have two modes

No FSHD

Basic research leads to better management of periodic paralysis Prof. Frank Lehmann-Horn from Germany reported on advances in understanding the muscle condition called hypokalemic periodic paralysis (HypoPP) at the International Congress on Neuromuscular Diseases. HypoPP causes intermittent attacks of muscle weakness and in the long term the overall strength of muscle can decline, leading to significant disability. Periodic paralysis is caused by problems with the pores which exist in the walls of

No activation signal

Activation signal Toxic DUX4 gene activated

No FSHD

FSHD

muscle cells. These pores or channels allow the passage of ions such as sodium, calcium, chloride and potassium into and out of the muscle cells, which allows muscles to conduct the electrical signals required for muscle contraction. The researchers studied the levels of potassium and sodium inside the muscle cells of HypoPP patients and found that although low potassium levels were associated with the intermittent attacks of weakness as previously thought, increased sodium levels may

contribute to the long-term muscle weakness. They found that treating patients with drugs that decrease the amount of sodium inside the muscle cells increased muscle strength. As a result of this research it is now recommended that levels of sodium inside the muscle cells are carefully monitored using a new type of MRI and treatment with sodium-lowering medications. In their experience, with this standard of care, long- term weakness leading to loss of mobility can be avoided or even reversed.

Big step forward for muscle stem cells What do muscle stem cells and Goldilocks have in common? It turns out that they are both very fussy about the surface that they lie on. Muscle stem cells – the cells that repair muscle when it is damaged – are notoriously difficult to keep alive once isolated from a person or animal. They also quickly lose their stem cell properties once in a Petri dish in the laboratory. Researchers in California led by Prof. Helen Blau investigated the conditions in which muscle stem cells are grown in the lab and discovered that the softness of the surface they are grown on is crucial. They grew mouse muscle stem cells on gel surfaces with various degrees of softness and found that they had a striking preference for the gel that best imitated the softness or pliability of muscle. The stem cells grown on this gel for one week retained their stem cell characteristics and when transplanted back into mice they were almost as good at regenerating muscle as freshly isolated cells. By contrast, those grown on hard plastic did not successfully transplant at all. This research is vital for the development of stem cell therapies because it allows muscle stem cells to be efficiently grown outside the body, increasing their numbers and giving an opportunity to manipulate them prior to transplant into a patient. However, the authors commented that the cells grown in the laboratory were not able to regenerate muscle to the same extent as freshly isolated cells, so there must be other factors that the cells require to maintain their properties outside the body. Further research is required into this and our researchers Dr Peter Zammit, Dr Jenny Morgan and Dr Lesley Robson (see p34) are all investigating various aspects of this area.

For more information go to www.muscular-dystrophy.org/research/news

Focus on hydrotherapy

Hydrotherapy is widely regarded as beneficial for people with neuromuscular conditions by patients, doctors and physiotherapists, but why is accessing it so difficult? We asked a specialist physiotherapist, our campaigns team and a service user for their points of view

The physiotherapist Hydrotherapy is one of the most well-liked therapeutic interventions for people with neuromuscular conditions. Exercising in water may be rehabilitative following a fracture or to improve mobility in the face of progressing weakness, or it may be primarily for maintenance and to provide the sense of well-being that is proven to be associated with regular exercise. Water provides both buoyancy and resistance at a level that can be individually controlled. For people with weak muscles and chronic pain the warm water is relaxing and enjoyable and enables muscles that are unable to move against gravity a freedom that cannot be experienced on land. For people without the strength to move against gravity this may be their only opportunity to actively exercise. The Government promotes exercise and currently actively encourages swimming by reducing the cost of entrance to swimming pools. However, these pools are frequently inaccessible for disabled people and wheelchair users and the water can be too cold for people to exercise comfortably. A warmer alternative is hydrotherapy but in many areas of the UK access to these facilities is limited. This poor provision is probably due to high running costs and a shortage of physiotherapists. All of this means that people with neuromuscular conditions

are unable to access therapy and leisure activities that may be beneficial. The rationale for the funding of treatment in the NHS is based on evidence. The best evidence for some types of treatment is provided by randomised controlled trials where an intervention is tested against a placebo and both the participant and the scientist are unaware of who is receiving the ‘real’ therapy. Clearly it is impossible to conduct such an experiment with hydrotherapy although there have been a handful of small studies without a placebo which have shown positive benefits. The NHS needs to rethink the kind of evidence required for funding a treatment such as hydrotherapy where a placebocontrolled trial is not possible. Comparing such things as the number of unplanned hospital admissions, number of falls and

Water provides both buoyancy and resistance at a level that can be individually controlled. For people with weak muscles and chronic pain the warm water is relaxing and enjoyable and enables muscles that are unable to move against gravity a freedom that cannot be experienced on land. quality of life (using questionnaires) between those people with access to hydrotherapy and those without, might demonstrate a significant benefit to patients and cost saving to the health service. This would however require a large number of patients

with each type of condition and since most neuromuscular conditions are rare or very rare, such a study may be prohibitively difficult and expensive. Perhaps the most effective way to improve facilities is by advocacy groups, such as the Muscular Dystrophy Campaign and patients and families lobbying their MPs and vocalising their support for this treatment. It is not only the NHS that needs to change its perception and provision of hydrotherapy but also local councils so that pools can be built with accessible facilities and warm water where people with neuromuscular conditions can exercise in their own time and at their leisure.

Dr Michelle Eagle, Consultant Physiotherapist, Newcastle Muscle Centre

The campaigner Bringing up access to hydrotherapy or specialist physiotherapy at any of our regional Muscle Group meetings always brings forward responses from members who tell us time and time again of being denied physiotherapy or hydrotherapy despite the huge benefits they feel it brings them in maintaining muscle strength, lung function and slowing the progression of their neuromuscular condition. We hear from young people who have their physiotherapy and hydrotherapy taken away when they turn 18 – just as their condition is deteriorating, from adults with neuromuscular conditions who go for years without the essential therapies that can keep them mobile and help to prevent falls, and from people who rely on chest physiotherapy to prevent infections and breathing problems. The results from our 2010 National Patient Survey – State of the Nation – back up

this evidence. Our survey showed that only one in 20 people with a neuromuscular condition has access to ongoing NHSfunded hydrotherapy. One Muscle Group member, Khurm Arshad from the South West, faced huge difficulties in finding a hydrotherapy pool for his brother Auzair, who has Duchenne muscular dystrophy. Khurm said, “I had been looking on the Internet for hydrotherapy pools and I was coming back with more and more results for animals but none for humans. When I first realised what the situation was, I thought it was an absolute joke.” Shocked by the lack of hydrotherapy pools for people with neuromuscular conditions, Khurm and his colleagues from the South West, decided to investigate further. They found that there are very few hydrotherapy pools available to families with neuromuscular conditions in the South West and where they could visit a pool, families faced high fees and in some cases, were required to supply their own lifeguard and public liability insurance. Building on the evidence of the members of the South West Muscle Group, campaigners from all over the UK are now investigating their own local hydrotherapy pools, to create a national picture of the availability of these pools and to fight for everyone with a neuromuscular condition to have access to ongoing hydrotherapy. To join the investigation, get in touch with the Policy and Campaigns Team on 020 7803 2865 or email campaigns@muscular-dystrophy.org

Alexandra Crampton, Senior Policy and Campaigns Officer at the Muscular Dystrophy Campaign

The service user

Baroness Celia Thomas of Winchester, Peer in the House of Lords, Vice President and Trustee of the Muscular Dystrophy Campaign, told us of her experience with physiotherapy and hydrotherapy: “Nearly twenty years ago, at the age of 46, I was diagnosed with adult onset limb girdle muscular dystrophy. The following year I met an American physical therapist, Meir Schneider, whose experience had taught him that the right gentle exercise and massage regime could greatly help people with all kinds of musculo-skeletal problems, including muscular dystrophy. I am still largely following his advice, although now at a reduced rate. I try to do water and landbased exercises at least once a week. In the swimming pool I use a flotation belt which enables me to “swim” in a bicycling position up and down the pool, exercising my legs without tiring them. I’m lucky as I don’t need very warm water. This kind of exercise seems to loosen tight muscles and helps to strengthen weak ones. My landbased exercises, done on a bed, involve loosening tight hip, ankle and knee muscles. Neither of these exercises takes more than twenty minutes in total – it’s important not to get tired. “If I led a less hectic life, I would exercise more, but as I still walk – albeit pushing a walker – I have to leave enough energy for that and especially for voting in the Lords!” Read the State of the Nation report www.muscular-dystrophy.org/stateofthenation Share your hydrotherapy experiences on the TalkMD forum www.muscular-dystrophy.org/talkmd

Translational research – the winding road to treatment Dr Marita Pohlschmidt, Director of Research, Muscular Dystrophy Campaign

Translational research has been the focus of research funding since the start of the new century, but when asked about it only a few people have an exact idea what translational research is. It seems important for almost everyone, but it means different things to different people. When put into a search engine on the Internet, one of the definitions that appears is: “Translational research is the process which leads from evidence based medicine to sustainable solutions for public health.” This is a rather complicated definition. For many, translational research means the ‘bench-to-bedside’ transfer of promising technology – an expression that starts to make sense. In 2006 the US National Institutes of Health (NIH) launched a Clinical and Translational Science Award programme that funds research in this area and also encourages the establishment of centres of translational research. The NIH envisages funding 60 centres by 2012 with a budget of $500 million per year. The UK is currently investing £450 million over five years for the establishment of translational research centres. One of them is the MRC Centre for Neuromuscular Diseases, a joint venture between the UCL Institute of Neurology, London and the University of Newcastle upon Tyne. Along with these Government initiatives, foundations, industry and the charitable sector have also started their own translational research programmes. Most neuromuscular conditions are inherited and the identification of the genes that harbour the mutations has been regarded as the gateway to develop treatments or even cures. Researchers now have a much better understanding of how muscles function than they did 20 years ago. The study of the genes that carry the mutations, the effect that these mutations have on the production of proteins and their interaction with each other in muscle fibres, has helped to unravel the underlying processes that lead to muscle wasting. However, although a number of promising technologies have demonstrated tremendous potential in preclinical experiments in the laboratory and some are now even being tested in clinical trials, to date there is no efficient treatment available for nearly all of the neuromuscular conditions that come under the umbrella of our charity. In order to translate promising technology into a clinical context it is not sufficient to only have the technology for a new therapeutic approach ready in the laboratory. There are a number of areas that need to be addressed to evaluate the effectiveness and safety of new promising technology in humans and all these activities are generally described as ‘translational research’. The following article aims to

From bench...

...to bedside

shed light on the different areas of this discipline and in particular what it means for people affected by muscular dystrophy or related neuromuscular conditions.

The translational triangle In order to be ready to test a new medicine or medical device in a clinical trial there are three essential elements that need to be in place – illustrated by the triangle below:

New Technology

Therapy

Patients

Outcome Measures

The translational triangle: A clinical trial not only requires promising new technology, it also requires locating patients fitting the criteria to take part in a trial and well-defined ways of assessing the potential benefits of a treatment. Firstly, preclinical studies need to be completed, including testing the technology in muscle cells grown in the laboratory and in animal models. The second crucial necessity is locating people who have the condition and are able to take part in clinical trials. This has been and still is a real challenge as most neuromuscular conditions are rare or even ultra-rare.

For example, the pharmaceutical company Genzyme developed a treatment for Pompe disease – the first effective treatment for an inherited muscle condition. In the UK there are about 100 people with this condition caused by a missing enzyme called acid alphaglucosidase (GAA). GAA normally breaks down excess glycogen – a form of sugar stored in the muscle. The missing enzyme leads to a build-up of glycogen that destroys the meticulously arranged order of proteins responsible for muscle contraction. In 2006 the company brought Myozyme to the market. This commercially produced enzyme is injected into the bloodstream and it makes its way into the muscles to prevent glycogen build-up. For the severe juvenile form of Pompe disease which is normally fatal within the first year of life, this treatment has been life-saving. The company spent $800m for its development and had to invest several million dollars and a considerable amount of time to find families affected by Pompe disease to participate in clinical trials. The technical term for the third requirement is ‘outcome measures’. These are defined ways of testing the benefit of a new potential treatment. For neuromuscular conditions this requires reliable ways to assess the changes in the muscles or nerves in response to a therapeutic intervention. And these have to be tested and agreed upon by all participating clinicians and health professionals conducting a clinical trial because otherwise it is not possible to compare results between different clinical trial sites. International harmonisation of outcome measures is particularly necessary for rare conditions as clinical trials at some stage have to be conducted in more than one country to allow inclusion of enough patients.

Clinical trial readiness The efforts that have been made to address these issues might be best illustrated by describing the activities of TREAT-NMD, a network of excellence funded by the European Union with 10 million euros over five years. It started in 2007 and aims to establish the international infrastructure that promotes clinical trial readiness. This initiative consists of 11 different work packages and establishing patient registries and driving international consensus for outcome measures are a large part of it. The coordination office is at the University of Newcastle upon Tyne

and Professors Kate Bushby and Volker Straub are leading the project. In the following paragraphs some of the activities are described in detail.

Patient registries Patient registries are databases that contain information about people affected by a condition with the focus on what is needed for clinical trials. The registries are generally designed for one specific condition and include basic information about the relevant characteristics and symptoms of those patients. TREAT-NMD aims to stimulate the development of national registries in each country which are then combined into a global registry. Prior to the transfer, patient data is encrypted so that only the curator (see below) of the national registry can make the association between the data and an individual’s name in the national registry. To ensure best legal and ethical practice TREAT-NMD has published the ‘TREAT-NMD Registry Charter’ to which all national registries have to adhere to. The Charter regulates the relationship between the national and global registries and defines minimum requirements to ensure international harmonisation. In most countries the patient registries are self-reported registries – the individual enters the data him/herself and the information is owned by the individual. The registry is usually monitored by a database curator who ensures the information is correct and up to date. For complex clinical and genetic information the individual is asked to give his/her permission for the curator to contact the treating clinician or genetic consultant. Individuals have access to all the data that is stored under their name and have the right to withdraw the data at any time. National and global registries have been developed for Duchenne muscular dystrophy and spinal muscular atrophy. The global registry for Duchenne muscular dystrophy now includes more than 10,000 patients from 36 countries. More than 2,000 patients have been entered into the global registry for spinal muscular atrophy from at least 30 national registries. The Muscular Dystrophy Campaign is currently involved in establishing a UK national registry for myotonic dystrophy and facioscapulohumeral muscular dystrophy.

For ultra-rare conditions such as the congenital muscular dystrophies (CMD) and limb girdle muscular dystrophies (LGMD) a slightly different approach is being pursued. These groups of conditions are very mixed and although clinically diagnosed as one disease, they can be caused by mutations in a number of different genes. Some forms within these groups are ultra-rare with less than ten known affected individuals in the UK, so international registries are being set up without the intermediate step of national registries. The Muscular Dystrophy Campaign has formed a partnership with an American patient organisation – CureMD – to support the international CMD registry. This registry can be accessed through our website (www.muscular-dystrophy.org/patientregistries). TREAT-NMD has set up an international registry for patients with defects in a protein called fukutin-related-protein (FRKP) which can be a cause for a form of LGMD and CMD. We also support this registry by funding a clinical fellowship at Newcastle University.

Care and Clinical Trial Site Registry

Natural history databases

The future

Natural history databases differ slightly from patient registries and sometimes the use of the two terms can be confusing. The purpose of natural history databases is to better understand the natural progression of a disease and hence they are designed to capture detailed clinical data that are entered by the treating clinician. This clinical data gives clinical trials baseline information against which the benefit of a therapy can be measured. For example, when the clinical trial for Pompe disease was designed it was regarded as unethical to conduct a placebo-controlled trial, because of the severity of the infantile form. Every baby that participated in the clinical trial received the drug and the benefit was measured by comparing the clinical trial data to natural history data. In addition, outcome measures – how to best assess improvements in muscle function – can be developed from the analysis of the data in natural history databases. The Muscular Dystrophy Campaign currently provides funding for the NorthStar natural history database to clinically monitor children with Duchenne muscular dystrophy and SMArtNET for individuals with spinal muscular atrophy. The set of databases was recently expanded to include congenital muscular dystrophies and myopathies and inclusion body myositis.

The initiatives described above are only a fraction of the activities that carry the label ‘translational research’. The work of TREAT-NMD also includes work packages that address the identification of the best animal models and international harmonisation of experiments that should be done to find out which are the most promising technologies to move forward to clinical trial. The remit for a further work package is the establishment of TACT – an Advisory Committee for Therapeutics. This committee gives researchers and companies the opportunity to seek the advice of world-renowned experts to evaluate the design of planned clinical trials. TREAT-NMD also played an important role in the standards of care recently launched for Duchenne muscular dystrophy and spinal muscular atrophy. These guidelines encourage clinicians worldwide to treat patients to the same high standard so that when clinical trials start, all patients are starting from the same baseline. The European Neuromuscular Centre (ENMC) is one of the biggest partners of TREAT-NMD. Its responsibility is to disseminate any outcomes to patient organisations and also provide training for clinicians and health professionals especially in Eastern European countries where health care services are lagging behind. In summary, the focus on translational research in the last decade has had a tremendous impact on the speed with which promising treatments for neuromuscular conditions can now move forward into clinical trial. The good news is that improvements in infrastructure have already attracted the interest of big pharmaceutical companies such as GlaxoSmithKline who are now willing to invest the funds necessary for clinical trials for neuromuscular conditions. However, although there is cautious optimism in the scientific community, it is very difficult to predict exactly when the first treatments for neuromuscular conditions will be brought to the market. All we are sure about is that it cannot be soon enough for the families whose lives are affected by them.

The therapeutic interventions that are currently being developed for neuromuscular conditions are often new technical approaches that require a high standard of clinical expertise from the muscle centres that take part in a clinical trial. A Clinical Trial Coordination Centre was established by TREAT-NMD that manages an international Care and Clinical Trial Registry. The registry collects information about existing muscle centres worldwide and evaluates their suitability for administering new potential treatments and carrying out the follow-up of individuals participating in the study. Considering the rarity of some neuromuscular conditions it is expected that a number of international study sites are necessary to have sufficient patients enrolled in a clinical trial. It is therefore of vital importance that every study site treats the participating individuals and assesses the benefit of a treatment in the same way. Only then is it possible to pool all the data and obtain robust and reliable results.

For further information about translational research and patient registries www.muscular-dystrophy.org/patientregistries and www.treat-nmd.eu.

Clinical trials update

AVI Biopharma exon skipping news Promising new results from the UK exon skipping clinical trial for Duchenne muscular dystrophy were released in June. This phase I/II trial involved delivering a molecular patch (AVI-4658) to the whole body via the bloodstream. This molecular patch is designed to skip exon 51 of the dystrophin gene (see p6) and could potentially be used to treat 13 percent of boys with Duchenne muscular dystrophy. The trial’s main aim was to assess the safety of body-wide treatment with AVI-4658. The 19 participants in the trial were treated with the molecular patch for 12 weeks at six different doses. They had muscle biopsies taken before and after treatment to allow measurement of the amount of dystrophin protein produced in the muscle. The eight participants given the two highest doses showed consistent skipping of exon 51 and all produced new dystrophin protein in the muscle fibres. Encouragingly, three participants had a strong response with 15 percent, 21 percent and more than 50 percent of the muscle fibres containing dystrophin after treatment with

AVI-4658. However, the response was variable from patient to patient with some boys producing very little dystrophin even at the higher doses. Importantly the molecular patch appeared to be well tolerated by all participants. No antibodies reacting to the newly produced dystrophin were detected in blood samples and indications of muscle inflammation were reduced. Muscle strength and lung and heart function measurements remained stable, but given the short duration of the study and the small number of participants, it may have been ambitious to expect to see an improvement. AVI Biopharma plans to start a phase II clinical trial in the US by the end of 2010 to test higher doses to try to achieve more consistent dystrophin production among patients. The company has also started preclinical development of a molecular patch to skip exon 50 which would be applicable to four percent of boys with Duchenne.

Prosensa/GlaxoSmithKline exon skipping news The Dutch pharmaceutical company Prosensa is testing a slightly different chemical formulation of molecular patch in the Netherlands, Belgium, Sweden and Italy and in partnership with GSK in the US. The company has been testing a molecular patch to skip exon 51 administered to the whole body by injection under the skin. A phase I/II trial of this patch was completed in May 2009. The company said that the treatment was well tolerated and dystrophin production was seen in muscle biopsies. Since then, the trial participants have received treatment for a further six months to gather more long-term data to prepare for a large international phase III trial which started this year. The phase III

trial will run for 12 months and will hopefully start to answer questions about whether these patches can alleviate or slow down the progression of the symptoms of Duchenne muscular dystrophy. This year Prosensa also initiated a phase I/ II clinical trial to test a second molecular patch to skip exon 44 – applicable to six percent of boys with Duchenne. This trial is being carried out in the Netherlands, Belgium, Sweden and Italy. A new phase I clinical trial opened this year in Ohio to test the safety of the molecular patch to skip exon 51 in older boys with Duchenne muscular dystrophy who are unable to walk. They will measure how quickly the patch is absorbed and broken down by the body. Since these boys are less active than those tested previously, the rate of absorption and breakdown of the patch could be different which may have implications for the dose required by them. This trial will provide initial dosage and safety information for any future clinical studies in this patient group and allow the development of methods to measure the effectiveness of the treatment. In June Prosensa and GSK announced new programmes to develop four new molecular patches in the laboratory prior to clinical trial in patients. These new patches will be designed to skip exons 45, 52, 53 and 55. The development of these four patches will allow the technology, if shown to be effective, to be applied to approximately 22 percent more boys with Duchenne muscular dystrophy.

Positive results from Pompe disease clinical trial A phase III clinical trial which tested Myozyme in 90 patients with late-onset Pompe disease for 18 months showed that walking ability and lung function were improved.

Pompe disease is a rare neuromuscular condition caused by the body not having an enzyme called acid alpha-glucosidase (GAA). In 2006 a treatment called Myozyme, which replaces the missing GAA enzyme, became available for babies with the severe infantile form of Pompe disease because initial clinical trials focused on this patient group. The infantile form of Pompe disease normally proves fatal within the first year of life and Myozyme proved to be life saving. Symptoms of the late-onset form of Pompe disease can appear at any age and vary greatly in severity and rate of progression. NHS funding for this very expensive therapy is not guaranteed for every patient and it can be difficult for some late-onset patients to access the therapy. The results of this clinical trial show that Myozyme modestly improves breathing and walking ability in late-onset Pompe disease patients. Importantly it prevents the deterioration of these abilities over time. This evidence will help patients around the world gain access to this treatment.

However, there is still the need for longer trials with more patients to help further understand the benefits of mexiletine for myotonia. A clinical trial has now been initiated in the US, Canada and the UK to test if mexiletine could also be effective in treating other types of myotonia such as myotonia congenita.

Disappointing results from BioMarin’s utrophin clinical trial for Duchenne BioMarin announced disappointing results from the Phase I clinical trial of ‘BMN-195’ and has halted development of the compound. BMN-195 is an oral drug with the potential to increase the amount of the protein utrophin in the muscles. Utrophin is thought to be able to substitute for the missing dystrophin protein in boys with Duchenne muscular dystrophy. The phase 1 clinical trial administered BMN-195 to a small number of healthy volunteers. Increasing doses of the compound were given orally to the volunteers, but even

at high doses only small amounts of BMN-195 entered the bloodstream. The concentrations of BMN-195 in the blood were much lower than those expected to be able to have an effect on utrophin levels in the muscle. BioMarin therefore concluded that BMN-195 is highly unlikely to be effective for Duchenne muscular dystrophy. All parties involved are still optimistic that this strategy of increasing levels of utrophin is a viable treatment approach for Duchenne muscular dystrophy and are working on alternative drug candidates which may be ready for clinical trial in the near future. Prof. Dame Kay Davies at the University of Oxford, who has been funded for over 25 years by the Muscular Dystrophy Campaign and was involved in the initial development of BMN-195, said, “The failure of the BMN195 in this phase I trial is obviously very disappointing as it looked so promising in the mdx mice. However, we have new screens coming along which should provide new and better candidates for increasing levels of

Heart drug shows promise for the treatment of myotonic dystrophy

How to keep up to date and get involved in clinical trials

Results from two clinical trials in the US have shown that the heart drug mexiletine is able to improve muscle stiffness for people with myotonic dystrophy type 1. The trials showed that patients taking mexiletine for seven weeks were able to relax their grip significantly faster than those taking a placebo. Myotonic dystrophy is the most common form of muscular dystrophy in adults and its symptoms include muscle weakness and wasting, cataracts and diabetes. In particular, individuals with myotonic dystrophy are unable to relax certain muscles, especially the hands, after use – this is called myotonia. During the trials mexiletine did not produce any serious side effects and none of the patients showed alterations in heart muscle function during the trials. The results from these trials help to confirm the benefit of using mexiletine to treat myotonia in patients with myotonic dystrophy. As mexiletine is already licensed for use in UK it could be available to myotonic dystrophy patients in a relatively short time.

Keeping up to date with all of the new clinical trial developments can be a daunting task. Here are some resources that can help:  On our website (www.muscular-dystrophy.org/clinicaltrials) you can find summaries of clinical trials for neuromuscular conditions written in layman’s terms.  The US National Institutes of Health clinical trials register (www.clinicaltrials.gov) contains information about trials worldwide for all diseases. It is often written in medical language so you may need to discuss it with your doctor or contact us for a translation.  If you have an email address you can join the mailing list for our monthly e-newsletter which contains research and clinical trial updates: www.muscular-dystrophy.org/enewsletter Although taking part in a clinical trial is not a guarantee of a successful treatment and carries risks that need careful consideration, there are significant benefits that you may wish to take advantage of. These include potentially gaining access to a treatment before it becomes widely available and extra clinical care during and after the trial which may help to better manage your condition. There are three main ways to increase your chances of being involved in a clinical trial:  Register your interest in taking part in trials with your doctor and remind him or her regularly.  Join national registries if they are available for your condition (see p13).  Directly contact the centre involved in the clinical study. They will get in touch with your local doctor whose involvement is essential.

utrophin. We, like BioMarin, remain committed to utrophin upregulation for the therapy of Duchenne muscular dystrophy.”

Update on Santhera’s Phase III trial of Catena®/Sovrima® for Duchenne muscular dystrophy Sovrima® (called Catena® outside Europe)* is a small molecule which can increase the production of energy by the “batteries” of the cell – the mitochondria. Swiss pharmaceutical company Santhera is currently developing this potential drug for five different conditions: MELAS syndrome, Friedreich’s Ataxia, Leber’s Hereditray Optic Neuropathy, multiple sclerosis and Duchenne muscular dystrophy. Results from the phase II trial (DELPHI) involving 21 Duchenne muscular dystrophy patients were released in 2008 and indicated that treatment with Catena® for one year improved heart and lung function. A phase III trial – called DELOS – is currently ongoing and will test the effectiveness of Sovrima® in a larger number of Duchenne muscular dystrophy patients treated for one year. In part one of the study, seven study centres in Europe (Austria, Belgium, France, Germany, Netherlands, Sweden and Switzerland) and one centre in the US are enrolling 40 patients who are not taking steroids. These trial participants are between the ages of 10 and 18 and can be walking or unable to walk independently. After analysis of the results part way through the treatment of this group of participants, part two of the study will be opened. This will aim to enroll a further 200 patients who may take steroids. At that time Santhera will open additional study sites in Europe and the US/ Canada. An announcement will be made to all Duchenne muscular dystrophy patient organisations when this second part of the study is open for patient enrolment. *Catena®/Sovrima® was previously known as SNT-MC17/idebenone and results from the phase II (DELPHI) trial were reported in the February 2009 issue of Target Research.

Ataluren clinical trial for Duchenne – less is more. Results from the clinical trial of ataluren (PTC124) have shown that unlike the high dose, a low dose may be able to reduce

the decline of walking ability in boys with Duchenne muscular dystrophy. The phase IIb clinical trial of ataluren involved 174 boys in 11 countries with Duchenne muscular dystrophy caused by a specific type of fault in the genetic code called a nonsense mutation. This type of mutation is the underlying cause of 10-15 percent of Duchenne cases. Ataluren is an oral drug with the potential to overcome nonsense mutations. The main measure of effectiveness of the treatment was the distance they could walk in six minutes. At the beginning of the trial the boys could walk about 360 metres on average. By the end of the 48-week trial there was no difference between the boys treated with placebo and those on high dose ataluren – the distance they could walk had reduced by approximately 42 metres in both groups. Surprisingly, the average distance the boys on low dose ataluren could walk reduced by only 13 metres by the end of the trial. Although this result was unexpected, this type of dose response has been observed for other drugs. Ataluren was generally well tolerated by the participants in the clinical trial and no patients dropped out of the trial due to side effects. PTC Therapeutics has now contacted the regulatory authorities to find out what further evidence is required to allow ataluren to be made available to patients. Even if this treatment is not a cure for Duchenne muscular dystrophy caused by a nonsense mutation, any treatment that slows down the deterioration in walking ability is of great interest to families affected by this condition.

Two new trials to test muscle-building drugs Myostatin is a protein naturally produced by muscle cells that prevents the muscles from growing bigger and stronger. Together with other proteins that promote muscle growth, myostatin contributes to the balance that helps keep muscles within a normal size range. However, scientists believe that blocking myostatin may help to improve the strength of muscles affected by neuromuscular conditions. Two different approaches to block myostatin are being developed.

The first approach involves a synthetic molecule called ACE-031 developed by Acceleron Pharma. ACE-031 binds to myostatin and related proteins preventing them from interacting with the muscle. ACE-031 has already been tested in healthy volunteers and a phase II clinical trial in Canada is aiming to recruit 76 boys with Duchenne muscular dystrophy. Meanwhile, researchers at Nationwide Children’s Hospital in Ohio have gained funding to carry out a phase I clinical trial of a gene therapy approach to block myostatin. They plan to inject an adeno-associated virus (AAV) containing the follistatin gene into the quadriceps muscle of volunteers with Becker muscular dystrophy or inclusion body myositis. Follistatin is a natural inhibitor of myostatin (for more information about gene therapy please see p4). Inhibiting myostatin is an attractive approach because it could increase muscle growth for a range of conditions. However, it does not repair the genetic defect or replace the missing protein that is the root cause of the condition, so one concern is that it might lead to the growth of large weak muscles. These clinical trials will assess the safety of this approach and start to understand if people with neuromuscular conditions, like mice, can gain muscle strength from these potential treatments. However, it is thought that these approaches may be of the greatest value when used in conjunction with other treatments that address the genetic defect. For more information on ongoing clinical trials for neuromuscular conditions visit our website www.muscular-dystrophy.org/clinicaltrials For clinical trial news see www.muscular-dystrophy.org/research/news

The puzzle of research funding

Funding our research

Dr Liz Philpots Head of Research Practice, AMRC

From the outside, it might seem that research is a doddle – turn up and work on interesting scientific questions, go to conferences, teach a few students – but the reality is much more complex. For every new research idea, the journey to bring it into reality is a long and complex one.

In the UK, medical research is funded by many different organisations, all with different priorities for what kinds of research are important. A typical university research group gets funding from many sources: the Government, charities and industry. This complex jigsaw is repeated in every university laboratory across the country. The Association of Medical Research Charities (AMRC) has 124 members who

Members of the Association of Medical Research Charities contribute approximately a third of all public expenditure on medical and health research in the UK*

National Institute of Health rsearch £819.2 million

Association of Medical Research Charities £935 million

Medical Research Council £704.2 million

*Figures are for 2008/09

are all charities that fund medical research in the UK; aimed at tackling diseases such as heart disease, cancer and diabetes, as well as rare conditions like cystic fibrosis and muscular dystrophy. Together, they contribute approximately one third of all the public money spent on medical research in the UK. AMRC membership is seen as a mark of quality. We support and guide our members to be effective funders of excellent research and work with them to make sure the UK is the best place for them to do research. All our members use peer review – independent scientific experts who recommend to the charity the best projects to fund. Charities provide a vital source of funding for medical research, with many, such as the Muscular Dystrophy Campaign supporting research into rare diseases that are often under funded by government agencies. They ensure that the research is really relevant to the condition they are interested in and work with researchers to communicate their discoveries to policy makers and the public. Recognising the value of charity-funded research, the Government introduced the Charity Research Support Fund in 2006. This fund contributes to the general running costs incurred by the university in conducting charity-funded research, for example, library access costs and human resources services. This ensures that every pound a charity grants to a project goes directly into the research. In the last decade, the money available to pay for research increased steadily, but the economic crisis of 2009 has led to less money being available. AMRC member charities spent more than £1 billion on research in the UK in 2009/10 but the recession is affecting donations to charities, with the Charities Aid

Foundation and the National Council for Voluntary Organisations reporting last year that the total amount of charitable giving was down by 11 percent from the year before. The Government has reacted to the recession and the budget deficit by saying it will spend less in all areas (except health and international development). In the recent comprehensive spending review, funding for scientific research was spared from the savage cuts seen in other areas, but with large cuts to the teaching budget, universities will have to make radical changes, which will undoubtedly have an impact on the research that they carry out. We should all be pleased that the Treasury has recognised the importance of science, but we need to work together to ensure the UK’s medical and health research is strong, so that patients can benefit from new thinking and treatments. Simon Denegri, Chief Executive AMRC, commented, “Is this a slam-dunk for science? No, of course not. Is this a good result in the circumstances? Yes, probably. The spending review announcement at least ensures continuity as well as stability in science funding for the foreseeable future. But I remain concerned about the added demands it will place on charity funding of research. As ever, the fine print will need careful reading.” In these difficult times, it is more important than ever that charities continue to fund research in the areas that they and their supporters feel is important. The AMRC will be there to support the charities in any issues that they face and will influence the Government, ensuring their needs are a priority. For more information please visit www.amrc.org.uk

Over the past 50 years the Muscular Dystrophy Campaign has invested more than £50 million into cutting-edge, high quality research projects. As a result, we now understand a lot more about the causes and effects of many types of neuromuscular conditions, and for the first time clinical trials are starting to happen.

Even in these hard economic times we need to keep this momentum moving forward so that this research can be translated into effective treatments for the benefit of people with neuromuscular conditions. On page 34 you can read all about the exciting research we currently fund. Many of these research projects would simply not happen without Muscular Dystrophy Campaign funding and the extraordinary dedication of our many fundraisers across the country.

The ultimate marathon This year a staggering 130 runners pulled on an orange t-shirt to take on the legendary London Marathon course and raise a stunning £250,000 for the Muscular Dystrophy Campaign. While all of our runners completed Paul crossing the finishing line

huge personal challenges in conquering the 26-mile route, twenty-year-old Paul McIntyre really went the extra mile with his attempt. Paul, from Perthshire, has Becker muscular dystrophy. Starting the race with the 40,000 other entrants on Sunday morning, Paul had aimed to walk the route over three days but instead kept on going to arrive at the finish line at 6pm on Monday. Paul refused to use a walking stick for the journey, conquering the course unaided to raise £4,000 in sponsorship. Completing the course, he said, “I’m absolutely shattered and think I’ll be aching for weeks but I’m so pleased to have completed my challenge.”

Great trek on the Great Wall In March this year, 100 trekkers braved subzero temperatures as they walked 80km of the Great Wall of China to raise a quarter of a million pounds. Among our amazing team, who walked seven hours a day for five days, was Muscular Dystrophy Campaign Director of Research Dr Marita Pohlschmidt. “Walking the Great Wall of China was an exhilarating experience and at the same time a real challenge for me. Starting the day without a shower and having to sleep in tiny tents at -5ºC is not something that I do in everyday life! Most importantly, it gave me the opportunity to spend time with our supporters and listen to their stories of how neuromuscular conditions have impacted their families.”

Thirty hours in a pod for ‘wheely’ tough challenge A team of five fundraisers spent an astonishing 30 hours trapped in a pod on Weston-Super-Mare’s London Eye-style big wheel this August to raise in excess of £2,500. Staying in the cramped confines of the pods for 30 hours would

Marita on the Great Wall of China be uncomfortable enough for most people, but especially so for participants Steve Ledbrook and Kim Randle, who have muscular dystrophy, and Lynette McMillan, who has cerebral palsy, all of whom are wheelchair users.

How you can help The Muscular Dystrophy Campaign organises and supports a vast array of fundraising events just like these throughout the year. If Marita’s experience has given you the taste for adventure, early next year sees us head to South America as our intrepid adventurers bid to reach the Incan city of Macchu Picchu on our trek of the Peruvian Andes (see p36). But fundraising doesn’t have to mean a huge physical challenge or a trip round the world. Our new fundraising initiatives Come Dine with Us and At the Movies mean that you can help raise money for our research programmes by holding a dinner party or a film night. We are always on hand to offer advice and support for any idea you would like to put into practice. If you’d like to get involved, head to www.muscular-dystrophy.org/get_involved or call 0845 872 9058.

Updates and highlights from research funded by the Muscular Dystrophy Campaign

New research uncovers potential method to avoid passing mitochondrial myopathy on to future generations Researchers led by Prof. Doug Turnbull (in collaboration with Dr Mary Herbert and Prof. Alison Murdoch) at the University of Newcastle upon Tyne have shown that it might be possible to prevent mitochondrial diseases being passed from mother to child. The majority of our genes are in the chromosomes located in a compartment in the centre of our cells called the nucleus. However, a very small proportion of genes – approximately 0.01 percent of our DNA – are located in structures called mitochondria. The mitochondria are the ‘batteries’ of the cell responsible for producing energy. Errors in these genes cause mitochondrial disorders such as mitochondrial myopathy. Since there are no treatments for these conditions and genetic counselling is extremely difficult, the researchers decided to look into how IVF techniques might be used to replace the faulty batteries with healthy ones from a donor egg during embryo development in the laboratory. To find out if this would be possible in humans, the researchers performed the

Researchers identify new mutation in myotonic dystrophy type 1 Prof. Darren Monckton’s research group in Glasgow has discovered a new type of mutation that affects the severity and inheritance of myotonic dystrophy type 1. This condition is caused by an increase in the length of a repetitive segment of the DMPK gene – instead of a piece of DNA code being repeated between five and 35 times it is repeated 50 to more than 1,000 times. The repeats are described as ‘unstable’ because they are prone to multiply from one generation to the next and over the lifetime of an individual. This is why the condition generally has an earlier age of onset from one generation to the next and worsens over the lifetime of an individual.

procedure on embryos donated to research after IVF. These embryos would have otherwise been discarded because they were abnormally fertilised and unsuitable for transfer back into the mother. The chromosomes were removed from the embryos and transferred into donor eggs. Embryos manipulated in this way had minimal amounts of mitochondria transferred to the donor egg and developed for several days in the laboratory. This proves that the technique is compatible with the onward development of human embryos. This represents a major breakthrough because in the future it may offer families affected by mitochondrial disease the chance to have healthy children. However, further research is required to test the safety of this technique before it can be considered safe for clinical practice.

This new research found that in about three to four percent of patients with myotonic dystrophy the DNA code of the repeats is slightly altered. This has the effect of making it more stable when transmitted from one generation to the next. This means that the age of onset for the condition does not become markedly younger from one generation to the next in these families. The patients with this unusual mutation also had additional symptoms such as early hearing loss, seizures and neuropathy (damage to the nerves, especially in the hands and feet). These new findings could help provide patients with more accurate advice on how their condition will progress and how future generations of their family may be affected.

New gene responsible for two types of adult-onset muscular dystrophy identified Research led by Dr Rumaisa Bashir in Durham has led to the identification of a gene which causes limb girdle muscular dystrophy type 2L (LGMD2L) and distal Miyoshi myopathy (MMD3). The researchers studied DNA from French-Canadian, Finnish and Dutch families with these conditions. By working together, scientists in the UK and Canada showed that the faulty gene in all of these families was ‘anoctamin 5’ (ANO5). At present, the role of ANO5 protein in muscle is not known, but it is thought that it might be involved in repairing torn cell membranes. The identification of this gene will allow more accurate diagnosis of the condition and help in the search for therapies in the future.

Research gives new insight into nemaline myopathy New research by Prof. Laura Machesky from the CRUK Beatson Institute for Cancer Research in Glasgow has provided new understanding of nemaline myopathy that is caused by mutations in the skeletal alpha-actin gene (ACTA1). Twenty percent of people affected by nemaline myopathy have mutations in this gene which contains the instructions for the production of alpha-actin protein – a major structural component of muscle cells. This new research showed that mutations in the alpha-actin gene not only weaken the structure of muscle cells but may also affect muscle development and growth. This is due to changes in the way it interacts with another protein called serum-response factor. The authors suggest that in the future drugs could be developed to overcome the alteration in this interaction which may be of therapeutic benefit to patients.

Effective treatment found for a type of congenital myasthenic syndrome In 2006 researchers at the University of Oxford led by Prof. David Beeson reported their discovery that faults in a gene called DOK7 can give rise to congenital myasthenic syndrome (CMS). This is the second most common cause of the condition and patients A stem cell attached to a muscle fibre

experience muscle weakness that starts in early childhood especially affecting the hips and shoulders. These patients often do not respond to drugs used for other types of CMS. In June this year Prof. Beeson reported that patients with mutations in the DOK7 gene can be effectively treated with a drug called ephedrine. This drug is sometimes used to treat asthma and bronchitis. This small study involved ten patients who took the drug for up to eight months and had their symptoms monitored. There was a profound improvement in daily activities especially the ability to raise the arms and legs. On the basis of this, clinicians now recommend ephedrine treatment for patients with DOK7 mutations. Further longer-term studies with more patients are required to find the best dose and determine what the long-term effects are. It is also not yet known how ephedrine works to improve the symptoms of CMS. We have granted funding for a new project this year to start to unravel this mystery and find out if this treatment could be used for other types of myasthenia – see p2.

New treatment strategy discovered for oculopharyngeal muscular dystrophy Research in Prof. Rubinsztein’s laboratory in Cambridge has found that a chemical called cystamine reduced the symptoms of oculopharyngeal muscular dystrophy (OPMD)

in cells grown in the laboratory and a mouse model of the condition. Human muscle cells grown in the laboratory and treated with cystamine had fewer of the protein clumps that are characteristic of the condition and less muscle cell death. In mice cystamine delayed the onset of muscle weakness and improved muscle strength. Drugs based on the action of cystamine may now be targeted for development to treat OPMD.

New insight into how muscle stem cells work Dr Peter Zammit’s research group at King’s College London published new findings in September about how muscle stem cells are regulated. Every muscle fibre has a number of muscle stem cells, also known as satellite cells, attached to its surface. These muscle stem cells are normally dormant, but become active and start to multiply when the muscle needs to grow or becomes damaged and requires repair. The muscles of people with muscular dystrophy progressively weaken and waste away and scientists think that it is the activity of the muscle stem cells that initially prevents the muscles from deteriorating. However, these muscle stem cells gradually lose their capability to replace the wasted muscle tissue. Scientists believe that replacing muscle stem cells or ‘convincing’ them to stay active could potentially be used to efficiently treat a range of muscular dystrophies. The researchers studied the function of a group of proteins called ‘bone morphogenetic proteins’ (BMPs) in mouse muscle stem cells. They found that these proteins help to maintain a healthy balance between the number of stem cells that develop into muscle cells and the number of cells that remain stem cells. This ensures that there are always sufficient muscle stem cells available to repair damage when the need arises. The results represent an important step towards understanding muscle stem cell biology which is vital for potentially developing stem cell therapy into a treatment. See also www.muscular-dystrophy.org/ research/news

Partnership project improves exon skipping and develops non-invasive muscle monitoring

could be collected. The data obtained from the scans of the boys with Duchenne muscular dystrophy also gave the researchers essential natural history data for this group – information on how the condition naturally progresses over time – providing an important baseline for clinical trials. As well as the work on boys with Duchenne muscular dystrophy, the team scanned various mouse models of neuromuscular conditions. This could be a useful tool to allow researchers to observe how different conditions progress in these animals over time and measure how effective prospective treatments might be prior to testing them in clinical trials.

Dr Julia Ambler, Head of Grants, Muscular Dystrophy Campaign

In 2003 the Muscular Dystrophy Campaign, the Duchenne Family Support Group and Action Duchenne worked with a group of top-class researchers to form the MDEX Consortium to develop treatments for Duchenne muscular dystrophy. In 2004, they won £1.6m worth of funding from the Department of Health (DoH) to test a revolutionary new type of gene therapy called exon skipping in boys with Duchenne muscular dystrophy.

Duchenne muscular dystrophy is caused by faults in the dystrophin gene which contains the instructions for making dystrophin protein – an important structural component of muscle. The principle of exon skipping is to encourage the cell to ‘skip over’ the faulty part of the dystrophin gene using small pieces of DNA called antisense oligonucleotides (AOs) or ‘molecular patches’ (for more information about how exon skipping works please see p6). The initial clinical trial planned by the MDEX Consortium involved injecting the molecular patches into a single muscle in the foot. The aim was to test whether or not the patches were safe, and secondly prove the principle of the technology, that is, if it was able to increase the amount of dystrophin in the muscle. Although the technology was ready for the DoH funded clinical trial, this was the first time that exon skipping had been tested in people and it was anticipated that further research would be required. In particular, studies in mouse models indicated that delivering enough of the molecular patches to the heart was going to be difficult and needed addressing. Being able to reliably assess the

effect the molecular patches were having on all of the muscles of the body was also crucial for the success of future clinical trials that would involve body wide administration of the patches via the bloodstream. Recognising the importance of this work, the Muscular Dystrophy Campaign along with Professors Volker Straub and Dominic Wells and the Duchenne Family Support Group made an application to the Big Lottery Fund and were delighted to receive a grant to the value of £469,497. The project which started in August 2006 had two strands: Prof. Wells’s work focused on improving the methods for delivering the molecular patches to all the muscles of the body and Prof. Straub assessed the potential of Magnetic Resonance Imaging (MRI) to track muscle damage and measure the effectiveness of the treatment. MRI images highlighting the variation between boys with Duchenne muscular dystrophy. The images are a cross-section through the mid-thigh of two boys aged 9 (top) and 8.6 (bottom). The dark areas are muscle.

The results of this Big Lotteryfunded project are influencing the design of further clinical trials of exon skipping therapy. Improving the delivery of molecular patches Knowing what factors effect delivery of molecular patches to the muscle is vital. So, Professor Wells and his team decided to investigate the cells that line the blood vessels – the vascular endothelium. These cells are a barrier that drugs need to cross in order to pass from the blood into tissues such as the muscle. In order to study these cells they used mdx mice – a useful model of Duchenne muscular dystrophy because, like boys with the condition, they produce little or no dystrophin protein in their muscles. They found that the vascular endothelium did affect how well the patches reach the muscle and work is now ongoing to devise ways to allow the patches to cross the vascular endothelium more efficiently. One of the methods for improved delivery being investigated by the team is the use of microbubbles. These are small gas-filled bubbles, less than one millimetre in diameter, normally used to increase the contrast on an ultrasound scan allowing clinicians to see organs such as the heart in more detail. They are already being investigated for delivering various different types of drug. The researchers mixed the molecular patches with microbubbles and injected them into mdx mice. Standard ultrasound equipment was then used to aid the passage of the molecular patch into the muscles. The exact way that the ultrasound works is unknown but it is thought

The Newcastle MRI scanner and researchers Prof. Volker Straub and Dr Penny Garrood. A model used to explain the scan to boys who took part in the research is shown on the scanner bed. that it causes the bubbles to vibrate and make small holes in the cell walls. This allows the molecular patch to pass from the blood stream into the muscle cells more easily. The microbubble and ultrasound treatment improved delivery of the patches to the muscles throughout the body of the mdx mice and especially to the heart. Repeated treatment improved the efficiency of this delivery with no ill effects. This is important because while exon skipping in mdx mice has been shown to be successful in the skeletal muscle (the muscles attached to the skeleton that move the body), there have been problems getting the patches into the heart. This is vital as many young men with Duchenne muscular dystrophy develop heart problems that are a major cause of death. An additional important result from these studies was an estimation of how much dystrophin is required in the muscles in order to have a beneficial effect on muscle function. They found that delivery of molecular patches in the mdx mice was beneficial if 20 percent of the muscle fibres produced dystrophin protein. This has provided the MDEX Consortium with a realistic goal for the amount of dystrophin that should provide some benefit to boys with Duchenne muscular dystrophy.

Non-invasive imaging Prof. Straub and his team set out to investigate the use of MRI to monitor the muscle damage in boys with Duchenne muscular dystrophy and mouse models of neuromuscular conditions. MRI is a diagnostic imaging technique that uses a magnetic field and radio waves to produce highly detailed images of the body. Conventional MRI allows the identification of scarred muscle (fibrosis). In combination with special dyes called ‘contrast agents’ the technique becomes even more powerful, allowing doctors to see the location and extent of muscle fibre damage. MRI could therefore be a very useful tool for use during clinical trials to monitor muscle changes over time and assess the effectiveness of potential therapies. Importantly, the technique is non-invasive so doesn’t require a painful muscle biopsy, and unlike a muscle biopsy, it can assess the muscles of the whole body rather than just one tiny area. Prof. Straub and his team recruited six healthy adults, five healthy children and 11 boys with Duchenne muscular dystrophy to the MRI study. They studied the advantages of two different MRI contrast agents and gained valuable understanding of the quality and quantity of information that

Future clinical trials The second MDEX exon skipping clinical trial sponsored by AVI Biopharma has now been completed. This trial involved injecting the molecular patch directly into the bloodstream. Although some boys produced impressive amounts of dystrophin in their muscles using this delivery method, the response was very variable from boy to boy and some produced very low amounts of dystrophin (see p15). Therefore, improvement to the delivery of the molecular patches is needed to increase the chances of exon skipping becoming an effective treatment for Duchenne muscular dystrophy – this is the challenge for the next clinical trials now being planned. The results of this Big Lottery-funded project are influencing the design of further clinical trials of exon skipping therapy. It will help researchers to develop ways to improve whole body delivery of molecular patches, giving this promising technology a greater chance of success in slowing or even halting the progression of Duchenne muscular dystrophy. It may also improve the monitoring of clinical trial success by assessing muscle changes in the whole body by MRI and reduce the need for invasive muscle biopsies. The use of MRI may be applicable to many different types of neuromuscular conditions, not just Duchenne muscular dystrophy. For more information on this and our other recently completed research projects see www.muscular-dystrophy.org/completed_grants

The Sequencing Revolution What impact will the recent developments in DNA sequencing capabilities have on the diagnosis and treatment of neuromuscular conditions? Dr Caroline Godfrey, Dubowitz Neuromuscular Centre, Institute of Child Health, University College London.

everyone unique). Since the Human Genome Project, major efforts have been made to understand the genetic differences between individuals but our knowledge is still very limited. Working out the way in which our genetic make-ups differ is key to understanding our susceptibilities to diseases such as diabetes and cancer and uncovering the genetic defects that cause conditions such as muscular dystrophy.

A change of pace

Human genetics is in the midst of a revolution. After the mammoth task of sequencing the first human genome in 2003 (involving nine countries, taking 13 years, and costing around £6 billion to complete), the promise of getting our own personal genome sequenced is tantalisingly close. Revolutionary new technology now exists to completely sequence an individual’s genome for a fraction of the price and in a matter of days. What does this mean for the field of human genetics and what impact will this have on the diagnosis and treatment of neuromuscular conditions?

Our similarities and differences Back in 2003 the Human Genome Project was big news. The three billion units (or ‘rungs’ on the DNA helix ladder) that make up all of our hereditary information had been determined for the first time (see box p 25). DNA contains only four types of unit abbreviated to A, T, C and G. The full sequence of the human genome is on display for all to see in an exhibition at the Wellcome Collection in London. It is housed on a bookshelf containing 120 volumes, each the size of a telephone directory. The streams of As, Ts, Cs and Gs had to be printed in font size 4.5 to squeeze them all in! This huge reservoir of data has transformed biological research, but whose genome was it? The DNA sequenced during the Human Genome Project was not from a single individual. DNA from a large number of volunteers was collected and a handful of these samples were then chosen anonymously. Therefore, the sequence published in 2003 was a composite human genome. Although our genomes are more than 99.9 percent identical, their vast scale means there are still millions of differences. A major challenge is to understand which of these differences cause disease and which ones do not (and are just part of what makes

The recent improvements in sequencing technology have been made possible through the use of a fundamental advance in chemistry. The ‘Sanger DNA sequencing’ method, first developed in the 70s and named after the man who first described it, has predominated until recently. This process involves determining the sequence of about 800 units of DNA at a time, which is time consuming and laborious. New technology, so-called ‘next generation sequencing’, overcomes these restrictions. A person’s whole genome (three billion units) is chopped into small pieces of about 100 units in length and attached to a small glass slide (see box p25). The DNA sequence of all of these fragments is determined simultaneously and assembled by powerful computer software. This new chemistry has brought about a staggering change of pace. In 2007 James Watson (of Watson and Crick fame, the scientists who discovered the helical structure of DNA) was the first person to have his complete genome sequenced using the new method. It cost less than £1.5 million and took just over two months (whereas the Human Genome Project took 13 years and cost £6 billion). At this time, next generation sequencing was only in its infancy and has since improved further. In 2009, biotechnology company Illumina launched its Individual Genome Sequencing service in the US. After consulting with a doctor or medical geneticist and receiving appropriate counselling, a blood and saliva sample is sent off for whole genome sequencing which takes about eight days. Actress Glenn Close and South African human rights activist Desmond Tutu have both paid to have their genomes sequenced. The price continues to fall and in June this year Illumina announced a new price of less than US$20,000 (£13,000) per genome. Within three to five years it’s expected that the cost of genome sequencing will be less than $1,000 (£650).

What can all of this information tell us? Unlike cancer or heart disease, neuromuscular conditions are usually caused by a mutation (or mutations) in a single gene. Finding a patient’s disease-causing mutation is important; it not only allows doctors to provide a more accurate diagnosis and prognosis to patients and

their families, helping them to plan for the future, but is vital in the development of treatments. With the prospect of gene therapies such as exon skipping (see p4-7), medicine is destined to become highly personalised and detailed information about a person’s genetic mutation will be required for its application. With around 26,000 individual genes in the human genome, analysing the correct ones in order to tease out the genetic root of disease is often far from straightforward. Already defects in over 100 genes have been identified as causing neuromuscular conditions and many more remain to be found. The current method for genetic diagnosis involves clinicians identifying the most likely candidate genes based on a patient’s particular symptoms. These genes are then sequenced one at a time using Sanger sequencing until the culprit is found. For some conditions the genes that need to be examined can be numerous, for example Charcot-Marie-Tooth disease can be caused by mutations in any one of more than 30 different genes. To add to the problem, for some conditions it is necessary to screen extremely large genes. This approach is both expensive and time consuming; finding disease causing mutations is often a long slog that can frequently take months, if not years. It is estimated that 20 to 25 percent of people with a neuromuscular condition remain without a genetic diagnosis despite the examination of all the genes known to cause their symptoms. Researchers are left to trawl through portions of the genome bit by bit in search of mutations; like looking for a needle in a haystack. Next generation sequencing is set to change things. Any DNA sequence variation detected across the whole of a patient’s genome

Sequencing: Unravelling our genetic code Our genetic code is contained within specialised structures known as chromosomes. These are stored in the centre of nearly every cell in the body. Tightly packed into each chromosome is the familiar double helical (spiral) structure of DNA. The code carries all the information necessary to maintain and propagate life. The code uses only four different types of units represented by the letters A, T, C and G. Reading or ‘sequencing’ our DNA involves determining the order or ‘sequence’ of these units along the helix. There are over three billion of these units in the human genome.

How does the new technology compare to the old? Of the billions of units in the human genome, the Sanger sequencing method ‘reads’ about 800 units of DNA at a time. The genome is sequenced bit by bit in individual 800 unit sections (one per experiment). This approach produces graphs with a series of colour coded peaks, each one representing a single unit (A, T, C or G) (below).

In contrast, next generation sequencing has the capacity to‘read’ the whole genome in one go. Thousands and thousands of overlapping 100 unit stretches of DNA are sequenced simultaneously. Specialised computer software then aligns these short units to compile the entire genome.

can be noted and investigated for disease-causing effects. Despite the challenges in interpreting this data, a flurry of high profile research papers published recently has given hints as to just how powerful this technology is. Towards the end of 2009, researchers in the US were the first to successfully use next generation sequencing to uncover the genetic basis of a rare disease. This technology was used to analyse the genomes of four individuals with Miller syndrome, which affects the development of the face and limbs. The researchers were able to identify defects in a gene not previously known to cause human disease. A number of large-scale next generation sequencing projects have recently been launched to investigate the millions of genetic differences between individuals and improve our understanding of the genetic causes of disease. In 2008 the ‘1,000 Genomes Project’ was launched as a collaborative project between hundreds of scientists around the world. Improvements in sequencing technology have enabled researchers to increase their initial target of sequencing 1000 people’s genomes to a total of 2,500 genomes. In June 2010 the Wellcome Trust launched the ‘UK10K Project’ focused on sequencing the genomes of 10,000 people over the next three years – one of the largest sequencing studies ever undertaken. This £10.5 million project is based in Cambridge in collaboration with clinical researchers from around the UK (including Prof. Francesco Muntoni, Prof. Mike Hanna and Prof. Mary Reilly who specialise in neuromuscular conditions). Next generation sequencing is changing the way researchers approach both basic and translational research. This new technology can also be applied to highly specialised techniques used to investigate the function of the gene and what goes wrong in the cell when the gene is faulty. It will allow researchers to collect data in ways that would have been completely impractical using older techniques. This information is important in understanding the disease process and is used in the development of appropriate therapies.

Perspective from a patient organisation We asked Alastair Kent, Director of Genetic Alliance UK – a charity which represents the interests of all people with genetic conditions – what the implications of new genome sequencing technology are. What are the opportunities that this new technology brings for people with genetic conditions? New sequencing technology offers exciting prospects for increasing the understanding of the flaws in our genome that cause or predisposes us to many different diseases. The way technology is advancing it will soon be easier and cheaper to sequence the whole of an individual’s genome than to pick out suspect bits and sequence these separately. This has tremendous potential. Can you envisage any problems? People having their genome sequenced, whether as part of a research project or in the future as part of a diagnostic service, need to be well informed about the possible consequences. If people aren’t well informed about the possible future uses of the

Challenges With literally millions of DNA sequence variations from person to person the challenge is identifying those changes that cause disease. As more information is collected on our similarities and differences, this will become easier but, in the meantime, geneticists are playing catch up in terms of interpreting the vast amounts of data this technology can generate. The storage of and access to this information also needs to be considered.

Completed human genomes: the milestones and celebrities Year

Completed human genomes

Time

Cost (US$)

2003

The Human Genome Project: This was the first time the entire human genome had been sequenced. DNA from several anonymous donors was used.

13 years

$10 billion

2007

Craig Venter: This American biologist and entrepreneur became the first person to have his entire genome sequenced.

5 years

$100 million

2007

James Watson: Nobel Prize Laureate; discovered the helical structure of DNA. This was the first genome to be sequenced using next generation sequencing technology.

4 months

$1.5 million

2010

Glenn Close: This American actress’ s genome was sequenced using Illumina’s newly launched Individual Genome Service.

8 days

$48,000

days

$1000?

2015?

data and are not confident that the regulatory framework is in place, they may be reluctant to volunteer to have their genome sequenced. An appropriate regulatory framework needs to be in place that covers issues such as:  access to samples and data  commercial exploitation of results (where appropriate)  feeding results back to sample donors (including decisions about how to handle the discovery of a significant risk of a disease unrelated to the original purpose of the study)  whose data is it, and who should have a say in its use? How can these issues be addressed? Many large scale sample collections have governance councils in place to provide such a regulatory framework that respects and protects the interests of sample donors. Such frameworks help prevent misunderstanding and provide a valuable support for the maintenance of trust now and in the future. This will help preserve the opportunity to undertake high-quality biomedical research and in doing so, move us forward towards the development of innovative therapies for many currently incurable diseases.

Whole teams of experts have been assembled, devoted to examining the ethical, legal and social issues related to sequencing our genomes. Although these issues are not new, having been implicated in past genetic research and clinical practice, as sequencing becomes more routinely available they will impact a much wider population on a much larger scale. As we learn more about how to interpret the changes between our individual genomes, sequencing may reveal susceptibilities to diseases for which there may or may not be treatments or preventable measures available. How this information is interpreted and used presents a huge challenge. To resolve these issues before genome sequencing becomes widely available, careful consideration and open debate is needed with the involvement of patients and/or their representatives.

Watch this space The Sanger DNA sequencing method was one of the most important technologies created in the 20th century, thoroughly transforming human biology. Next generation sequencing methods are now tipped to rival the significance of Sanger sequencing. Once scientists understand more about the significance of the variations in our genomes, next generation sequencing methods could allow more neuromuscular patients than ever before to receive a genetic diagnosis. This will also be achieved more efficiently – in terms of both time and money. Its use will also dramatically increase our understanding of disease processes which will feed into the pipeline of information for the development of new treatments and therapies. It’s no quick fix but it is certainly something to watch out for. Contact us if you have any questions at research@muscular-dystrophy.org

Ask a scientist

The Muscular Dystrophy Campaign research department is always available to answer any questions about research. Questions we don’t know the answer to, we refer to our network of scientists and clinicians working in the field. In this article we posed some of the questions we have received recently to top researchers for further expert opinions. Q1. How much exercise is too much for boys with Becker muscular dystrophy? Many boys are able to have an active lifestyle compared to, for example, boys with Duchenne muscular dystrophy, but does this put extra strain on the cardiac muscle and increase the chances of developing cardiomyopathy? Helen Stockdale A. For many years the general notion was that exercise accelerates the disease process in patients with muscular dystrophies. In particular, dystrophin – the protein affected in Becker muscular dystrophy (BMD) – is thought to have a structural role in the skeleton of the cell, so that abnormal dystrophin would leave the muscle cell vulnerable to stress when the muscle contracts. Exercise training has not been studied systematically in children with BMD but aerobic (endurance) training in adult BMD patients for a year was highly beneficial, improving not only endurance and maximal oxygen uptake, but also muscle strength. Importantly, these benefits can be obtained without damaging the muscle. Similar studies in other muscular dystrophies have also documented the beneficial effect of physical training, thus

dismissing the old scepticism about training in these conditions. Exercise training is also a well-recognised therapy for people with heart disease (cardiomyopathy) on its own. In accordance with this, studies have shown that BMD patients with cardiomyopathy had improved heart function with training. There is no reason to believe that these findings in adults cannot be applied to children with BMD. A general recommendation is to train three to five times a week, for 30 minutes each time, performing aerobic exercise (cycling, rowing, jogging, swimming, etc) at a target heart rate above 150 if you are a teenager, above 140 if you’re below 50 years old and above 130-135 if you are older. However, you should always consult your doctor before embarking on a new exercise programme. Prof. John Vissing, Consultant Neurologist and Professor of Neurology at the University of Copenhagen, Denmark.

Q2. Further to the latest findings (see p9) on facioscapulohumeral muscular dystrophy (FSHD) what would be the best way of de-stabilising DUX4 or making it unreadable? What sort of timescale would we be looking at for these potential treatments? Colin Lucas A. FSHD is caused by changes to a region of DNA on chromosome 4 called D4Z4 that has the same piece of DNA code repeated many times. In healthy individuals the number of repeats varies between 11 and 100. People with FSHD have less than 11 repeats and this causes the activation of a gene called DUX4. At the moment, the use of antisense oligonucleotides to destabilise or ‘silence’

Exercise training has not been studied systematically in children with BMD but aerobic (endurance) training in adult BMD patients for a year was highly beneficial, improving not only endurance and maximal oxygen uptake, but also muscle strength. (see p7) the RNA copy of the DUX4 gene looks to be the most likely strategy. However, there are a number of considerations to take into account during development of such a therapy as we don’t as yet understand what DUX4 does. For example, we need to ensure that the normal function of DUX4 – whatever that might be – is not affected by such treatment. We also don’t know when, during someone’s lifetime, it might be most effective to destabilise this RNA. One of the ways that DUX4 is activated has to do with changes to the way this piece of DNA is folded when the number of repeats is reduced. This exposes the DNA code to be read by the cell, like opening a book. Other approaches to therapy might be to close up the D4Z4 DNA and turn off DUX4, but it is difficult at the moment to see how these could be made to act specifically on D4Z4 and not interfere with other essential regions of the genome. Another note of caution is that currently there are no animal models that properly mimic the FSHD mutation (unlike the situation in Duchenne muscular dystrophy for example where the mdx mouse model is widely used). This lack of a natural mouse model will make it difficult to test how well potential therapies work in a whole animal.

The timescale is really difficult to predict, and although we now have a clear signpost to direct our research there is still a lot that we don’t understand and it will probably be several years before we even have developed techniques that will work in the laboratory. Taking any of these into the clinic is likely to take much longer. One very good thing, however, is that antisense oligonucleotide therapies are currently being developed for other diseases such as Duchenne muscular dystrophy. This means that many general aspects, such as how to make these molecules more stable and more effective, and how to deliver them to the appropriate muscle cells are already being developed and optimised. So for FSHD, we can learn a huge amount from the Duchenne work that is currently taking place. This will really help to speed up development of potential antisense therapeutic approaches. So although we need to proceed carefully with caution, there are reasons to be optimistic. Prof. Jane Hewitt, University of Nottingham.

Q3. Is the decision to start a research project decided on the most pressing need or the highest chance of success? Who decides? David Vaughan A. Both, the quality of the research grant application and the immediate relevance for people affected by a neuromuscular condition are being considered throughout the very thorough application procedure that we have in place. Every research application undergoes a rigorous peer review during our annual competitive grant round. We generally invite researchers to apply for funding during the autumn months. Initially they submit a two page proposal that explains the purpose and aim of the research and broadly describes the planned experiments. It is then checked whether it fits into the strategic priorities that are set out in our Research Strategy.

Our charity supports research into more than 60 different conditions every one at a different milestone on the route to therapy. For Duchenne muscular dystrophy, for example, incredible scientific advances have been made in recent years and a number of clinical trials are on the way to test promising potential treatments. For other conditions such as some forms of limb girdle muscular dystrophy and congenital muscular dystrophy the genetic defects are not well understood. About 20-25 percent of people affected by neuromuscular conditions have not yet received a genetic diagnosis and finding the causative gene to provide genetic counselling and an accurate prognosis will be high on their list of priorities. The strategic priorities are therefore kept relatively broad and state that we fund basic science as well as research dedicated to speed up the translation of promising technology into a clinical context. Potential applicants are then invited to submit a full application which includes detailed information about the project, but also contains information about, for example, whether the researcher has the legal requirements in place to conduct animal research, if that is what they are proposing to do. The applications are then sent to international experts who provide written feedback on the quality and appropriateness of the project. The application and the written reviews are evaluated by a committee of independent scientists who then make recommendations to the Board of Trustees who make the awards. This year, for the first time, the opinions of lay members were included in the decision of which research to fund. Applicants had to fill in a lay application form to explain their research in a language understandable by everybody. A lay committee reviewed the applications and fed back their priorities for funding to the research committee. About 20 percent of the vote now consists

of people whose lives are affected by neuromuscular conditions to ensure that the most pressing need as well as the quality of the research we fund is taken into account. Dr Marita Pohlschmidt, Director of Research, Muscular Dystrophy Campaign.

Q4. Is there any research into whether Duchenne muscular dystrophy affects the muscle used for digestion? I am interested to know if there is any evidence that a physical inability to digest food due to deterioration in muscle can lead to lack of appetite and eventually to malnutrition. Ann Patterson A. The muscle around the gut, the bladder and the larger blood vessels is slightly different from the other muscles and is called smooth muscle, but it also contains dystrophin. So far, there is very little evidence that the deficiency of smooth muscle in the blood vessels causes problems in individuals with Duchenne muscular dystrophy. However, there is some indication that in older Duchenne patients a lack of dystrophin can lead to dysfunction in the gut and bladder. In the oesophagus the smooth muscle causes wavelike contractions that push food into the stomach. In the stomach, the smooth muscle helps to mix the stomach contents and propel food to the small intestine. In the large intestine smooth muscle helps to push the stools towards the anus. When the smooth muscle becomes less efficient, each of these steps will be slower. This can occasionally lead to distension (swelling) of the stomach, but much more frequently to constipation which in turn gives rise to reduced appetite.

While these complications are common in young men with Duchenne muscular dystrophy they can most of the time be managed by appropriate intervention; generally by preventing the stools becoming too hard with the regular use of laxatives. If properly managed it is rare for these complications to lead to malnutrition. In older individuals with Duchenne muscular dystrophy, malnutrition is more often due to reduced chewing and swallowing abilities and can be helped both by dietary modification and supplements, and, if necessary gastrostomy feeding – insertion of a feeding tube directly into the stomach. Assessment by a multidisciplinary team is essential in these cases including a dietician, a speech and language therapist and, if necessary, a gastroenterologist. Prof. Francesco Muntoni, Head of the Dubowitz Neuromuscular Centre, University College London.

Q5. What’s the relationship with overseas research teams – competition or collaboration? David Vaughan A. Most research groups have a healthy respect for their counterparts overseas. For rare diseases it is often important to collaborate since it is only through a joint effort that enough patients can be grouped together to get answers to scientific questions. Moreover, it is rare for one laboratory to have expertise in all aspects of the multidisciplinary methods required for cutting-edge research. Thus, collaboration is often necessary; the European Union has been helpful in facilitating this and in general most European research teams interact well together. However, the currency of the research scientist is the publication of research papers, and if you do not publish you lose your job, as simple as that. As such, there is still fierce competition to publish first in the top journals. The position of the researcher’s names in the list of authors is also considered very important which can cause stress in collaborations. The last thing you want is for a group from the USA to pip you at

the post in reporting something that has taken you five years to work out! However, this fierce competition is important since it gives the drive and the edge to make breakthroughs and publish the results as quickly as possible. Prof. David Beeson, University of Oxford.

Q6. With the advent of genetic testing will neuromuscular conditions eventually be wiped out so that no babies are born with these conditions? Anonymous A. Genetic conditions are inherited in one of three ways: 1. Autosomal dominant inheritance results in a condition being passed down through successive generations with a risk of 50 percent for each child of an affected parent. Thus, there is usually a family history of the condition. 2. Autosomal recessive inheritance occurs when the affected person has inherited an abnormal copy of a gene from each parent. The parents are otherwise healthy and there is usually no family history of the condition. We all carry abnormal recessive genes, but it is pure chance whether or not we meet a partner who also carries a copy of the same abnormal gene. 3. Sex-linked inheritance occurs when a copy of the faulty gene is inherited on the X chromosome from the mother. There is usually a family history where sons are affected and mothers are carriers. Thus, where there are known to be affected individuals in a family, parents can make choices about whether or not to go for pre-natal testing, sperm or egg donation or pre-implantation diagnosis. But it is important to remember that genetic misprints causing any muscle condition can arise spontaneously in an individual without an abnormal gene being inherited from a parent. We call these de novo mutations and they cannot be predicted or prevented. This is why concentrating on treatment for neuromuscular conditions is so important.

Dr Ros Quinlivan, Dubowitz Neuromuscular Centre, Great Ormond Street Hospital, London.

Q7. FSHD can be inherited or can be ‘spontaneous’ – up to a third of cases fall into the latter category, I believe. It is surely inconceivable that spontaneous cases are the result of random DNA corruption; something must be causing them. What are the current theories for this, which is most likely, and how might it be proved? David Vaughan A. From what we know about how the FSHD mutation occurs, in fact it does seem to be a random event. The nature of the DNA sequence in this region means that it is very likely to change in length by increasing or decreasing the number of copies of the D4Z4 ‘unit’. This happens by a perfectly normal cellular process; other regions of our genomes show similar changes in copy number. Unfortunately, because of special features of this part of chromosome 4, a reduction in D4Z4 copy number below 12 most often results in FSHD. Previous work by the group at Leiden in the Netherlands showed that this change in D4Z4 copy number tends to occur at a very, very early stage in development of human embryos. The mutation will then be present in the eggs or sperm of that individual and so they can then pass the mutation on to their children. Thus, a person in whom FSHD occurs spontaneously can give rise to inherited FSHD if they have children. This means that the inherited and spontaneous forms have the same underlying defect, which does mean that the same therapeutic approaches are equally applicable to both forms of the disorder. Prof. Jane Hewitt, University of Nottingham.

If you have any research questions please get in touch by calling 020 7803 4813 or emailing research@muscular-dystrophy.org

Spotlight on two of our long-term supporters Professor Alan Emery and Lord John Walton Dr Kristina Elvidge, Research Communications Officer, Muscular Dystrophy Campaign

Professor Alan Emery and Lord John Walton of Detchant were honoured at The International Congress on Neuromuscular Diseases in Naples in July for their contribution to the better understanding of neuromuscular conditions. The five-day conference, held every four years, brought together over 1,500 scientists and clinicians working on neuromuscular conditions. We took this opportunity to interview them both.

Professor Alan Emery Emeritus Professor Alan E. H. Emery is a Vice President of the Muscular Dystrophy Campaign. Emery-Dreifuss muscular dystrophy is named after Prof. Emery and American neurologist Fritz Dreifuss who first described the condition in the 1960s, and later identified the defect in the protein emerin. Prof. Emery is also credited with being instrumental in setting up the European Neuromuscular Centre (ENMC).

What made you want to get involved in muscular dystrophy research? It was chance. I went to medical school after military service and when I qualified I had no idea what I was going to specialise in – my professor suggested that I do genetics. In 1960 that was seen as a bit of a weird subject! We hadn’t had any lectures on it at medical school and two of our professors at Manchester didn’t believe in genetics at all! So my professor sent me to America to work with the world-renowned geneticist Victor McKusick in Baltimore. Next door to our genetics clinic was the neurology department – the people there were very nice and I expressed to them an interest in neurology. They said I could work with some of their patients with muscular dystrophy and that’s where it all started. I studied the original family with, what became later referred to as, Emery-Dreifuss muscular dystrophy. You have had a long and distinguished career in research; what has kept you motivated? To me as a clinician it’s the patients and the families. As a doctor when you’re seeing patients you just wish you could do something for them. That’s what has motivated me all of my life. I’m so glad now that new treatments are just on the horizon. I just hope they’re going to work because for so long we’ve been saying that we will have a treatment one day. Do you think you’ll see a treatment for muscular dystrophy in the near future? Yes, but how it might occur I couldn’t tell you – it could be gene therapy, antisense oligonucleotides, upregulating utrophin or something else. You have to be careful –

talking about science is easy but talking to families it is more difficult, you don’t want to give false hope. You were instrumental in setting up the ENMC in 1989, and it is still going strong. Why do you think it is so important? I feel that if I have made any significant contribution to the field of neuromuscular conditions it would have to be establishing the ENMC. I was appointed as the first Research Director of the ENMC in 1989 and had the support of a number of eminent neuroscientists throughout Europe which ensured its success. I had a very clear idea right from the beginning about how I wanted the ENMC to work. I wanted people to apply for funding for workshops with a very clear idea about what they wanted to cover over the weekendlong meeting and it had to happen in a way that people would be willing to share their ideas and material from patients – biopsies, DNA and so on. I wanted to encourage people to collaborate, not go to meetings and give well-defined talks, which is what happens at most meetings. Instead workshop participants present their ideas and data which might take 20 minutes and then it is open for discussion and questioning which might take two hours. The first objective I wanted the workshops to achieve was to agree on diagnostic criteria for each neuromuscular condition. The next thing was to look at the genetic defects. Once you’ve got the gene then you can start to think about therapies, which is ongoing now. To date more than 180 workshops have been held and it has worked very well indeed. I suspect that much of what is discussed at the international congress would not have been possible without the collaboration that occurred through the ENMC.

You’re still working in Oxford as an Honorary Fellow; how do you spend your time now? I do a bit of tutoring to senior medical students and I write a lot. My wife and I have just finished new revisions of two of our books – ‘The history of a genetic disease’ which is about the history of Duchenne muscular dystrophy and ‘Muscular Dystrophy: the facts’ for patients and families. I have always enjoyed writing, I also enjoy reading and music and I paint of course and exhibit in a gallery in Devon. I write poetry too. In my career as a doctor I worked every hour God sent, but now that the pressure has eased off it’s nice to have time to do the other things I enjoy.

Lord John Walton Lord Walton, Honorary Life President of the Muscular Dystrophy Campaign, founded the charity in 1959 with Prof. Fred Nattrass and Mr Joseph Patrick. He continues the fight against neuromuscular conditions in the House of Lords today. Lord Walton specialised in neurology and together with Nattrass published an influential paper on the classification of muscular dystrophies in 1954. Their success helped establish the muscle centre in Newcastle – a centre of excellence for research and care. You have retired from clinical practice now but you are still very active – how do you spend your time now? I attend the House of Lords regularly as a cross-bench Life Peer when the House is sitting – contributing to debates on medicine, science and education. I also try to keep up to date with developments in research especially in my own field of neurology and neuromuscular conditions by reading the relevant literature. At the last count I am also president, vice-president, patron or vicepatron of about 15 medical charities, but the Muscular Dystrophy Campaign remains the most important to me. What has kept you motivated to keep fighting neuromuscular conditions? I started working on neuromuscular conditions with Professor Nattrass when

I came out of the army in 1949 and I was particularly affected by the plight of patients and families affected by muscular dystrophy; in particular by boys with Duchenne muscular dystrophy. That inspired me to devote my research interests over many years to these condtions. You were honoured at the conference for your contribution to the neuromuscular field – what do you think your biggest contribution has been? I suppose it was because when working with Nattrass in the1950s we published in the journal ‘Brain’ in 1954 a major paper based upon my examination and analysis of all the patients with muscular dystrophy and related conditions that I identified in the North East region of England. On the basis of that study, I was able to introduce a new classification system based on clinical and genetic characteristics. I think that a lot of subsequent work, which has greatly modified this classification in the light of new knowledge, was in part based on that early work. With the aid of grants from not only the Muscular Dystrophy Campaign but also from the American and Canadian Muscular Dystrophy Associations, the Medical Research Council and Wellcome Trust, I established the neuromuscular research group in Newcastle, which has continued to flourish and has made major contributions to the field long after I left it. You have done a lot of campaigning for better care in the NHS for people with neuromuscular conditions, with ‘The Walton Report’ and so on; do you think they’re starting to listen? What has the impact been? I have no doubt at all that the Government and particularly health service bodies are beginning to listen. We had many discussions with commissioning groups in the parts of the country where the services for neuromuscular conditions were, in my opinion, defective as testified in ‘The Walton Report’. Already in the South West and West Midlands steps have been taken to recruit specialist consultants, physiotherapists and all the other staff needed to improve the care of patients with neuromuscular conditions.

What issues are you hoping to raise in the House of Lords in the future? I’m going to take note of what developments take place in improving the services for those with neuromuscular conditions and their families and where there are areas of continuing concern I will raise these issues. At the same time I have a continuing interest in medical education and the training of doctors and scientists and will be keeping an eye on whether budget cuts will impact on this area or impinge on the services available for patients with neuromuscular conditions. In the era of expensive clinical trials, how do you think the Muscular Dystrophy Campaign research grants can maximise their impact? The great virtue of having a charity like the Muscular Dystrophy Campaign is its ability to raise funds specifically to support care and research so as to improve the lives of people living with neuromuscular conditions. There is no doubt that it is through these research grants that several clinical trials have been initiated into treatments for various types of these condtions. Do you think you’ll see a treatment for muscular dystrophy in the near future? I think that treatments are now emerging, and although as yet none of them can be regarded as a cure, there is little doubt that the treatments under trial at the moment are likely to improve the lives of patients with various neuromuscular conditions.

Workshops bring international scientists together

(maternally inherited). Changes to this DNA can prevent the mitochondria from producing energy efficiently which particularly affects the muscles because they need a lot of energy and have thousands of mitochondria. Mitochondrial disease can also affect many other parts of the body including the brain and the eye.

Dr Kristina Elvidge, Research Communications Officer, Muscular Dystrophy Campaign

The European Neuromuscular Centre (ENMC) is a highly successful organisation that has funded and organised more than 180 workshops since 1989 to bring international scientists and clinicians together to join forces in the fight against neuromuscular conditons. The ENMC is funded and steered by European neuromuscular patient organisations, including the Muscular Dystrophy Campaign. In 2010 eleven workshops were held and here we summarise some of this year’s highlights.

FSHD standards of care and clinical trial readiness Twenty-four international expert clinicians and scientists met in January to discuss standards of care for facioscapulohumeral muscular dystrophy (FSHD) and develop strategies to prepare for future clinical trials. Diagnosis Getting the right diagnosis is the first step in providing good care for patients. Genetic testing, including prenatal and pre-implantation diagnosis were discussed. The limitations and difficulties were documented and recommendations made as to how clinicians can most effectively provide these services. They agreed that best practice guidelines for FSHD genetic testing should be developed so that everybody worldwide gets the same quality of advice. Clinical management The care required by people with FSHD can vary greatly and it was agreed that it should

be tailored to their needs. Multidisciplinary care is vital, involving specialists such as physiotherapists, orthotists and occupational and speech therapists. In addition, all patients with FSHD should be referred to an opthamologist to check for a treatable eye condition called retinal vasculopathy, and children with FSHD should have hearing tests. Preliminary evidence has shown that exercise can be of benefit for people with FSHD; improving fitness and strength. It is recommended that under the supervision of a health professional, 30 minutes of aerobic exercise (such as cycling or swimming) should be undertaken at least three times per week. Pain and fatigue are common and often underestimated symptoms of FSHD. Appropriate management such as physiotherapy, pain medications and energy conservation strategies were discussed. The benefits of surgery on the shoulder blades (scapular fixation) were also considered and recommendations made. However, there are risks involved and careful consideration of the timing of the surgery needs to be undertaken. Pregnancy for women with FSHD is generally considered to be safe with good outcomes but there have been conflicting reports of increased risk of preterm birth and emergency caesarean delivery. Therefore, it was recommended that obstetricians should, as a precaution, treat these pregnancies as high risk and delivery should be in a hospital with appropriate care available. More comprehensive studies on pain, surgery and pregnancy in FSHD patients were identified as goals for the future. Clinical trial readiness It is critical that the components needed to efficiently conduct clinical trials in FSHD patients are in place in time for any potential

treatments that may come along in the near future. Firstly, outcome measures need to be defined. These are ways to reliably measure if a treatment is working. Some reliable outcome measures have been established but scanning methods such as MRI were considered by the workshop attendees to warrant further investigation. Quality of life questionnaires specific for the symptoms of FSHD also need to be developed. The establishment of patient registries is essential for clinical trial readiness. Currently there are large registries in the USA and Italy as well as other smaller informal FSHD registries in other countries. It was agreed that developing a global registry using the model set up by TREAT-NMD to combine data from existing registries would help to facilitate future clinical trials for FSHD. The workshop attendees agreed to meet in approximately one year to re-examine outstanding issues.

Preimplantation diagnosis for mitochondrial diseases This workshop took place in March with 18 attendees including clinicians, scientists, an ethicist and a patient representative. Pre-implantation genetic diagnosis (PGD) involves eggs being fertilised by sperm in the laboratory (IVF) and testing the resulting embryos to identify low-risk embryos to start a pregnancy. What are mitochondrial diseases? Mitochondrial diseases are caused by faulty mitochondria which are the energygenerating batteries of the cell. Mitochondria have their own small piece of DNA – separate from the DNA contained in our chromosomes – which is passed down from mothers to their sons and daughters

Progress in PGD for mitochondrial diseases PGD for mitochondrial diseases is more difficult than for other genetic diseases because patients usually have a mixture of normal and abnormal mitochondria in their cells. The proportion of healthy mitochondria can vary from cell to cell and can change over a lifetime. Symptoms occur when the level of normal mitochondria – without a mutation in their DNA – falls below a critical level. This critical level is not well defined for a lot of conditions which makes it difficult to predict whether the symptoms of mitochondrial disease will develop. The mitochondrial diseases where PGD is considered possible include some types of maternally inherited Leighs disease (MILS or NARP), maternally inherited diabetes and deafness (MIDD) and myoclonic epilepsy with ragged red fibres (MERRF). A reasonable amount is known about the cause of these conditions and the proportion of abnormal mitochondria needed to cause symptoms.

Nine families worldwide with these conditions have undergone this experimental PGD treatment over the past five years resulting in the birth of three apparently healthy children and one pregnancy which was still ongoing at the time of the workshop. The workshop attendees created detailed guidelines outlining when PGD can be considered for mitochondrial disease, the ethical considerations and the best way to go about the procedure. The workshop report concluded that although PGD for mtDNA diseases remains challenging, there have been significant advances that may help some affected families to have healthier children in the future.

Moving towards therapies for the dysferlinopathies In January a workshop with 17 international participants was dedicated to dysferlinopathies, a group of conditions due to mutations in the dysferlin gene, the best known of which are limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. The workshop was organised to consider how the diagnosis of these conditions should be made and what needs to be done to understand why the condition develops. Potential therapies currently being developed in the laboratory were also discussed.

Prof. Kate Bushby and the other attendees of the dysferlinopathy workshop

Diagnosis The current procedures used and the difficulties of diagnosing the dysferlinopathies were discussed. This allowed the identification of central points to be included in diagnostic guidelines. An informal Network on Dysferlin Mutational Analysis was formed to develop these guidelines. This session also included an overview of a new gene called anoctamin 5, which has been found to cause a condition similar to the dysferlinopathies. Understanding the cause of dysferlinopathy Recent research by Prof. Kate Bushby in Newcastle (funded by the Muscular Dystrophy Campaign) showed that inflammation is of central importance in causing the symptoms of the dysferlinopathies. This research was outlined and discussed along with other new data on other processes that contribute to causing these conditions. The need for standardisation of both animal and cellular models was identified and the group was committed to working together to make this happen. Therapeutic approaches The final session was dedicated to therapeutic approaches. Preliminary data from a clinical trial in Germany has indicated that corticosteroids do not improve muscle strength for individuals with dysferlinopathies – important information that needs to be disseminated to clinicians worldwide. Several innovative gene therapy based strategies currently under development and the potential of drugs targeting inflammation were discussed. The workshop was concluded by a general discussion on efforts to be made towards harmonising national patient registries so that the data can be easily combined into an international registry. A planned natural history study was also discussed which will document the natural progression of the symptoms of dysferlinopathy. These are essential steps towards clinical trial readiness for the dysferlinopathies. For more information go to www.enmc.org.

Research projects currently funded by the Muscular Dystrophy Campaign Principal Investigator

Institution

Prof. David Beeson

Improving diagnosis and investigating new avenues for treatment for people with myasthenia gravis and congenital myasthenic syndromes. Investigating how the drug ephedrine has such a University of Oxford positive effect on certain people with congenital myasthenic syndromes and if it would be suitable for some individuals with myasthenia gravis (see p2). Identifying new gene mutations that cause University of Newcastle Upon Tyne Identifying novel molecular pathways Bethlem myopathy and Ullrich congenital and therapeutic targets for Bethlem muscular dystrophy. This may lead to the myopathy and Ullrich congenital identification of potential therapies for muscular dystrophy these conditions. University of Newcastle Upon Tyne Dysferlin deficient muscular dystrophy, Understanding more about the underlying causes and potential avenues for treatment of limb muscle regeneration and neutrophil girdle muscular dystrophy type 2B and Miyoshi recruitment myopathy, collectively known as dysferlinopathies. University of Oxford Upregulation of utrophin for Duchenne Continuing with research to find drugs to increase muscular dystrophy therapy the amount of the protein utrophin in muscle. This protein may be able to substitute for the missing dystrophin protein in Duchenne muscular dystrophy. Investigating the use of viruses to deliver antisense University of Oxford Optimisation of U7 snRNA vectors oligonucleotides to all the muscles of the body for therapy of Duchenne muscular with the aim of improving on current exon dystrophy skipping technology for Duchenne muscular dystrophy (see p7). Studying how gene interactions affect the University of Oxford Dynamic chromatin structures direct development of the specialised structure where developmental control of AChR and the nerve meets the muscle - the neuromuscular NMJ-related loci in muscle junction - to gain new insight into congenital myasthenic syndromes (see p3). Investigating ways to optimise a gene therapy Royal Holloway, University of Enhancing the therapeutic London functionality of adeno-associated Virus approach which uses a virus to deliver a shortened version of the dystrophin gene to muscle cells (AAV) vectors encoding dystrophin which is a potential treatment approach for for preclinical Duchenne muscular Duchenne muscular dystrophy (see p7). dystrophy gene therapy studies Investigating how the powerhouses of the University College London Exploring the roles of mitochondria cell - the mitochondria - might be involved in in the pathophysiology of core the progression of central core disease and myopathies* multiminicore disease (see p2). Identifying new gene mutations that can cause University College London Genetic heterogeneity and periodic paralysis, myotonia congenita and mechanisms of phenotypic paramotonia congenita, collectively known variability in human skeletal muscle as channelopathies. This will lead to a better channelopathies* understanding of the conditions as well as more accurate diagnoses (see p2). University of Nottingham Investigation of molecular mechanisms Investigating further the mutations causing facioscapulohumeral muscular dystrophy (FSHD). in facioscapulohumeral muscular Understanding the underlying mechanism behind dystrophy * FSHD will help towards the development of potential therapies in the future. Investigating why symptoms can vary so much University of Glasgow Complex repeats in myotonic from person to person in myotonic dystrophy by dystrophy type 1 and Charcot-Marieexamining the different genetic mutations causing Tooth disease the condition. This could help provide patients with more accurate advice on how their condition will progress (see p20 for an update).

Prof. David Beeson

Prof. Kate Bushby

Prof. Dame Kay Davies

Prof. George Dickson

Prof. Michael Duchen

Prof. Michael Hanna

Prof. Jane Hewitt

Prof. Darren Monckton

*= PhD Studentship

University of Oxford

Project Title Targeting ColQ as a cause of disease and as a novel therapy in myasthenic disorders * Therapy to stabilise synaptic structure for the treatment of both genetic and autoimmune disorders at the neuromuscular junction*

Description

For more information got to www.muscular-dystrophy.org/currentgrants

Total project cost £91,950

Principal Investigator

Institution

Project Title

Description

Dr Jenny Morgan

Imperial College London

The role of extracellular matrix components on satellite cell function.

£158,507

Dr Jenny Morgan

Imperial College London

Factors effecting the self-renewal of mouse satellite cells

Dr Ros Quinlivan

RJAH Orthopaedic Hospital

Dr Charles Redwood

Pilot project investigating the use of novel ankle foot orthosis and footwear combination to improve walking stability in children with Duchenne muscular dystrophy Analysis of the effects on contractile function of mutations in betatropomyosin that cause different inherited myopathies

In a group of congenital and limb-girdle muscular dystrophies known as the dystroglycanopathies the muscle stem cells are impaired. Understanding this impairment could help scientists to develop a therapy using muscle stem cells. Investigating the muscle environment so that scientists can determine which factors can be used to improve muscle stem cell regeneration of damaged muscle. Investigating the use of new orthoses and other footwear to improve walking and stability in children with Duchenne muscular dystrophy

Investigating how mutations in the betatropomyosin gene alter the stability of muscle fibres and their ability to contract. This may be able to explain how the different mutations cause conditions such as nemaline myopathy and cap disease. Studying a gene called Mrf4 to investigate if it Institute of Cancer Research Development of a mouse model to study the newly discovered function of could be used to develop ways to improve or promote muscle growth after injury or wasting MRF4 in adult muscle hypertrophy caused by neuromuscular conditions (see p3). Barts and The London SMD Polycomb gene family member Bmi1 in Investigating the role of a protein called Bmi1 in muscle stem cells with the aim of determining if the specification and maintenance of it can be used to improve the efficiency of muscle myogenic satellite cells * regeneration. University of Newcastle Upon Tyne Prevention of transmission of Investigating the use of IVF techniques to mitochondrial DNA disease prevent transmission of mitochondrial myopathy (see p20 for an update). University of Newcastle Upon Tyne Exercise therapy for patients with Investigating if exercise therapy is beneficial for mitochondrial myopathies people with mitochondrial myopathy. King's College Hospital Trust Changing adverse illness beliefs in Exploring how negative perceptions and other those with neuromuscular conditions. * social factors can impact upon quality of life of people with neuromuscular conditions with the aim of devising a programme of cognitive behavioural therapy. University of Sheffield Regulation of dystroglycan function * Investigating the function of a protein called dystroglycan in order to further understand its role in healthy muscle and also in Duchenne muscular dystrophy. Examining the role that a group of genes known University of Oxford Role of miRNAs in the pathology of as microRNAs (miRNAs) have in the progression Duchenne muscular dystrophy and of Duchenne muscular dystrophy and an potential as biomarkers of disease investigation as to whether they can be used as progression biomarkers to indicate the severity of the disease (see p3). Exploring ways to increase the amount of University of Oxford Novel exon skipping peptide-PMOs antisense oligonucleotides delivered to the heart for correction of heart dystrophin to improve on current exon skipping technology expression and function in mouse for Duchenne muscular dystrophy (see p3). models of Duchenne muscular dystrophy.* King’s College London What controls the efficiency of muscle Studying the factors that affect how well muscle regeneration? stem cells can regenerate muscle. Manipulating these factors could provide a basis of potential therapies for muscular dystrophy (see p21 for an update). Exploring the role that muscle stem cells have in King’s College London Does perturbed satellite cell function the progression of facioscapulohumeral muscular contribute to facioscapulohumeral dystrophy. Funded in partnership with Kings muscular dystrophy?* College London.

£150,097

£94,286

£95,485

£123,809

£179,036 Prof. Peter Rigby

£170,045

£29,672

Dr Lesley Robson

Prof. Douglass Turnbull

Prof. Douglass Turnbull Prof. John Weinman £130,931

Prof. Steve Winder £69,864 Dr Matthew Wood £121,156

Dr Matthew Wood £98,280 Dr Peter Zammit £184,007 Dr Peter Zammit

*= PhD Studentship

University of Oxford

Total project cost

£195,100

£32,418

£29,350

£116,179

£199,993

£153,502 £61,000

£100,772

£24,000

£117,480

£190,464

£60,000

Glossary

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This glossary is intended to help with some of the scientific and technical terms used in this magazine.

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Animal model – a laboratory animal such as a mouse or rat that is useful for medical research because it has specific characteristics that resemble a human disease or disorder, for an example see mdx mouse below.

Embryo – a fertilised egg that has the potential to develop into a foetus.

Antisense oligonucleotides – see p6.

Exon – genes are divided into regions called exons and introns. Exons are the sections of DNA that code for the protein and are interspersed with introns, which are also sometimes called ‘junk DNA’.

Antibodies – proteins made by the body to protect itself from foreign substances, such as bacteria or viruses Adeno-associated virus (AAV) – see p4. Cells – the structural and functional unit of all known living organisms. They are often called the building blocks of life. Humans have an estimated 100 trillion cells.

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Cellular model – cells grown in the laboratory and used to study biological processes and diseases. For example, potential therapies could be tested on muscle cells from a patient with muscular dystrophy grown in the laboratory. Chromosome – cylindrical shaped bundles of DNA found in the cell nucleus. They consist of long, threadlike strands of DNA coiled upon themselves many times. Corticosteroids (glucocorticoids) – a type of drug that reduces inflammation and suppresses the immune response. They are often prescribed to boys with Duchenne muscular dystrophy and may stabilise or even improve muscle strength for a period of time but not all boys respond to treatment. These are not ‘anabolic steroids’ which is what athletes use illegally to build up muscle. DNA – (deoxyribonucleic acid) is the molecule that contains the genetic instructions for the functioning of all known living organisms. DNA is divided into segments called genes. Dystrophin – the protein missing in people with Duchenne muscular dystrophy and reduced in those with Becker muscular dystrophy. Dystrophin is important for maintaining the structure of muscle cells.

Enzyme – a protein that can initiate, facilitate or speed up a chemical reaction in the body.

Exon skipping – see p6. Genes – Genes are made of DNA and each carries instructions for the production of a specific protein. Genes usually come in pairs, one inherited from each parent. They are passed on from one generation to the next, and are the basic units of inheritance. Any alterations in genes (mutations) can cause inherited disorders. Genetic testing – the examination of an individual’s genes to identify defects causing a disorder. Genome – all of the genetic information or DNA contained within an organism. Glucocorticoid – see corticosteroid. Inflammation – the body’s reaction to injury or infection resulting in redness, pain, swelling and warmth. It is a protective attempt by the body to remove whatever is causing the injury or infection (for example a splinter in your finger or a virus in your lungs) as well as initiate the healing process. In-vitro fertilisation (IVF) – a process by which the egg is fertilised by sperm outside the womb.

Mouse model – a strain or breed of mouse which has a disease that is similar to a human disorder. MRI (Magnetic resonance imaging) – a non-invasive body imaging procedure that uses powerful magnets and radio waves to construct pictures of the internal structures of the body. Multidisciplinary – a term used to describe a team of doctors and other health care professionals working together. For example, pediatricians, physiotherapists and respiratory specialists. Mutation – the alteration of a gene. Mutations can be passed on from generation to generation. Nonsense mutation – a change in the DNA which causes a premature stop signal to occur in a gene. When this happens protein is either not produced at all or does not function properly. Outcome measures – a way to reliably measure the effectiveness of a treatment or therapy. Phase I clinical trial – a small study designed to assess the safety of a new treatment, often using healthy volunteers. If it is conducted in patients some preliminary data on the effectiveness of the treatment may be gathered which is called a phase I/II trial. Phase II clinical trial – a study to test the effectiveness of a treatment on a larger number of patients. Participants are usually divided into groups to receive different doses or a placebo. Sometimes divided into phase IIa and phase IIb.

mdx mouse – a mouse model of Duchenne muscular dystrophy. These mice have a mutation in the dystrophin gene - the gene that is mutated in boys with Duchenne. The muscles of these mice have many features in common with the muscles of boys with Duchenne.

Phase IIa clinical trial – a study to determine the best dose of the drug.

Mitochondria – the batteries of the cell. They are structures found within most of the body’s cells and have their own DNA that is passed down from the mother.

Phase III clinical trial – involves a larger number of patients and follows the same process as phase II. This step can take two to three years. The aim is to gain

Phase IIb clinical trial – a study which aims to find out how well the drug works at the dose determined in the phase IIa study.

a more thorough understanding of the effectiveness and benefit of the drug. Phase IV clinical trial – evaluates the long-term risks and benefits of the drug once it’s available on the market. Placebo – an inactive substance designed to resemble the drug being tested. It is used as a control to rule out any benefits a drug might exhibit because the recipients believe they are taking it. Preclinical research – testing of an experimental drug in the laboratory or in animals prior to human testing. Preimplantation diagnosis – involves eggs being fertilised by sperm in the laboratory (IVF) and testing the resulting embryos to identify low-risk embryos to start a pregnancy. Prenatal testing – testing for a genetic condition in a foetus before it is born. Protein – large molecules required for the structure, function, and regulation of the body’s cells, tissues, and organs. Our bodies contain millions of different proteins, each with unique functions. The instructions for their construction are contained in our genes. RNA – ribonucleic acid, a substance very similar to DNA. When a gene is ‘switched on’ RNA carbon copies of the gene are made which move to the protein producing machinery of the cell. Skeletal muscle – muscle which applies force to bones and joints to move the body. Other types of muscle include cardiac (heart) and smooth muscle (blood vessels, stomach, intestines). Stem cells – cells that have not yet specialised to form a particular cell type, and can become other types of cell such as muscle cells. They are present in embryos (embryonic stem cells) and in small numbers in many adult organs and tissues, including muscle. Utrophin – a very similar protein to dystrophin. Low levels of utrophin are present in everyone – including people with Duchenne muscular dystrophy - but in insufficient amounts to compensate for the loss of dystrophin.

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Send your own seasonal message and let someone know you are supporting the Muscular Dystrophy Campaign this Christmas with a stylish and unique eCard. You can create your own or send a pre-drawn card to family and friends from just £1. This year, our pre-drawn eCards feature a Christmas pudding card drawn specially by Rick Bridger who has Duchenne muscular dystrophy. To send yours visit www.muscular-dystrophy.org/ecards

Supported by the Big Lottery Fund

The Big Lottery Fund distributes half of the National Lottery good cause funding across the UK. The Fund is committed to bringing real improvements to communities and the lives of people most in need. Registered Charity No. 205395 and Registered Scottish Charity No. SC039445

Front cover image: ‘What Can Science Do For Us?’ a line drawing by renowned contemporary artist Jacqueline Donachie from Glasgow. She has illustrated her niece Rhona, 11, and nephew Fraser, 15, who both have myotonic dystrophy, against a street background. The drawing illustrates the importance of not just scientific research to patients and their families, but also the significant role that families themselves have in research. ResearchComms/Dec10/9k