Development and Validation of a Transgenic Rabbit Model of Dravet Syndrome

Dravet 综合征转基因兔模型的开发和验证

• 批准号: 10574719
• 负责人: Lori L. Isom
• 金额: $ 37.21万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10574719
• 关键词: Action Potentials; Age; Animal Genetics; Animal Model; Animals; Antisense Oligonucleotides; Apnea; Arrhythmia; Autonomic Dysfunction; Behavioral; Biological Markers; Brain; CRISPR/Cas technology; Calcium; Cardiac; Cardiac Myocytes; Cardiovascular system; Cause of Death; Cells; Cessation of life; Clinical; Complex; Data; Development; Dissection; Drug Combinations; Epilepsy; Frequencies; Future; Generalized seizures; Genes; Genetic; Genetic Models; Goals; Heart; Human; Incidence; Knowledge; Link; Michigan; Modeling; Mus; Neurons; New Zealand; Organism; Oryctolagus cuniculus; Patients; Pharmaceutical Preparations; Phase; Phenotype; Physiology; Rattus; Risk; Rodent; Role; SCN1A protein; SCN8A gene; Seizures; Sodium; Sodium Channel; Testing; Tissues; Transgenic Mice; Transgenic Organisms; Translating; Translational Research; Universities; Validation; Variant; Video Recording; Work; clinical diagnosis; clobazam; cohort; density; dravet syndrome; effective therapy; epileptic encephalopathies; gain of function; gene therapy; heart innervation; human disease; human old age (65+); induced pluripotent stem cell; insight; loss of function; mouse model; nerve supply; novel marker; novel therapeutic intervention; premature; prevent; respiratory; risk variant; stem cell model; success; sudden unexpected death in epilepsy; translational model; translational therapeutics; two-dimensional; voltage

ABSTRACT 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. Our work in mice led to the hypothesis that cardiac arrhythmias contribute to the mechanism of SUDEP in channelopathy-linked genetic epilepsies. We demonstrated altered cardiac myocyte (CM) ionic currents, calcium handling, and action potentials (APs), as well as cardiac arrhythmias in mouse models of SCN1A-, SCN8A-, and SCN1B-linked developmental and epileptic encephalopathy (DEE). We showed that induced pluripotent stem cell (iPSC)-derived CMs from Dravet syndrome (DS) patients have substrates for arrhythmias. Importantly, no animal or iPSC model can completely replicate the human DS phenotype. Because mouse and human cardiac APs are very different, we used human iPSC-CM models to investigate cell autonomous effects of SCN1A haploinsufficiency. However, cells in 2-dimensional culture cannot replicate complex cardiac tissues, cardiovascular changes, or cardiac innervation. Here, we will develop and validate a large animal model in which the role of cardiac arrhythmias, in addition to seizures, in SUDEP can be investigated. Rabbits closely replicate the human cardiac AP and provide a complete organism to translate to the clinical setting. Our objective is to develop and validate a transgenic rabbit model of SCN1A-linked DS. We generated a New Zealand White (NZW) rabbit Scn1a deletion model using CRISPR-Cas9 gene editing. However, NZW Scn1a+/- rabbits showed neither seizures nor cardiac arrhythmia. We later found that F1: NZW x Dutch Belted Scn1a+/- rabbits have seizures, cardiac arrhythmia, and premature death. These exciting results suggest that we may have generated the first transgenic large animal model of a DEE, although this model must now be rigorously validated. If validated, this work will be a significant advance over currently available DS models. R61 Phase Specific Aims: 1. To characterize seizure onset, seizure types, seizure frequency and duration, and determine the rate of SUDEP in DS rabbits. 2. To characterize arrhythmia types, frequency, and duration, whether arrhythmias occur independently of seizures, and whether cardiac arrhythmia is associated with SUDEP in DS rabbits. R33 Phase Specific Aim: To validate the model using the ASO drug STK-001 or the drug combination stiripentol + clobazam to reduce seizures and SUDEP. Establishing a genetic rabbit model of DS will better inform the translatability of neuro-cardiac mechanisms of SUDEP to human disease.

抽象的 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。我们在小鼠中的工作导致了心律不齐有助于该机制的假设 在通道疗法连接的遗传癫痫中的SUDEP。我们证明了心肌细胞（CM）离子改变 电流，钙处理和动作电位（AP）以及小鼠模型中的心律不齐 SCN1A-，SCN8A-和SCN1B连接的发育和癫痫性脑病（DEE）。我们表明了这一点 来自Dravet综合征（DS）患者的诱导多能干细胞（IPSC）衍生的CMS具有底物 心律不齐。重要的是，没有动物或IPSC模型可以完全复制人类DS表型。因为 小鼠和人类心AP非常不同，我们使用人IPSC-CM模型研究细胞 SCN1A单倍不足的自主效应。但是，二维培养中的细胞无法复制 复杂的心脏组织，心血管变化或心脏神经神经。在这里，我们将开发并验证 大型动物模型，除了癫痫发作外，心脏心律不齐的作用可以是 调查。兔子密切复制人类心脏AP，并提供完整的生物体以翻译成 临床环境。我们的目标是开发和验证SCN1A连接DS的转基因兔模型。我们 使用CRISPR-CAS9基因编辑产生了新西兰白（NZW）兔SCN1A缺失模型。 但是，NZW SCN1A +/-兔子既没有癫痫发作也不表现出心律不齐。后来我们发现F1：NZW X 荷兰腰带SCN1A +/-兔子有癫痫发作，心律不齐和过早死亡。这些令人兴奋的结果 表明我们可能已经生成了DEE的第一个转基因大动物模型，尽管该模型必须 现在要严格验证。如果得到验证，这项工作将是当前可用DS的重大进步 型号。 R61相具比目的：1。表征发作发作，癫痫发作类型，癫痫发作频率和 持续时间，并确定DS兔子中的SUDEP速率。 2。表征心律失常类型，频率和 持续时间，心律不齐是否独立于癫痫发作以及心律不齐是否相关 与DS兔子一起使用Sudep。 R33相特定的目的：使用ASO Drug STK-001验证模型或 药物组合搅拌 + clobazam，以减少癫痫发作和SUDEP。建立一个遗传兔模型 DS将更好地告知SUDEP神经心脏机制为人类疾病的转化性。