EnCRISPrx - A Permanent CRISPR-based Approach To Rescue Dravet Syndrome By Enhancing Scn1a Promoter Activity

Dravet Syndrome is a severe catastrophic neurological disorder affecting young children. Every day 3 or 4 children are diagnosed with Dravet syndrome worldwide, with symptoms including epilepsy, autism, movement disorders and sleep disturbances. To date, the majority of therapies are ineffective or poorly tolerated. Sodium channel drugs which are otherwise effective in many forms of epilepsy may even worsen Dravet syndrome.
In approximately 80% of cases, Dravet syndrome is caused by one of several hundred heterozygous mutations in a single gene, SCN1A, which result in loss of function. These are either spontaneous or inherited, and lead to insufficient expression of the protein. Our aim is to rescue all of these mutations with a single treatment that increases the expression of the protein. To do so, we propose to modify SCN1A gene to make it more active. We will increase the activity of both copies of the gene, but only the non-mutant one will make new proteins. We are aiming for a therapy that will restore the normal functioning of the brain, which is unlike conventional drugs that target proteins that underlie communication in the brain and can dampen activity overall. We will test several possible approaches in an animal model of Dravet Syndrome, using neuronal excitability, epilepsy and behavioural changes as readouts.
This proposal will be useful not only for understand if a general treatment for Dravet syndrome is achievable, but also will give insight into the potential of these techniques to cure other neurological diseases caused by similar mutations in different genes.
We stress that the approach proposed here is not gene editing as such, which can rescue only a single mutation at a time. Instead, we aim to correct the effects of all the mutations leading to a common pathology, and so the strategy can be generalized and have a substantial impact for a wide range of diseases that share a common genetic mechanism.

Dravet Syndrome is a severe childhood epilepsy affecting 1:15000 live births, and is characterized by prolonged seizures, autistic features, movement disorders and sleep deficits. 80% of the mutations causing Dravet are in the sodium channel gene SCN1A, which codes for Nav1.1. These are mainly nonsense mutations leading to SCN1A haploinsufficiency. Dravet syndrome is resistant to anti-epileptic medication, and sodium channel targeting drugs may even worsen it, probably because inhibitory neurons do not tolerate SCN1A haploinsufficiency as well as principal neurons. At the moment gene therapy is not realistic because the gene is too big for Adeno-Associated viral vectors that have been used in clinical trials for widespread CNS transduction.
We aim to find a general approach to rescue haploinsufficient mutations in SCN1A. We will increase the activity of the endogenous SCN1A promoter. Using this strategy, our goal is to increase the transcription of both the wild type and the mutant alleles, thus restoring Nav1.1 expression to a normal level.
We will test the therapy in an animal model of Dravet Syndrome at different developmental stages, examining their efficiency in suppressing spontaneous and febrile seizures, and correcting behavioural deficits such as social interactions.
This proposal will be useful not only to understand if and when we can rescue Dravet syndrome, but will yield proof of principle of a generalizable method for treating other CNS diseases arising from haploinsufficiency.
We think this approach will help to take gene editing closer to clinical translation for neurological diseases. Moreover, it will have an impact for basic neuroscience because, for the first time, we will be able to study the consequences of modifying genes in vivo.

The goal of this proposal is to develop an innovative therapy for Dravet Syndrome that can be transferred to other diseases caused by haploinsufficiency. In addition to direct benefits to the scientific community, this project may eventually be life-changing for Dravet patients.
While rare, Dravet is a truly devastating syndrome. Severe epilepsy and autistic features are often observed early in patient's life (as young as 6 months - 1 year of age), and early mortality is common. Most of the mutations causing Dravet are in the sodium channel SCN1A gene. The development of a strategy to upregulate SCN1A expression would address a major unmet need, since this disorder is highly drug resistant.
Given the severity of patient symptoms and the frequent early mortality observed in patient cohorts, finding a therapy is essential. Gene editing, particularly with CRISPR/Cas9, is at the moment the best option for untreatable genetic diseases. Currently, however CRISPR/Cas9 has not been used for the treatment of neurological disease because DNA editing in post-mitotic neurons is difficult. Another limitation of gene editing for Dravet is the potential cost of developing and validating different tools to correct each of several hundred mutations that have been identified. Here, we develop a new tool able to rescue the deficit in expression for all of the mutations.
Our study will provide a fundamental tool to the community of neuroscience researchers, to achieve gene modification. This tool will help to expand the range of possible treatments for haploinsufficiency diseases, and also enhance our knowledge of gene function.
Finally, the post-doctoral researcher will gain advanced knowledge of molecular genetics, gene editing, ex-vivo and in vivo electrophysiology, and behavioural tests. Since there is a high demand for skilled electrophysiologists with molecular knowledge to develop new gene therapy approaches, we anticipate that the techniques acquired during this project will significantly help their future career.