Disease mechanisms and RNA-based therapies for pathogenic mutations at the neuromuscular synapse

Lead Research Organisation: University of Oxford
Department Name: Clinical Neurosciences


Nerves communicate through the transfer of electrical signals, achieved largely by opening and closing ion channels in their cell surface at specialised sites called synapses. Recent research into inherited brain diseases suggests that defects in the opening and closing of ion channels or in their precise positioning may contribute to some of the more common brain disorders such a migraine and epilepsy. We believe an understanding of how ion channels malfunction at synaptics sites will help both in finding the causes of many neurological disorders and in devising therapies. We study inherited diseases called congenital myasthenic syndromes that cause severe disability in children and adults and affect ion channels, called acetylcholine receptors, located at the connection between nerves and muscles. Inherited mutations affecting these receptors in different ways, causing disease that may or may not be treatable. Understanding how these mutations cause disease enables us to give patients correct treatments and counselling, and provides the knowledge base required for exploring new therapies. In this project we will use fluorescently-labelled receptors to visualise how the mutations are causing disease and we will explore new methods of treatments that should prevent the production of the harmful disease-causing proteins .

Technical Summary

Congenetial myasthenic syndromes (CMS) stem from genetic defects that afect transmission of information from nerves to muscles at the neuromuscular junction (NMJ) and result in fatiguable muscle weakness. They comprise a disabling - and sometimes potentially lethal - set of disorders with subtly different clincal features that arise from different underlying molecular mechanisms. An understanding of the different disease mechanisms enables appropriate treatments to be given to patients, will facilitate investigations of more common CNS disorders, and provides model examples of synaptic dysfunction that may be used to evaluate new treatment strategies such as RNA -based gene therapy. Gene silencing or gene repair that targets mRNA is a rapidly evolving area of biology that has been given a powerful boost by the discovery of RNA interference (RNAi) as a mechanism for turning off gene expression. Acetylcholine receptors (AChR) are the ion channels that receive the signal for muscle contraction. The majority of mutations in CMS patients are in the AChR, but recently we have detected a series of mutations in another protein, rapsyn, in which clusters and anchors AChR at the NMJ.

The objective of this proposal is twofold i) to set up disease models of rapsyn mutations to investiate the molecular mechanisums that underlie the muscle weakness, and ii) to explore the potential of RNAi and catalytic nucleic acids for allele-specific gene silencing and gene repair in an inherited neurological disorder.

We have recently shown that green flourescent protein can be integrated into human AChR without affecting its function. We shall use this new experimental tool in a combination of microscopic and genetic techniques to determine if rapsyn mutations affect the stability of AChR‘s in the surface membrane or the process of insertion into the membrane, and to determine effects on the fetal form of AChR that cause deformities at birth. Previously we have characterised AChR mations that have dominant excitotoxic functional effects or are null mutations. We will compare the ability of RNAi, DNAzymes and hammerhead ribozymes to give allele-specific down regulation of mutant-AChR mRNA, and investigate repair of mutant e-subunit mRNA by group I intron ribozymes . Efficacy will be established inHEK 293 cells, muscle cell lines, mutant muscle cell lines from patients, and in vivo models. We will continue to screen CMS patients for mutations in new candidate genes by PCR and automated sequence analysis. Effects on AChR function will be assessed using biochmical and electrophysiological techniques.


10 25 50
publication icon
Abdelgany A (2005) Selective cleavage of AChR cRNAs harbouring mutations underlying the slow channel myasthenic syndrome by hammerhead ribozymes. in Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research

publication icon
Abdelgany A (2005) Design of efficient DNAzymes against muscle AChR alpha-subunit cRNA in vitro and in HEK 293 cells. in Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research

publication icon
Abdelgany A (2005) Selective DNAzyme-mediated cleavage of AChR mutant transcripts by targeting the mutation site or through mismatches in the binding arm. in Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research

publication icon
Abdelgany A (2007) Hairpin DNAzymes: a new tool for efficient cellular gene silencing. in The journal of gene medicine

publication icon
Beeson D (2006) Dok-7 mutations underlie a neuromuscular junction synaptopathy. in Science (New York, N.Y.)

publication icon
Beeson D (2008) Congenital myasthenic syndromes and the formation of the neuromuscular junction. in Annals of the New York Academy of Sciences

publication icon
Burke G (2004) Distinct phenotypes of congenital acetylcholine receptor deficiency. in Neuromuscular disorders : NMD

Description MRC Programme Grant
Amount £1,482,000 (GBP)
Funding ID G0701521 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2008 
End 09/2013
Title Expression plasmids 
Description cDNA clones expressing CHRNA, CHRNB, CHRNG, CHRND, CHRNE, RAPSN, AGRN, DOK7, 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Provided expression constructs to numerous laboratories throughout the world for studies of the neuromuscular junction, both pre and post 2006. 
Title Model of AChR deficiency syndrome 
Description Mouse model of AChR deficiency syndrome 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Cossins et al., 2004. Used to establish optimum therapeutic strategies for difficult to treat patients. 
Title cell line 
Description Cell lines from patients with congenital myasthenic syndromes 
Type Of Material Model of mechanisms or symptoms - human 
Provided To Others? Yes  
Impact Used in determining underlying disease mechanism and in determining if DNA variants are pathogenic 
Description OXION Wellcome Trust ion channels initative 
Organisation University of Oxford
Department Department of Physiology, Anatomy and Genetics
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution I am on OXION management committee, and collaborated in writing grant application. As part of the consortium my group was chosen by one of the research fellows.
Collaborator Contribution Provided a 3 year fully-funded post-doctoral research fellow
Impact A series of papers involve members of the OXION consortium.
Title ephedrine 
Description We demonstrated this commonly available drug has a remarkable beneficial effect for patients with DOK7 CMS. 
Type Therapeutic Intervention - Drug
Current Stage Of Development Refinement. Clinical
Year Development Stage Completed 2006
Development Status Under active development/distribution
Impact Use of ephedrine in DOK7 CMS patients results in a dramatic improvement in disability scores. For example patients who were in wheel chairs are now often able to run. Ephedrine and salbutamol (which acts in a similar way) are now therapies of choice for DOK7 CMS 
Description Congenital myasthenic syndrome open day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact Patient day. Patients are given a series of lectures on scientific research updates, and then in the afternoon there are a series of workshops for particular patient groups where needs that have been identified or areas of special interest are addressed. Typically this might involve treatments, exercise, benefits, future therapy updates.

Patients and researchers are both enthused about the research effort
Year(s) Of Engagement Activity 2006,2008,2010,2013,2014
URL http://www.ouh.nhs.uk/services/referrals/neurosciences/myasthenia.aspx
Description Lectures to patient groups 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact Regularly give talks to small patient groups and parents who have an obvious interest in inherited myasthenic disorders

Useful patient researcher contact.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2013
Description Oxford science week 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Open lecture to the public

Useful conversations
Year(s) Of Engagement Activity 2009
Description Work experience 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Schools
Results and Impact Individual students spend a week in the laboratory learning about scientific research.

School comes back each year requesting further pupil experience.
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012