Development and Validation of a Transgenic Rabbit Model of Dravet Syndrome

摘要 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。我们在小鼠身上的工作导致了心律失常与这一机制有关的假说 SUDEP在经络病连锁遗传性癫痫中的作用。我们证明了心肌细胞(CM)离子的改变 电流、钙离子处理和动作电位(AP)，以及心律失常 SCN1A、SCN8A和SCN1B连锁的发育性和癫痫性脑病(DIE)。我们向大家展示了 Dravet综合征(DS)患者的诱导多能干细胞(IPSC)来源的CMS具有以下底物 心律不齐。重要的是，任何动物或IPSC模型都不能完全复制人类DS的表型。因为 小鼠和人类的心脏AP非常不同，我们使用人类IPSC-CM模型来研究细胞 SCN1A单倍体功能不全的自主效应。然而，二维培养中的细胞不能复制 复杂的心脏组织、心血管改变或心脏神经支配。在这里，我们将开发和验证 除癫痫发作外，心律失常在SUDEP中的作用可以是一种大型动物模型 调查过了。兔子紧密复制人类心脏AP，并提供一个完整的有机体来翻译 临床环境。我们的目标是建立和验证SCN1A连锁DS的转基因兔模型。我们 利用CRISPR-Cas9基因编辑技术建立新西兰白兔Scn1a缺失模型。 而NZW Scn1a+/-兔未出现惊厥和心律失常。后来我们发现F1：NZW x 荷兰腰带Scn1a+/-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转基因的大型动物DIE模型，尽管这个模型必须 现在要进行严格的验证。如果得到验证，这项工作将比目前可用的DS有很大的进步 模特们。R61阶段的具体目标：1.表征癫痫发作、癫痫类型、癫痫发作频率和 持续时间，并测定DS兔SUDEP发生率。2.确定心律失常的类型、频率和 持续时间，心律失常是否独立于癫痫发作发生，以及心律失常是否相关 在DS兔体内应用SUDEP。R33阶段特定目标：使用ASO药物STK-001或 联合用药以减少癫痫发作和SUDEP。建立遗传性兔模型的研究 DS将更好地说明SUDEP的神经心脏机制对人类疾病的可译性。

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.