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.

摘要 癫痫猝死（SUDEP）是癫痫患者死亡的主要原因。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， 心律失常我们在小鼠中的工作导致了心律失常有助于机制的假设 SUDEP在通道病变相关遗传性癫痫中的作用。我们证明了心肌细胞（CM）离子 电流，钙处理和动作电位（AP），以及小鼠模型中的心律失常。 SCN 1A-、SCN 8A-和SCN 1B-连锁的发育性和癫痫性脑病（DEE）。我们发现 来自Dravet综合征（DS）患者的诱导多能干细胞（iPSC）衍生的CM具有用于 心律不齐重要的是，没有动物或iPSC模型可以完全复制人DS表型。因为 小鼠和人类心脏AP非常不同，我们使用人类iPSC-CM模型来研究细胞 SCN 1A单倍不足的自主效应。然而，二维培养中的细胞不能复制 复杂的心脏组织、心血管变化或心脏神经支配。在这里，我们将开发和验证一个 大型动物模型，其中心律失常的作用，除了癫痫发作，在SUDEP可以 研究了兔子密切复制人类心脏AP，并提供一个完整的生物体来翻译 临床设置。我们的目标是开发和验证SCN 1A连锁DS的转基因兔模型。我们 使用CRISPR-Cas9基因编辑产生新西兰白色（NZW）兔Scn 1a缺失模型。 然而，NZW Scn 1a +/-兔既没有癫痫发作，也没有心律失常。我们后来发现，F1：NZW x 荷兰带Scn 1a +/-兔有癫痫发作，心律失常和过早死亡。这些令人兴奋的结果 这表明我们可能已经产生了第一个DEE的转基因大型动物模型，尽管这个模型必须 现在已经得到严格验证。如果得到验证，这项工作将是对目前可用DS的重大进步 模型R61阶段具体目标：1.描述癫痫发作、癫痫发作类型、癫痫发作频率和 持续时间，并测定DS兔的SUDEP率。2.描述心律失常类型、频率和 持续时间，心律失常是否独立于癫痫发作发生，以及心律失常是否与癫痫发作相关。 在DS兔中的SUDEP。R33阶段特定目标：使用阿索药物STK-001或 药物组合司替戊醇+氯巴占，以减少癫痫发作和SUDEP。兔遗传模型的建立 DS将更好地告知SUDEP的神经心脏机制对人类疾病的可翻译性。