Abstracts

Rationale: Dravet syndrome is a severe childhood epilepsy marked by frequent seizures, developmental delays, and an increased risk of sudden unexpected death. Most patient cases are caused by loss-of-function mutations in the SCN1A gene, resulting in decreased expression of the alpha subunit of the voltage-gated sodium channel NaV1.1. Within the brain, decreased NaV1.1 function reduces GABAergic inhibitory neurotransmission, driving neuronal network hyperexcitability and contributing to symptoms. To study this, we have created a laboratory model using neurons derived from patient stem cells and grown on high-density microelectrode arrays that allow us to record electrical activity at the neuronal network and single-cell level at scale. We have combined this approach with single cell RNA sequencing to understand the effects of reduced NaV1.1 on RNA expression. This model enables studies of Dravet syndrome pathophysiology from human neurons in vitro and evaluation of therapeutics on relevant electrophysiological endpoints.

Methods: GABAergic cortical neurons (SCN1A^+/- and SCN1A^+/+) were derived and differentiated from human iPSCs. Neuronal firing and voltage gated sodium currents were assessed using manual patch clamp recordings, and relevant markers of maturity through qPCR. Using high-density microelectrode arrays (HD-MEA), electrophysiological recordings were performed on functional neuronal networks capable of spontaneous firing and network oscillations. From these recordings, a computational pipeline was used to identify and characterize the firing properties of single neurons and the diversity of neuronal population functional activity. In parallel, single cell sequencing was used to identify the transcriptome of the neuronal populations. We used this model system to evaluate the effects of potentiators of Na_V1.1 on neuronal function.

Results: In characterization by manual patch clamp, SCN1A+/- neurons showed a decrease in spiking frequency and voltage-gated sodium current compared with SCN1A+/+ neurons. Using the high-density microelectrode platform, the SCN1A+/- neuron cultures showed an increase in hyperexcitable network activity compared with SCN1A+/+ culture. Single cell level analysis of the array recordings showed that the neurons exhibited a wide profile of spontaneous firing activity like the diversity of neuronal gene expression observed in the single cell sequencing data. SCN1A+/- neurons showed a decrease in spontaneous firing rate compared with SCN1A+/+ neurons that could be rescued with treatment of a NaV1.1 potentiator.

Conclusions: These results demonstrated that human SCN1A^+/-  neurons recapitulate the neuronal physiology deficits and neuronal network abnormalities observed in Dravet syndrome.  Potentiation of Na_V1.1 in SCN1A^+/-  neurons rescued neuron physiology deficits recorded at the single cell level at scale using high-density microelectrode array technology. Our results support the use of human neurons as a viable translational model to enable therapeutic discovery in Dravet syndrome.

Funding: NA