Development of a Rabbit Model of SCN1A-linked Dravet Syndrome

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Sudden Unexpected Death in Epilepsy (SUDEP) is a leading cause of death in patients with epilepsy. SUDEP mechanisms are not understood, although there is evidence to implicate apnea, autonomic dysfunction, and cardiac arrhythmias. The majority of SUDEP patients die during sleep and, by definition, autopsy findings are largely unremarkable. Here we will generate a novel animal model of genetic epilepsy to investigate the role of cardiac arrhythmias in this devastating outcome. Loss-of-function variants in SCN1A are identified in patients with Dravet syndrome (DS). DS patients have the highest SUDEP risk, up to 20%. SCN1A is expressed in both the heart and brain of humans and mice. Because of this, we proposed that cardiac arrhythmias contribute to the mechanism of SUDEP in DS. We were the first group to show evidence for altered cardiac myocyte (CM) sodium current (INa) density and action potentials (APs), as well as cardiac arrhythmias in mouse models of SCN1A-linked DS. We were also the first to show that induced pluripotent stem cell (iPSC)-derived CMs from DS patients have substrates for arrhythmias. Importantly, no single animal or iPSC model can completely replicate the human DS phenotype. Because cardiac APs in mice are very different than in humans, we used human iPSC-CM models to investigate cell autonomous effects of SCN1A haploinsufficiency to predict cardiac arrhythmias. In spite of our success with iPSC-CMs, cells in 2-D culture cannot replicate complex cardiac tissues, cardiovascular changes, or cardiac autonomic innervation. Thus, we propose to add a transgenic rabbit model to our work because rabbits more closely replicate the human cardiac AP than mice and provide a complete organismal system with which to work. Adding a rabbit model is critical to our ability to fully understand SUDEP and to develop biomarkers for SUDEP risk in the future. The goal of this application is to develop a rabbit model of Scn1a-linked DS that can be used to more accurately replicate human cardiac physiology to ultimately understand the mechanisms of SUDEP in the genetic epilepsies. Using donor funds, we generated a New Zealand White (NZW) rabbit Scn1a deletion model using CRISPR-Cas9 gene editing techniques. We found that Scn1a-/- rabbits seize and die by postnatal day 11, similar to Scn1a-/- mice, physiologically confirming gene deletion. However, because DS patients are haploinsufficient for SCN1A, it is critical to develop a reliable Scn1a+/- rabbit DS model. We propose 3 Aims to characterize our new model: 1. To record EEGs in NZW Scn1a+/- rabbits to determine whether the animals have electrographic seizures. 2. To determine whether hyperthermia- induced seizures in NZW Scn1a+/- rabbits progress to DS-like spontaneous seizures. 3. To determine whether NZW Scn1a+/- rabbits have cardiac arrhythmia. Accomplishment of this work will establish an important, new model for use in SUDEP research that can be shared with other investigators and provide critical guidance for the future generation of other rabbit models of genetic epilepsy.