Functional significance of neuronal sodium channel splicing in epilepsy and pain

Lead Research Organisation: University College London
Department Name: Institute of Neurology

Abstract

Many neurological disorders, including epilepsy and chronic pain, are associated with changes in the functioning of nerve cells. These cells become more active than in healthy tissue, and can even start sending signals when they should remain quiet. It is thought that these changes can lead to the uncontrolled activity in seizures, or the chronic sensation of pain, even when there is no painful stimulus present. But why do the cells become excessively active?

We are seeking to answer this question by exploring the changes in the processing of molecules which drive neuronal activity: the sodium channels. Many sodium channels exist in two versions, one predominates in healthy adult neurons, the 'adult variant', the other, almost exactly the same, predominates in very young neurons, the 'neonatal variant'. However in some diseases, such as epilepsy, the neonatal variant is increased, even in mature nerve cells. How does this increase in neonatal channels alter the behaviour of adult nerve cells?

We are especially interested in the change in the sodium channels because we know that many common drugs bind to these channels. These include common anti-epileptics, most local anaesthetics, and other drugs used to treat a growing number of disorders, including chronic pain. At present, many of these drugs are thought to bind to *all* sodium channel variants, but some drugs may show some preference for the neonatal or the adult channels. If the neonatal variant is being up-regulated in disease, is it helpful to specifically target this variant - to knock it back down? Could deliberately suppressing only the neonatal variant reduce the side-effects caused when these drugs bind to healthy sodium channels?

An additional line of evidence suggesting these variants are important is that they are extremely highly conserved in evolution, but as yet it is not known what function they play in any animal, from flies to humans. Such conservation is usually a sign that a variation is important to healthy functioning of the organism. We will investigate the roles of these highly-conserved variants healthy nerve cells, and also in models of both epilepsy and pain, and will use our findings to recommend whether pharmacological companies should consider developing compounds which are specific for either variant.

Our findings will offer new perspectives on how these fundamental channels are regulated, and whether there is an avenue for treatments that are better tolerated in humans.

Technical Summary

While it has been known for decades that sodium channels are subject to highly conserved alternative splicing in their voltage sensing paddles, the functional role and adaptive significance of this splicing has been obscured by the challenge of expressing, clamping and comparing these channels in neurons. The sensitivity of the channels to the intracellular modulation, and their specialisation for neuronal functions, means that heterologous expression is severely limited, and even over-expression of cDNAs in neurons may not recapitulate the roles of these highly similar variants.
We have recently established tools that enable us to ask, in neurons, the functional consequences of selectively manipulating splice variants. We have developed shRNA sequences that can significantly alter the proportion of each splice variant in neurons. Using our lentiviral-delivered shRNAs we have shown in vitro, that reducing each of the two variants of SCN8A leads to distinct effects, with reducing the 'neonatal' variant significantly altering resting membrane potential, while reducing the 'adult' variant disrupts regular firing, and appears to reduce the contribution of the axon initial segment to action potentials.
We have developed four parallel approaches for measuring the functional roles and distribution of splice variants in neurons. In addition we have two models of clinically-important disease (epilepsy and pain) that are strongly associated with altered sodium channel activity. We will test whether key drugs that modulate sodium channels can distinguish between the functions of splice variants, and whether the changes in this voltage sensing paddle may be a potential target for rationally-designed drugs. Finally we will return to the question of how a human polymorphism that changes splicing might lead to altered dosage of drugs to ask how splicing impacts drug response.

Planned Impact

Impact on Health: Epilepsy and pain are two serious neurological disorders that can have devastating effects on patients. Both are often resistant to existing pharmaceutical treatments. One problem is that many of the treatments to both of these disorders target sodium channels, but fail to distinguish between 'healthy' and 'abnormal' channels. By probing one of the physical changes that occurs in sodium channels during the progression of diseases, we will raise the possibility of treatments with fewer off-target effects, by demonstrating the impact of selectively manipulating the 'abnormal' channel splice variants. Improved treatments to chronic and serious neurological disorders would offer an improvement in quality of life.
The third sector: SS currently serves on the Science Advisory Committee of the charity Epilepsy Research UK, and this involvement means our findings will be transmitted quickly to the patient bodies and other researchers in this sector, where other researchers may adapt it to generate new hypotheses and to exploit findings for new treatment strategies.

Timescales: During the course of the project we will present data at charities and symposia that will reach our academic and clinical beneficiaries. Our publications will fall towards the end of the project, and shortly thereafter. By the end of the project (3 years) we hope that our findings will be informing other studies of drug response in epilepsy and pain, and any data that suggest an effective approach to mitigating seizures or excess excitability of pain sensing neurons will serve to underpin applications for pre-clinical studies (4 years). In the longer term, our findings may lead to rationally targeted novel anti-epileptic drugs and pain therapy.

Skills for staff on project: The staff will benefit from the development of advanced skills in slice patch clamp and use of GM viruses, a growing field for gene therapy treatments. We have found skilled electrophysiologists to be in high demand (both when we try to recruit, and when students graduate from the group), thus we anticipate that the skills they hone will place them in high demand for future positions.
 
Description Appointed to Epilepsy Research UK Science Advisory Committee
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Appointed to board of Trustees for Epilepsy Research UK
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Member of SACGM(CU)
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Queen Square award for implemenation of 3Rs in Research
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
Impact We are working to change our local attitudes towards working with animals in research. We presented this award to the then Cabinet Minister, and it was recognised as a high level local engagement with the 3Rs in animal work. All students presenting their scientific posters at the symposium are now aware that they must also explain their measures to improve 3Rs to our NACWO, including justifying the model used.
 
Description UCL Celebrates the 3Rs
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
 
Description MRC DPFS Award
Amount £2,200,000 (GBP)
Funding ID MR/R015333/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2018 
End 03/2022
 
Description ONO Pharmaceuticals Pilot funding
Amount £70,000 (GBP)
Organisation Ono Pharmaceutical 
Sector Private
Country Japan
Start 04/2017 
End 10/2017
 
Description Renewal of University Reserch Fellowship
Amount £317,000 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 09/2018
 
Title COMBINED USE OF A VECTOR ENCODING A MODIFIED RECEPTOR AND ITS EXOGENOUS AGONIST IN THE TREATMENT OF SEIZURES 
Description The invention provides methods and materials for treating a seizure disorder such as epilepsy in a patient which employ a vector encoding a modified receptor, the so-called "DREADD" receptor being characterised by (i) a decreased responsiveness to its endogenous activating ligand (ii) a retained or enhanced responsiveness to an exogenous agonist. The modified receptor is expressed in neurons of a seizure focus in brain of the patient, and an exogenous agonist is administered which activates the modified receptor to reversibly alters the excitability of the neurons in the seizure focus leading to synaptic silencing or other inhibition. 
IP Reference US2016375097 
Protection Patent application published
Year Protection Granted 2016
Licensed No
Impact This licencing is part of a portfolio of approaches to developing gene therapy to treat epilepsy
 
Title EXPRESSION VECTORS COMPRISING ENGINEERED GENES 
Description The invention provides expression vectors, nucleic acids, vector particles and methods of treatment involving these vector particles, comprising an engineered KCNA 1 gene encoding an edited Kv1.1 potassium channel, as well as methods of confirming the presence of engineered KCNA 1 mRNA in a cell. The features of the engineered KCNA 1 gene combine to advantageously enhance the translation and activity of the Kv1.1 protein and improve detection of KCNA 1 gene expression in a cell and can be used for example in the treatment of epilepsy and similar neurological disorders. 
IP Reference WO2018229254 
Protection Patent application published
Year Protection Granted 2018
Licensed No
Impact This patent is helping support our award to translate our gene therapy to a first in human clinical trial
 
Description DIS visits from students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact International undergraduate students (20-30) with the DIS programme in Copenhagen visit ION at Queen Square, hear a presentation and tour the Department, and discuss careers in science with the team.
Year(s) Of Engagement Activity 2015,2016,2017
 
Description Presentation at Royal Society summer science festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact I gave a presentation "Brain Hacking With Viruses" introducing the concepts of Gene therapy for epilepsy at the Royal Society Summer Science Festival. The talk was repeated 4 times, and was well attended by students from around the UK. I also raised some of the ethical and safety concerns of the approach and got very strong feedback in the questions that students were interested even if the testing involved use of animals in research.
Year(s) Of Engagement Activity 2016
URL https://royalsociety.org/science-events-and-lectures/2016/07/schools-talk-schorge-tuesday/
 
Description Royal Society Summer festival presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact This was a summer science 'lates' which meant running a both with a hands on activity for people attending the Royal Society Summer Science Festival. We presented an origami hands-on demonstration of how gene therapy uses viral shells to carry instructions for making healthy genes. The event was very well attended with over 3000 at the Royal society, and my personal booth (going by how many sheets of origami paper we gave out) had over 100 visitors, mostly from the general public but also some schools and university students.
Year(s) Of Engagement Activity 2018
URL https://royalsociety.org/science-events-and-lectures/2018/07/summer-science-lates/
 
Description School Visit (Lenox) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact This was a presentation on Gene Therapy and treatments of epilepsy to a High School audience in the states.
Year(s) Of Engagement Activity 2016,2017
 
Description Work experience at ION 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Both students were well-engaged with basic and clinical research at the Institute of Neurology.

Social Mobility Fund and the Worshipful Company of Pewterers have both indicated they'd be delighted to carry on with the scheme in future years.
Year(s) Of Engagement Activity 2014