UCL’s Targeted Gene Therapy Proves Promising Against Drug-Resistant Epilepsy

• It is reported by WHO that some 50 million people worldwide suffer with epilepsy. Around 70% of this patient population are able to control their occurrences of seizures, the hallmark of this neurological disorder, with anti-seizure medication.
• However, this leaves around a third of patients who do not respond to current treatments. Currently the only option to control seizures in epileptic patients who are drug resistant, is brain surgery, which comes with significant risks. 
• Scientists at UCL are exploring other avenues. Dr Gabriele Lignani and Professor Dimitri Kullmann are part of a team investigating the potential of a gene therapy developed to target neurons in the brain specially involved with epileptic pathology.
• Promising results in mice and lab grown human brain cells shows the treatment causes a well targeted, closed-loop system – successful in sensing neuronal excitability and subsequently preventing seizures. 

Could you please introduce yourselves and tell me a little bit about what you do?

GL: I’m Gabriele Lignani, I am a Professor at UCL Queens Square Institute of Neurology. I studied biotechnology and then did a PhD in experimental neurosciences. I studied epileptic mechanisms and now, along with Dimitri and other colleagues, we are developing new genetic therapy tools for neurological diseases.

DK: Hi, I’m Dmitri Kullmann. I’m a neurologist and neuroscientist – I still see patients, but I’m mainly in the laboratory. I’m interested in all aspects of how synapses work in the brain and in the peripheral nervous system and have an interest in the mechanisms of neurological diseases including epilepsy. Together with Gabriele and a couple of other colleagues, we have been working on developing gene therapy for epilepsy for a while because of the enormous unmet needs that are recognized in this field.

Can you please tell us more about the novel gene therapies that you are developing to target epilepsy?

DK: Epilepsy is a multifactorial disease and there are a lot of different sub-types of this disease. We have been working on developing tools that will work across different types of epilepsy and these are based on adjusting the excitation and inhibition in the brain.

Typically, we try to reduce neurotransmitter release from excitatory neurons. Therefore, in a way our gene therapies work in a similar way to current anti-seizure medication, but we have the advantage of targeting the neurons specifically in the area of the brain where the seizures arise, and also to bias the expression of the gene therapies to excitatory neurons. It is a more rational approach than exposing the entire brain, or the entire body, to an anti-seizure medication. We are not trying to undo the underlying genetic abnormality, scar, tumor, or any other cause of the seizures, we’re just trying to improve the ability to stop the seizure.  What we’re doing is helping the brain’s intrinsic mechanisms that regulate excitability and inhibition.

Over the years, we’ve tried a number of different technologies. This includes overexpressing potassium channels, which reduces neuronal excitability and reduces neurotransmitter release. We’ve also used a method that inserts an inhibitory receptor into neurons. This receptor has been made insensitive to endogenous neurotransmitters, it is only activated with an exogenous drug. This technology is called chemogenetics and we’ve shown that it is also effective. We have also put in a receptor for the main excitatory neuron glutamate, which was an inhibitory receptor. Coupling it to a chloride channel means that when glutamate goes up during a seizure, it then opens a chloride channel, which has an inhibitory effect. More recently, with Gabriele, we’ve been working on some more modern cutting-edge methods to manipulate endogenous ion channels.

Our latest invention was published in the journal ‘Science’, and we’re very excited by it – Gabriele can tell you more about that!

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GL: As Dimitri says we have been working with new technologies, for example, a CRISPR that cannot cut DNA but can be fused with transcription activators to upregulate the expression of any gene. Recently, we have developed a new way of thinking about therapies to treat epilepsy, as featured in our piece in ‘Science’. 

Instead of thinking about decreasing the overall excitability of all excitatory neurons at all points, we thought to acknowledge the dynamic pathology of epilepsy. The brain is not always seizing in these patients, so instead, we looked to decrease the excitability of the specific excitatory neurons participating in the triggering of a seizure and only at the time of seizure onset. The healthy neurons in surrounding or intertwined areas that do not impact the disease pathology are not affected.

In response, the tool we developed is based on activity-dependent promoters that can sense an increase in activity, coupled with potassium channels that can decrease the excitability. This creates a closed-loop system – if a neuron increases in excitability, when it receives more inputs, then its coupled potassium channel increases expression to decrease excitability. This is a closed loop in both space and time – only the neurons that need to be treated will have this tool activated when the activity is increased.

How is this therapeutic development striving to meet the unmet need for refractory epilepsy?

DK: There are two parts to this gene therapy – the promoter and the transgene. The promoter determines how much of a gene is transcribed in any given cell, and the transgene is something, typically a protein, that is added to the genome. In our case this is a potassium channel. The potassium channel keeps neurons relatively unexcited, reducing the propensity of neurons to generate action potentials and neurotransmitter release from the neuronal terminals. Previously we’ve shown that by constitutively overexpressing this potassium channel, we can reduce excitability and the tendency to have seizures in experimental models. The novel thing we have done here is to put the potassium channel gene under a promoter that is not active constitutively but is only active in response to intense pathological activity in cells.

This promoter belongs to a family of genes called immediate early genes. More specifically it comes from a particular type of immediate early gene, a c-Fos gene, which switches on in response to intense stimuli such as seizures. The promoter of the c-Fos gene is used to drive the expression of the potassium channel. This creates a closed loop mechanism. If a seizure is detected in the form of neuron excitability then it switches on the c-Fos promoter, which then leads to the expression of the potassium channel, the potassium channel then reduces neuronal excitability and reduces neurotransmitter release. The result is that the propensity for seizures is suppressed without having any effect on cells that aren’t involved in seizure pathology.

Other cells may have been infected by the virus that we use as a vector to introduce the DNA package into cells. However, only those involved in specific seizure pathology will be triggered. This allows us to differentiate between bystander or intermingled neurons, which are not firing pathologically, from those which we want to treat.

What test results have you seen so far?

GL: We have been able to demonstrate an 80% decrease in spontaneous seizures in a chronic model of intractable, drug-resistant epileptic mice.

Following network dynamics very closely we have also been able to validate that when this tool is activated it can decrease neuronal excitability, and act as a closed-loop and self-regulated system.

Lastly, we have also collected data from laboratory-grown ‘mini brains’ grown from stem cells derived from healthy human donors. When you induce epileptic activity in these mini-brains, our gene therapy has been shown to decrease their excitability and the severity of this epileptic activity. This was proof that this therapeutic tool is effective in human neurons.

So what are the next steps for your research?

DK: The translational process as you can imagine is not straightforward. There are a lot of safety issues that need to be verified, and many hurdles to be overcome, such as making a clinical-grade virus, finalizing the design of the definitive viral vector to be used, verifying that we have the correct dose, and, are targeting the correct position in the brain. Most importantly we need a large amount of money to progress things!

What have the main challenges been so far in this project?

DK: Having good models of epilepsy is a challenge. Spontaneous seizures, the hallmark of epilepsy, occur unpredictably. As a result, you have to have methods to monitor experimental models in real-time, over a long period of time.

We also have to take into consideration welfare issues – our experiments are very tightly regulated. Technology is used to detect these seizures and verify that they are genuinely seizures, not some sort of artifacts. Having these models puts us in a strong position, even though they take a long time to validate.

Another challenge is working with our miniature model of the human brain. Growing these models in Petri dishes takes around 250 days of incubation, and they must be stored under controlled conditions – one incubator disaster and the whole experiment can wrong.

These challenges mean it has taken a long time to get where we are, and as you can tell from the number of authors on the paper, a lot of people have put a lot of work into Gabriele’s original idea. The results we are seeing are a testament to his ability to get people to work hard!

GL: Indeed! It has taken 6 years to get from the original idea to get to the point we are now – with real results validating the potential of gene therapy as a treatment for refractory epilepsy.

How does this gene therapy have the potential to change the lives of people living with refractory epilepsy?

DK: The most exciting aspect of this project is this therapy’s intrinsic ability to normalize neuronal function, which is really quite unique. Every other gene therapy to date permanently alters the properties of the neurons, whereas here it’s an on-demand closed-loop manipulation. So far we have only validated this therapy in models of epilepsy, but in principle it could be used in other neurological disorders where you have populations of neurons that become overactive and generate symptoms as a result of that pathological over activity. Of course, we have to be cautious as we haven’t tested this, but we do speculate about that in the paper. This is a really important advance over other gene therapies.

GL: I agree that we have to be cautious in our claims before we test this therapy in other diseases, but also that  in epilepsy we have seen significant potential. A benefit of our gene therapy is that because it’s not a permanent change it should have less impact on cognition and normal brain function. Current epileptic medicines have quite a large number of side effects. By targeting just the area where where the seizure starts with this gene therapy it can reduce and control the side effects for  patients. 

DK: We have to avoid the impression that this is going to replace all medications for epilepsy. Most people with epilepsy have their seizures very well controlled by medication. This therapy has been developed for a proportion of people with refractory or drug-resistant epilepsy – many of whom have focal epilepsy. This type epilepsy is primarily generalized by seizures generated in a specific part of the brain where there may be an abnormality that you can see on an MRI scan, they are the patients who this therapy could potentially benefit in the future. 

Find out more about Dr. Gabriele Lignani, Proffesor Dimitri Kullmann, and, the rest of the team at UCL Queen Square Institute of Neurology’s research here.

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