In vivo CRISPRa alleviates epilepsy

crispr to treat epilepsy

Date: 10th March 2020

Epilepsy is a central nervous system (neurological) disorder in which brain activity becomes abnormal, causing seizures or periods of unusual behaviour, sensations, and sometimes loss of awareness.  Now scientists have used CRISPRa in vivo to decrease seizures and rescue cognitive deficits in a rodent model of epilepsy.

Around 50 million people worldwide have epilepsy, making it one of the most common neurological diseases globally. Approximately 30% of patients continue to experience seizures despite the use of medication.

Focal epilepsies (those that affect one hemisphere of the brain) account for 60% of all forms of epilepsy and traditionally have been regarded as largely acquired disorders, however, there is a growing body of evidence suggesting that genetic factors also play a significant role. The majority of drug-resistant epilepsies fall into this focal category and treatments remain limited.

Now a team of scientists led by Gabriele Lignani, from University College London, UK, have used an activating variant of CRISPR, CRISPRa, to modulate the expression of endogenous genes by directly targeting their promoters in vivo.

The CRISPRa system consisted of a nuclease-defective Cas9 (dCas9) fused to a transcription activator and a small guide RNA (sgRNA) that targeted dCas9 to the promoter of the gene of interest.  In this case the team wanted to increase endogenous gene expression in glutamatergic neurons to decrease their excitability.

As a proof-of-principle, they chose the Kcna1 gene encoding the Kv1.1 channel, which is important for the regulation of neuronal action potential firing and synaptic transmission.  Previous work by others had shown overexpression of this gene administered by lentivirus or adeno-associated virus reduced neuronal excitability, and could suppress seizures in rodent models of epilepsy.

The team initially tested the CRISPRa in vitro, and showed an increase in endogenous Kcna1 expression which reduced neuronal excitability in primary neurons.

In vivo CRISPRa use

To translate the system for in vivo use, they used a dual adeno-associated virus (AAV) system.  The CRISPRa elements were separated into two AAV viral vectors, one carrying the dCas9-VP64 (transcription activator) and the other a single guide RNA (sgRNA) controlled by a specific neuronal promoter.  The whole system was inducible and only active after doxycycline (dox) administration (fed to the mice).

Following co-injection of both AAVs into the hippocampus of mice, and activation of the system (by dox) the mice brains displayed a decrease in neuron firing rate in the brain and increased current threshold when compared with control mice for a 3-week period.  This supported the systems use as a candidate antiseizure gene therapy.

To really put the system to the test, the authors then injected the Kcna1-dCas9A AAVs into the hippocampi of a mouse model of acquired epilepsy.

From the 11 injected mice, 2 failed to express dCas9 and were excluded from the analysis.  The remaining 9 all expressed dCas9 (of those 5 controls, and 4 Kcna1-dCas9A).  A 50% increase in Kcna1 expression was seen in the mice transduced with Kcna1-dCas9A compared with the controls.

To investigate the ability of Kcna1-dCas9 to treat chronic temporal lobe epilepsy the team then analysed the frequency of generalised tonic-clonic seizures in each animal before and after dox administration.  A significant decrease in the number of seizures per day after doxycycline administration was seen in the Kcna1-dCas9A treated animals.

However, other EEG parameters did not change, suggesting that Kcna1-dCas9A reduced the probability of tonic-clonic seizure initiation, but otherwise did not change seizure properties recorded in the cortex.

Finally, the team wanted to assess cognitive co-morbidities which are associated with epilepsy.  They therefore assessed various behavioural paradigms after treatment.  The data showed rescue of behavioural deficits in the epileptic mice which had been treated with Kcna1-dCas9A.  Furthermore, gene expression analysis showed that out of the 388 common deregulated genes that were identified in the epileptic mice, 165 were rescued by Kcna1-dCas9A treatment.


Conclusion and future applications

The notion of CRISPRs as potential genetic disease treatments is currently well established, however, their role in non-genetic disease has been somewhat overlooked until now.  As the majority of epilepsy cases are not thought to be due to single gene mutations and are often acquired during life, the use of CRISPRa here is paving the way for a new treatment for epilepsy and many other neurological disease associated with altered transcription.

By controlling the activity of the gene themselves, CRISPRa offers advantages over other systems, such that multiple mutations do not have to be repaired, genes irrespective of length can be controlled, and DNA cleavage does not occur.  In theory, the latter may reduce the amount of off-target effects.

Whilst, it is still early days for CRISPRa, we are beginning to harness their potential.  Late last year scientists from the University of North Carolina, developed a dose-dependent, gene-specific and reversible CRISPRa system.  Here chemical epigenetic modifiers (CEMs) recruited components of the endogenous chromatin-activating machinery to drive expression of target genes via dCas9.

From a clinical viewpoint activating gene expression could be a potential source of preventing many diseases.  In this paper, Gabriele Lignani and the team are taking the next step forward and translating the tech into an in vivo setting.  For effective translation into a clinical setting the system may have to be simplified, for example, both CRISPR elements could be placed in one AAV to simplify delivery.

However, the team have shown compelling evidence that CRISPR-mediated control of gene expression can be successfully exploited to modulate neuronal activity and to mitigate seizures and behavioural co-morbidity associated with epilepsy.


Colasante, G., Y. Qiu, L. Massimino, C. Di Berardino, J. H. Cornford, A. Snowball, M. Weston, S. P. Jones, S. Giannelli, A. Lieb, S. Schorge, D. M. Kullmann, V. Broccoli and G. Lignani (2020). “In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy.” Brain.