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（SUDEP）突然出现意外死亡是癫痫患者死亡的主要原因。 Sudep 尽管有证据表明呼吸暂停，自主神经功能障碍和 心律不齐。大多数SUDEP患者在睡眠期间死亡，从定义上，尸检结果是 在很大程度上并不引人注目。在这里，我们将生成一种新型的遗传癫痫动物模型，以研究 在这种毁灭性的结果中心律不齐。在患者中鉴定出SCN1A的功能丧失变体 与Dravet综合征（DS）。 DS患者的SUDEP风险最高，高达20％。 SCN1a在两者中都表达 人类和小鼠的心脏和大脑。因此，我们提出心律不齐有助于 DS中SUDEP的机制。我们是第一个展示改变心肌细胞（CM）证据的组 在小鼠模型中，钠电流（INA）密度和动作电位（AP）以及心律不齐 SCN1A连接的DS。我们也是第一个表明诱导多能干细胞（IPSC）衍生的CMS的人 DS患者具有心律不齐的底物。重要的是，没有任何动物或IPSC模型可以完全 复制人类DS表型。因为小鼠的心AP与人类大不相同，所以我们使用了 人IPSC-CM模型研究SCN1a单倍性的细胞自主效应以预测心脏 心律不齐。尽管我们在IPSC-CM上取得了成功，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 我们的工作是因为兔子比小鼠更紧密地复制人类心脏AP，并提供完整的 可以使用的有机系统。添加兔子模型对于我们充分理解SUDEP的能力至关重要 并开发未来的生物标志物来供SUDEP风险。该应用的目的是开发兔子模型 可用于更准确地复制人类心脏生理的SCN1A连接DS的DS 了解遗传性癫痫中SUDEP的机制。使用捐助资金，我们产生了一个新的 使用CRISPR-CAS9基因编辑技术的Zealand White（NZW）Rabbit SCN1A缺失模型。我们发现 SCN1A - / - 兔子在产后第11天抓住和死亡，类似于SCN1A - / - 小鼠，生理上确认基因 删除。但是，由于DS患者对SCN1A的单倍弹性不足，因此开发可靠的 SCN1A +/-兔DS模型。我们建议3个旨在表征我们的新模型：1。在NZW SCN1A +/-中记录EEG。 兔子确定动物是否具有电图。 2。确定是否高温 - NZW SCN1A +/-兔子中的诱发癫痫发作发展为DS样自发癫痫发作。 3。确定是否 NZW SCN1A +/-兔有心律不齐。完成这项工作将建立一个重要的，新的 可以与其他研究人员共享的SUDEP研究模型，并为 遗传癫痫的其他兔子模型的未来一代。