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MDEX Consortium Update:

Restoring Dystrophin Expression in Duchenne Muscular Dystrophy

A UK Consortium for Preclinical Optimisation and a Phase I/II Clinical Trial Using Antisense Oligonucleotides

The MDEX Consortium was formed to develop and test a treatment for Duchenne muscular dystrophy using patches of genetic material to restore production of the essential muscle protein dystrophin. Funding from the Department of Health enabled the project to begin in January 2005.

There has been considerable progress over the past 20 months in developing and testing molecular patches, preparing for the clinical trial and in obtaining some additional funding to start addressing some of the long term issues that this technique will pose.

The project is divided into several sections of work, each led by a different research group but with all members of the Consortium sharing results and meeting regularly. The work is progressing extremely well with several of the milestones achieved ahead of schedule. The project also benefits from having a Scientific Board of experts who meet every 4-6 months with the Consortium to discuss progress and offer advice.

1. Chemistry of the molecular patches:

Developing new patches:

Professor George Dickson and Dr Ian Graham at the Royal Holloway London are involved in developing and testing new molecular patches (antisense oligonucleotides) and optimising the chemistry of existing patches. Using a molecular patch to cause exon skipping allows the genetic error causing DMD to be bypassed and some dystrophin protein to be made. However, predicting and developing effective sequences is a complex and time-consuming task.

The most important goal of the team's first year's research has been to collaborate with other members of the Consortium in the task of comparing and evaluating both existing and newly developed patches for exon 51. In collaborative studies the functional activity of the morpholino patch selected by the MDEX Consortium was independently validated in the laboratories of our Dutch colleagues. Following these experiments the MDEX Consortium is now confident that the patch for exon 51 that will be used in the UK clinical trial is the most effective one for our purposes. The sequence is one developed by the Australian group and has a different backbone chemistry compared to the one that will be used in the Dutch trial, as the MDEX Consortium will be using a morpholino backbone. The fact that there are differences between the patches used in the UK and Dutch trials means that comparisons on effectiveness between different backbone chemistries can be drawn from the 2 studies but there will be other differences such as age which will have to be taken into account when the results are analysed. Approximately 20% of all patients with
deletions in the gene for Duchenne muscular dystrophy would benefit from the skipping of exon 51 and for this reason it was chosen for the first clinical trial.

Now that the decision has been made regarding the sequence to be used in the clinical trial, the MDEX Consortium has moved to developing and testing molecular patches for exon 53. A series of new patches have been developed and are in the process of being tested.

Improving the ability of molecular patches to enter muscle cells:

Dr Matthew Wood at Oxford University is also involved in the design of molecular patches but is focussing on joining molecules (called ligands) to the genetic sequence of the patch to try and improve or increase the ability to deliver the patches to muscle cells especially in difficult areas such as heart muscle. This will be very important when moving to systemic delivery, i.e. getting the patches to all affected muscle via the bloodstream. The Consortium have always been clear with those funding this research that whilst the aim of the first trial is to prove the technique works after intramuscular injection and is safe to use in humans, ultimately the treatment needs to be delivered systemically to all part of the body and this is where Matthew’s work is focussed. Various different molecules have been joined to existing patches and then tested to see whether there is an improved effect in firstly getting the patch into muscle and secondly whether it is possible to target areas that are hard to reach such as heart muscle. Some promising results have been obtained in animal models and future experiments will look in their effectiveness in different ages and over different periods of time. As mentioned before all this type of laboratory work takes considerable time because each new molecule has to be evaluated many times in different situations before a decision can be made on the effectiveness of a particular design.

2. Testing how effective molecular patches are in model systems:

Testing in animal models:

Each new patch or change to an existing patch has to be fully evaluated to determine whether it will work and if it is more effective than existing ones. This testing is performed by several laboratories within the Consortium and abroad to check results are consistent.

In evaluating the effectiveness of this therapy it is important to test for production of dystrophin protein but also to test whether the muscle is stronger after treatment. Professor Wells has set up special equipment which is able to repeatedly stretch muscles and measure their ability to withstand the force applied to the muscle. Using this machinery the team is able to compare normal muscle to treated and untreated DMD animal muscle. So far the results in animal models have been promising demonstrating that treatment with molecular patches is able to improve muscle strength in the mouse model of DMD.

Testing molecular patches in human cells:

The patches are also tested in human muscle cells. Muscle biopsies are routinely taken at the time of diagnosis and the Hammersmith team have used part of this muscle tissue to isolate human muscle cells and grow these in culture. These stocks of human muscle cells are then frozen so that they can be used in experiments to test the effectiveness of different patches as they are developed. The team led by Dr Jenny Morgan at the Hammersmith has compared several different exon 51 patches in terms of their ability to skip exon 51 but also to produce dystrophin protein after exposure to the patch.

Skin cells are also being collected from boys with DMD as it is easier to collect these and in the laboratory they can be converted into muscle cells and used to test the patches.

Revertants:

Although boys with DMD carry genetic errors that prevent dystrophin protein from being produced, there are often a small number of cells called revertants (usually less than 5%) which do produce a tiny amount of dystrophin. These are thought to occur by exon skipping that occurs naturally in a small percentage of cells. It is important that the Consortium is able to distinguish the dystrophin produced by these cells from the dystrophin due to the treatment so that the effectiveness of the therapy can be evaluated. Work in this area has focussed on looking at biopsies taken in the past at the time of diagnosis and comparing them to more recent biopsies to determine the level of revertants and note whether this figure changes over time. Results suggest that these numbers do not vary significantly and that with specialised tests they will be able to distinguish the effect of administering the patches in the human clinical trial.

Preparation for the clinical trial:

Professor Francesco Muntoni leads the Consortium and together with Professor Kate Bushby is responsible for the clinical aspects of the project.

Recruitment to the clinical trial:

A comprehensive database is being developed by the Senior Clinical research Fellow Dr Kinali at the Hammersmith to store patient information and details such as the genetic error causing the disease and results of any muscle biopsies performed. Consultants around the UK have been contacted regarding the trial so that details of their patients can be entered into this database and to date 19 Centres are supplying information. Building up a comprehensive database is important for clinicians as it enables them to have a clearer picture of the numbers of patients with particular types of genetic errors, their ages and where they are currently receiving treatment. When recruitment begins, this information will allow clinicians to approach suitable patients who may wish to participate in the trial.

The muscle to be injected in the clinical trial:
During the clinical trial the molecular patches will be injected into a small muscle called the extensor digitorum brevis (EDB) on the top of the foot. To ensure this is a suitable site for injection, preparatory studies were done to investigate the level to which this foot muscle is affected in boys of various ages having Duchenne muscular dystrophy. Ethical approval was obtained to perform muscle biopsies in boys undergoing surgery for various reasons and muscle MRI (magnetic resonance imaging) to image the EDB muscle. Since December 2005, 17 DMD patients have undergone MRI studies and 7 have had muscle biopsies. These studies have shown that this small foot muscle is relatively well preserved compared to other muscles in boys up to the age of around 15, although there is some variation depending on the level of severity of the condition. It has been agreed that the EDB muscle is suitable and will be the target area for injection of the molecular patches in the clinical trial.

Safety (toxicology) studies:

Toxicology studies investigate how compounds such as a molecular patch enter cells and their effect inside cells. In particular they look for negative side effects which might cause harm to patients. Clinical trials will not be approved until substantial toxicology data is available showing both effectiveness and the safety profile of the patch to be used in the clinical trial. This work has been coordinated by Professor Nic Wells who in consultation with the relevant regulatory authorities, is looking for the most appropriate sets of toxicology studies to allow the trial to be finally approved. In addition the group lead by Professor Wells has been developing a specialised mouse model called a SpliceOmouse to track the genetic patches inside the body and look for adverse side effects.

There has also been a lot of work around determining the most effective and safe amount of molecular patch to administer to get the optimum effect. Again this work is extremely time consuming as each experiment needs to be repeated many times. However this information is essential in designing the clinical trial and forms part of the information requested by the regulatory authorities.

Professor Wells has also spent time investigating how effective molecular patches are in older animals. This work enables the Consortium to make more informed judgements on the age at which molecular patch therapy will be effective in DMD. The excellent news from these studies is that even in older mice the patches have some effect although the extent to which it is possible to restore function is a lot less than in younger mice. This gives some hope that in older boys it may be possible to deliver patches to prevent further muscle breakdown.

Production of clinical grade molecular patches is being handled by an American specialist biotechnology company (AVI Biopharma); the company has been closely involved in the trial design and the development of the assays to help and address some of the safety issues. The Consortium is extremely grateful for the commitment of AVI Biopharma to the MDEX Consortium. The clinical grade molecular patches to be used in the trial are currently being manufactured.

Regulatory approval for the trial:

There are two regulatory authorities that have to approve the clinical trial; these are GTAC (Gene Therapy Advisory Board) and the MHRA (Medicines and Healthcare Products Regulatory Agency). There are for obvious reasons very tight regulations in place to safeguard as far as possible those participating in clinical trials. GTAC’s role is to consider and advise on any human clinical trials where genetic material (e.g. molecular patches) is being used. They primarily look at whether the study is ethical and weigh up the potential benefits and risks of the proposed clinical trial and agree the design of the trial, the actual methods that will be used and factors such as the age of those who may participate in the trial. The MHRA assess the safety and efficacy of the molecular patch to be used in the clinical trial and require data from toxicological studies that look at factors such the safety profile of the molecular patch and how effective it is at different doses in producing dystrophin protein in muscle.

The amount of preparatory work necessary before submissions can be made to GTAC and the MHRA is enormous but without their approval trials cannot begin. The excellent news is that the Consortium has received GTAC approval and is shortly due to submit their application to the MHRA. It is impossible to give accurate timings on when recruitment to the clinical trial will begin but it is likely to be the early part of 2007. This will however become clearer once the Consortium has submitted
their application to the MHRA and received feedback. The best outcome would be an immediate approval but we have to anticipate that additional
information may be required before it is finally approved.

Clinical Trial Information:

Details of the trial have been entered on two sites, clinical trials.gov and Orphanet. The website addresses for further information is: www.clinicaltrials.gov and www.orpha.net The clinicaltrials.gov website gives information on eligibility, inclusion and exclusion criteria, the protocol to be used and details of participating
centres. In addition, Orphanet has detailed information on clinical trials; the different phases and what their purpose is in testing a compound as well as information about this trial.

Many parents may ask how to enrol their children into this trial. The first thing to know is if the affected child has one of the deletions which make him eligible for the inclusion criteria (i.e. deletions of exons 45-50; 47-50; 48-50; 49-50; 50; 52; 52-63). In all these instances the removal of exon 51 is predicted to allow the production of dystrophin.

Most children in UK are being followed by paediatricians and paediatric neurologists who are fully informed about the trial. Parents should discuss with their local doctor the intention for their child to be considered for the trial, and ask the doctor to forward relevant information to either the Hammersmith Hospital or Newcastle, the 2 clinical coordinating centres.

Many UK centres have already agreed to be part of the network which will recruit children for this trial, and the appropriate ethical approval is in place, so from most places the local physicians will be able to send us the required information. Another thing that parents could do if their child has one of the relevant deletions is to let us have copy of the DMD registry (DMD PPUK has generated a registry for patients) and if you have one with all the relevant information, you could send it to Dr Maria Kinali, Department of Paediatrics, Hammersmith Hospital, Du Cane Road,London W12 ONN.

MDEX Consortium
November 2006