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)是癫痫患者的主要死亡原因。SUDEP 机制尚不清楚，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患者在睡眠中死亡，根据定义，尸检结果是 这在很大程度上并不引人注目。在这里，我们将建立一种新的遗传性癫痫动物模型，以研究 心律失常在这一毁灭性的后果中。在患者中发现了SCN1a功能缺失的变异 患有德雷维氏综合征(DS)。DS患者的SUDEP风险最高，高达20%。SCN1a在两种基因中均有表达 人类和老鼠的心脏和大脑。正因为如此，我们提出了心律失常对 弥漫性眩晕的SUDEP机制我们是第一个证明心肌细胞(CM)改变的小组。 钠电流(INa)密度和动作电位(AP)，以及心律失常的小鼠模型 SCN1a连锁的DS。我们也是第一个证明诱导多能干细胞(IPSC)起源于CMS的人 DS患者有心律失常的底物。重要的是，没有单一的动物或IPSC模型可以完全 复制人类DS表型。由于小鼠的心脏AP与人类的非常不同，我们使用了 人IPSC-CM模型研究SCN1A单倍体功能不全的细胞自主效应预测心脏 心律不齐。尽管我们在iPSC-CMS上取得了成功，但二维培养的细胞不能复制复杂的心脏组织， 心血管改变，或心脏自主神经支配。因此，我们建议增加一个转基因兔模型 因为兔子比小鼠更接近复制人类心脏AP，并提供了一个完整的 与之共事的有机系统。添加一个兔子模型对于我们完全理解SUDEP的能力至关重要 并在未来开发SUDEP风险的生物标志物。这个应用程序的目标是开发一个兔子模型 可以用来更准确地复制人类心脏生理，最终 了解SUDEP在遗传性癫痫中的机制。利用捐赠者的资金，我们产生了一个新的 应用CRISPR-Cas9基因编辑技术建立新西兰白兔Scn1a缺失模型。我们发现 SCN1a-/-兔在出生后第11天发病并死亡，类似于Scn1a-/-小鼠，生理确认基因 删除。然而，由于DS患者的SCN1a基因单倍性不足，因此开发一种可靠的 SCN1A+/-兔DS模型。我们提出了三个目标来描述我们的新模型：1.在NZW Scn1a+/-中记录脑电 以确定动物是否有脑电惊厥。2.确定体温过高是否- NZWScn1a+/-兔诱发癫痫进展为DS样自发性癫痫发作。3.确定是否 NZW Scn1a+/-兔出现心律失常。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，可与其他研究人员共享，并为 下一代其他遗传性癫痫兔模型。