Development of a Rabbit Model of SCN1A-linked Dravet Syndrome

SCN1A 相关 Dravet 综合征兔模型的开发

• 批准号: 10062010
• 负责人: Lori L. Isom
• 金额: $ 42.9万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-15 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10062010
• 关键词: Action Potentials; Acute; Animal Model; Animals; Apnea; Arrhythmia; Autonomic Dysfunction; Autopsy; Behavioral; Biological Markers; Brain; CRISPR/Cas technology; Calcium; Cardiac; Cardiac Myocytes; Cardiovascular system; Cause of Death; Cells; Childhood; Complex; Contracts; Data; Development; Dissection; Early Infantile Epileptic Encephalopathy; Electrocardiogram; Electrodes; Electroencephalogram; Epilepsy; Febrile Convulsions; Funding; Future; Future Generations; Gene Deletion; Genes; Genetic; Genetic Models; Genotype; Goals; Heart; Human; Implant; Incidence; Induced Hyperthermia; Link; Michigan; Modeling; Mus; Neurons; New Zealand; Operative Surgical Procedures; Oryctolagus cuniculus; Outcome; Patients; Phenotype; Physiological; Physiology; Property; Rattus; Research; Research Personnel; Risk; Role; SCN8A gene; Seizures; Sleep; Sodium; Sodium Channel; Surface; System; Techniques; Telemetry; Testing; Therapeutic; Time; Tissues; Transgenic Mice; Transgenic Organisms; Translational Research; Universities; Variant; Ventricular; Work; clinical Diagnosis; cohort; density; dravet syndrome; effective therapy; gain of function; high risk; human disease; indium arsenide; induced pluripotent stem cell; insight; loss of function; medical schools; mouse model; nerve supply; novel; novel marker; novel therapeutics; postnatal; premature; prevent; programs; risk variant; stem cell model; success; sudden unexpected death in epilepsy; translational model; two-dimensional; voltage

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.

癫痫猝死（SUDEP）是癫痫患者死亡的主要原因。南苏丹发展计划 尽管有证据表明呼吸暂停、自主神经功能障碍和 心律失常。大多数 SUDEP 患者在睡眠期间死亡，根据定义，尸检结果是 基本上不起眼。在这里，我们将建立一种新的遗传性癫痫动物模型来研究遗传性癫痫的作用 心律失常造成了这种毁灭性的后果。在患者中发现 SCN1A 功能丧失变异 患有 Dravet 综合征 (DS)。 DS 患者的 SUDEP 风险最高，高达 20%。 SCN1A 在两种细胞中均表达 人类和老鼠的心脏和大脑。因此，我们提出心律失常会导致 DS中SUDEP的机制。我们是第一组显示心肌细胞 (CM) 改变的证据 钠电流（INa）密度和动作电位（AP），以及小鼠模型中的心律失常 SCN1A 连锁 DS。我们也是第一个证明诱导多能干细胞 (iPSC) 衍生的 CM DS 患者有心律失常的基础。重要的是，没有任何一种动物或 iPSC 模型能够完全 复制人类 DS 表型。由于小鼠的心脏 AP 与人类的心脏 AP 有很大不同，因此我们使用 人类 iPSC-CM 模型研究 SCN1A 单倍体不足的细胞自主效应以预测心脏 心律失常。尽管我们在 iPSC-CM 方面取得了成功，但二维培养中的细胞无法复制复杂的心脏组织， 心血管变化或心脏自主神经支配。因此，我们建议添加转基因兔模型 对我们的工作来说，因为兔子比小鼠更接近地复制了人类心脏 AP，并提供了完整的 与之合作的有机系统。添加兔子模型对于我们充分理解 SUDEP 的能力至关重要 并开发未来 SUDEP 风险的生物标志物。该应用程序的目标是开发兔子模型 Scn1a 相关的 DS 可用于更准确地复制人类心脏生理学，最终 了解 SUDEP 在遗传性癫痫中的机制。利用捐助资金，我们产生了一个新的 使用 CRISPR-Cas9 基因编辑技术的新西兰白 (NZW) 兔 Scn1a 缺失模型。我们发现 Scn1a-/- 兔子在出生后第 11 天抓住并死亡，与 Scn1a-/- 小鼠相似，生理学证实基因 删除。然而，由于 DS 患者的 SCN1A 单倍体不足，因此开发可靠的 SCN1A 至关重要 Scn1a+/- 兔 DS 模型。我们提出 3 个目标来描述我们的新模型： 1. 在 NZW Scn1a+/- 中记录 EEG 兔子以确定动物是否有电图癫痫发作。 2. 确定是否出现过热 NZW Scn1a+/- 兔子的诱发癫痫发作进展为 DS 样自发癫痫发作。 3. 判断是否 NZW Scn1a+/- 兔子有心律失常。这项工作的完成将建立一个重要的、新的 用于 SUDEP 研究的模型，可以与其他研究人员共享，并为以下研究提供关键指导： 下一代其他遗传性癫痫兔模型。