Modelling neuronal dysfunction in early onset epilepsies; a patient-centric approach

Many babies present with epilepsy in the first year of life, sadly resulting in severe disability and shortened lifespan. One form of epilepsy is known as "Epilepsy of Infancy with Migrating Focal Seizures (EIMFS)" in which affected babies have very frequent seizures, often up to sixty per day, which usually do not respond to currently available medications.

Abnormalities in three genes, known as KCNT1, SLC12A5 and SCN2A can cause EIMFS. These genes make important proteins in the brain that, when abnormal, cause seizures in young babies.

However, it is not clear how they lead to epilepsy. Using a new state-of-the art brain cell model made from skin cells taken from patients in the study, I will investigate how abnormalities in these genes lead to epilepsy and developmental problems in patients.

Skin cells from each patient will be converted into stem cells. Stem cells have the potential to convert into any of the cell types in the body. Once the stem cells are formed, gene editing technology will be used to correct the mutation in some of the cells. This will allow me to verify that the effect we see in the abnormal neurones is just due to the abnormality in KCNT1, SLC12A5or SCN2A and not due to other genes.

The stem cells will be converted into three dimensional structures or organoids which after maturation for several months will be made up of layers of neurons, effectively mini-brains. These min-brains contain mostly excitatory neutrons. Excitatory neutrons cause brain excitation, whereas inhibitory neurons slow down or prevent brain excitation. I will convert some of the stem cells into 3D collections of inhibitory neurons which will be fused together with the organoids. This is to ensure all the correct types of neurons are present and that they can form connections with each other. I will test the electrical properties of the brain cells and how they connect with other brain cells.

If we can work out precisely how the abnormal genes cause seizures, this may help us identify better drugs for both this form of epilepsy and other epilepsies.

As part of the project I will also test a form of gene therapy, where a normal copy of the gene is replaced in the brain cell where it is missing. In addition, I will also be testing a different treatment, "antisense oligonucleotides". An oligonucleotide is a short synthetic DNA strand which has the mirror image code (antisense) of the DNA it is trying to bind to. The antisense oligonucleotide is designed to block and inactivate the abnormal mutated DNA.

My aim is to improve our understanding of how these abnormal genes lead to epilepsy and development problems which will help in the development of new treatments, with the ultimate aim of improving quality of life for my patients and their families.

Aim: to create a patient-derived 3D neuronal model of EIMFS which will give insight into the temporal development of the disease phenotype and act as a platform for novel therapy discovery and testing.
Objectives:
1. Establish patient-derived induced pluripotent stem cell (iPSC) and isogenic control lines
2. Create patient-derived 3D neuronal models
3. Investigate the neuronal phenotype of EIMFS
4. Rescue neuronal and network phenotype with novel therapeutic approaches

Methodology:
1. Fibroblasts from EIMFS patients with mutations in SLC12A5, KCNT1 or SCN2A will be reprogrammed into iPSCs. Isogenic controls will be created by CRISPR/Cas9 genome editing.
2. iPSC lines from patients, isogenic controls and age-matched controls will be differentiated into 3D cerebral organoids. Using patterning factors, medial ganglionic eminence-like organoids will be generated and fused with cerebral organoids.
3. Cell surface expression will be studied with immunoblotting, immunohistochemistry and confocal microscopy. Patch clamping and multielectrode arrays will assess cellular and network electrophysiological phenotype. Synaptic integrity will be assessed with confocal microscopy for synaptic markers. scRNAseq will examine gene expression differences.
4. SLC12A5 lentiviral gene therapy and KCNT1 antisense oligonucleotides will be assessed using the assays described above.
Elucidation of neuronal dysfunction in EIMFS organoids will provide insights into disease mechanisms and allow testing of novel treatments.

Healthcare impact: this fellowship has the potential to lead to the development of novel therapies for patients with severe early onset epilepsies. These conditions are currently very difficult to treat and result in frequent out-patient appointments and hospital admissions. These are distressing to families and extremely costly in terms of healthcare resources. Findings in these rare disorders may have implications for more common epilepsies. Currently, 30% of people with epilepsy in general do not respond to standard medications, therefore development of new therapies would have a significant impact. For example KCC2 is thought to be involved in brain tumour related epilepsy and temporal lobe epilepsy. KCC2 dysfunction is also implicated in neuropathic pain. Identification of novel modulators may have relevance to this debilitating condition which currently requires multiple, costly medications with a significant side-effect profile.

Industrial/commercial impact
Research findings from this project may contribute to further investigation of the role of antisense oligonucleotides (AON) for genetic epilepsies. So far, AONs have been successful in spinal muscular atrophy and Huntington disease. This fellowship could lead to a future partnership with industry. Assays that are employed in this project may have relevance to drug discovery in other rare diseases. The use of patient-derived 3D models which are amenable to high throughput testing will be of great interest to the industry as a way of reducing animal testing and assessing drug tolerability in a human model.

Economic impact: more effective epilepsy treatments would reduce disease burden and this have significant economic effects in reducing time off work for patients and their families. This fellowship would contribute to a skilled workforce by providing employment and training in a stem cell project. Stem cell technologies are likely to be important to the UK economy and it is vital that there is a skills base to support this.

Public perception of science: a successful and impactful fellowship will improve the public perception of research, particularly if the results can be translated into real benefit for patients. Visible success will also encourage young people of all backgrounds to consider careers in medicine, science or technology.