Disease mechanisms and therapy for inherited disorders of the neuromuscular synapse.

This programme of work will study a group of inherited diseases called congenital myasthenic syndromes (CMS) in which the transfer of the signal for muscle contraction from nerve to muscle is disrupted. These are rare disorders, present in around 1 in 100,000 of the UK population, that may be life-threatening and can cause severe disability in children and adults, but we are frequently able to work out what is going wrong and as a result devise appropriate therapy. In the last two years we have found mutations in four new genes that cause CMS and have been at the forefront of showing that treatments usually used for asthma can result in remarkable improvement for our patients. Research success led our laboratory to be the basis for a National Referral Centre for CMS, and through this service our goal is to provide tailored therapy for each of our patients. To move towards this goal this programme of work will address the following questions:

i) Which genes are responsible for additional cases of CMS? We have around 40 cases in which we have not been able to find the genetic cause of the patient's condition. Patients, above all, want a definitive diagnosis. We will use the latest DNA sequencing techniques to search for the genetic defects in these patients, and new patients who are referred to our clinic.

ii) How do the genetic defects in the genes that we recently identified cause disease? If we can work out how the mutations are disrupting the information transfer from nerves to muscle we can often devise appropriate therapy. Therefore if we can find out how the mutations in these four new genes result in disease it should help in their treatment.

iii) Can we find new available treatments? We have found that certain drugs commonly used for asthma can work exceptionally well for some of our patients. We want to find out exactly how these are having their effects and then see if there are other similar but potentially even better drugs available.

iv) Can we experiment to find novel treatments? The site where information is passed from nerve to muscle is called a synapse. We have noted that when one of the proteins we study, DOK7, is produced at high levels in muscles is generates greatly enlarged synapses in their correct location, which we call 'super synapses'. When we generated these 'super synapses' in a disease model for DOK7 CMS is completely cured the disease. We want to perform studies on the super synapses to define exactly why they work so well and also we want to try this novel therapy in other CMS disease models. Moreover, it could potentially be applicable to some other more common diseases that feature defects in the neuromuscular junction synapse such as motor neurone disease or spinal muscular atrophy.

Congenital myasthenic syndromes (CMS) stem from genetic defects that affect signal transmission at the neuromuscular synapse. They provide a rare example of genetic muscle disorders that are treatable if the underlying molecular mechanisms are understood. At least 19 causative genes have been uncovered with mutations identified in proteins involved in signal transfer, synaptic stability, or glycosylation.
There are four objectives to this proposal:
i) to use next generation sequencing techniques to resolve the genetics behind our residual undefined CMS cases;
ii) to find out the mechanisms through which glycosylation mutations impair neuromuscular transmission;
iii) to study molecular mechanism to explain how treatment with beta2-adrenergic receptor (beta2-ADR) agonists results in dramatic clinical improvement;
iv) when over-expressed in muscle DOK7 generates correctly located but greatly enlarged neuromuscular junctions or 'super synapses'. We will test the potential of these 'super synapses' as a novel gene therapy.
Within the laboratory there is expertise for the analysis of neuromuscular junction proteins. Next generation sequencing techniques will be used to screen for genes with new CMS-causing mutations. Molecular genetic (including CRISPR/Cas9 genome editing), biochemical and fluorescence microscopy techniques will be used to generate and study disease models in fibroblast or muscle cells. Effects of beta2-ADR agonists will be studied in transgenic models, in myotube cultures, and within AChR clusters. Detailed analysis of molecular dynamics within AChR clusters will use super-resolution microscopy in combination with temporal and spatial optogenetic stimulation. Therapy, including adeno-associated virus delivery of DOK7 to generate 'super synapses', will be given to transgenic mouse disease models and analysed in vivo by strength tests and electromyography and ex vivo by electrophysiology, microscopy, and biochemical analysis.

Impact statement:
The research proposed in this application is predicted to have a direct and immediate impact on patients with congenital myasthenic syndrome and their care. It may have longer term but significant implications for patients with autoimmune myasthenia gravis, and patients with some less common neurological disorders such as some forms of limbic encephalitis.
At an immediate level the results of a genetic diagnosis will be fed back to patients through the Oxford-based National Diagnostic and Advisory Service for Congenital Myasthenic Syndromes.
For patients and their families this provides:
i) A definitive diagnosis
ii) A basis for genetic counselling, prenatal diagnosis, and prognostic advice.
iii) Inappropriate diagnosis and treatments will be avoided. For challenging cases, prior to genetic analysis, the patient will often have had immunosuppression and undergone a thymectomy.
There are at least six different effect drugs that can be used either independently or in combination for the congenital myasthenic syndromes. Knowledge of the disease mechanism for particular mutations provides the basis for appropriate therapy. Correct therapy can have a dramatic beneficial effect both on muscle strength and quality of life. For instance, we first defined DOK7 mutations as a cause of congenital myasthenic syndrome and by treating these patients appropriately we are often able to get patients who were wheel chair-dependent leading near normal lives. A tailored treatment service will continue to provide
iv) Dramatic improvement in quality of life for patients and their families.
For the broader UK economy appropriate therapy for a disabling and sometimes life-threatening disorder will:
i) Reduced clinical burden of these disorders
ii) Reduce or prevent ITU admissions which are frequent for patients with certain subtypes of congenital myasthenic syndrome, but may occur for all forms.
iii) Improved performance and quality of life in employment.
iv) Reduce burden of caring performed by family members and social services
v) Facilitate employment
vi) Reduce the overall costs in benefits that are required for a disabled person
vii) Reduce the requirement for one-to-one support in school or outside
Other NHS benefits:
i) The NHS will benefit from definitive diagnosis
ii) Improved education of clinicians on managing these disorders
iii) Increased understanding of principle of translating basic research into clinical solutions e.g. 'bench to beside'

These benefits will be rapidly transferred from laboratory to patients through the National Service for Congenital Myasthenic Syndromes, where patients are quickly offered consultations once the genetics and disease mechanism of their condition is defined.

Patients are regularly updated on advances from the laboratory through Patient open days, or through the Myasthenia Gravis Association and Muscular Dystrophy Campaign charities.
Named researchers have been part of this programme for many years and are uniquely qualified to educate clinical fellows and research students on these specialised disorders and the techniques used for functional analysis of mutations.

We are exploring novel therapeutic approaches based on the biology of the DOK7 protein. Though at an early research stage, this approach may potentially have applications in a wide variety of neuromuscular disorders that feature defective neuromuscular junction structure during disease progression such as ALS, SMA, or sarcopenia which are intractable and multifactorial diseases.