Development of a Genetic Rabbit Model of Kcnh2-Mediated Epilepsy, SUDEP, & Long QT Syndrome Type 2

Kcnh2 介导的癫痫遗传兔模型的开发，SUDEP，

• 批准号: 10728653
• 负责人: David Scott Auerbach
• 金额: $ 41.79万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10728653
• 关键词: Action Potentials; Animal Model; Anticonvulsants; Arrhythmia; Biological Assay; Brain; CRISPR/Cas technology; Cardiac; Cause of Death; Cessation of life; Clinical; Complement; Data; Development; Disease; Dose; Drug Modelings; Electrocardiogram; Electroencephalography; Epilepsy; Exhibits; Face; Family member; Frameshift Mutation; Future; Genes; Genetic; Genotype; Goals; Heart; Heterozygote; High Prevalence; Human; Ions; Link; Long QT Syndrome; Measures; Mediating; Membrane Potentials; Modeling; Muscle Cells; Neurons; Oryctolagus cuniculus; Outcome Measure; Pathology; Patients; Pattern; Pentylenetetrazole; Persons; Pharmaceutical Preparations; Phenotype; Phenytoin; Potassium Channel; Predisposition; Prevalence; Proteins; Recording of previous events; Research; Rest; Risk; Rodent; Rodent Model; Safety; Seizures; Sudden Death; System; Testing; Type 2 Long QT syndrome; Validation; Variant; cell type; clinically relevant; drug testing; genetic variant; high risk; in vivo; mutant; patient registry; prevent; screening; sudden unexpected death in epilepsy; translational model; translational study

Sudden Unexpected Death in Epilepsy (SUDEP) is the leading cause of death in epilepsy. Many SUDEP cases had genetic variants linked to cardiac arrhythmias, particularly Long QT Syndrome (LQTS). Our lab and others showed that LQT2 patients are at a >2-fold higher risk of epilepsy, compared to genotype negative family members. LQT2 is caused by KCNH2 variants that encode a K+ channel protein (Kv11.1), which produces K+ current (IKr). It is critical for cardiac repolarization, stabilizes the neuronal resting membrane potential, and suppresses repetitive firing. People with KCNH2-mediated epilepsy and LQT2 need safe and effective anti- seizure medications (ASMs). A critical barrier is the lack of a clinically relevant animal model of LQT2 with epilepsy. We will develop the first translational model of Kcnh2-mediated epilepsy, SUDEP, and LQT2. As LQT2 patients with KCNH2 pore-domain variants are at the highest risk of seizures, we used CRISPR-Cas9 to generate rabbits with a heterozygous frameshift mutation in the Kcnh2 pore-domain (Kcnh2(+/mut)). This model has superior construct validity. Due to rodent vs. human differences in cardiac electrical function, and unphysiological Kcnh2 expression patterns, present models are not appropriate for translational studies of Kcnh2-mediated neuro- cardiac pathologies. Many rodent models fail to reproduce the natural progression of clinical epilepsy, include non-seizure related neuronal damage, require triggers that are not physiologically relevant, and have low predictive validity for ASM screening. Rabbits are established models for drug testing, and studying seizures, arrhythmias, and sudden death. In contrast to rodents, neuronal cell-types are similar in humans and rabbits. R61: External Face Validation: Using quantifiable and clinically relevant endpoints, we will test if Kcnh2(+/mut) rabbits reproduce the neuro-cardiac pathologies seen in LQT2 patients with epilepsy. Preliminary data indicates that we generated a clinically-relevant rabbit model of Kcnh2-mediated epilepsy, SUDEP, and LQT2. There is reduced Kv11.1 expression in the brain and heart, QTc prolongation, spontaneous noncardiogenic epileptic seizures, and spontaneous seizure-mediated sudden death. R33: Despite LQT2 patients being at a high risk of epilepsy and SUDEP, there are no established ASMs for LQT2 patients with epilepsy. We demonstrated that LQT2 patients are at an increased risk of arrhythmias when on vs. off ASMs, particularly Na+ channel blocking ASMs (e.g., phenytoin). External Face Validation: (1) We will demonstrate that myocytes and cortical neurons from Kcnh2(+/mut) rabbits have reduced IKr and are hyperexcitable. (2) Similar to LQT2 patients, cellular and in vivo assays will test if phenytoin has adverse cardiac effects in Kcnh2(+/mut) rabbits. Preliminary data indicates phenytoin blocks IKr and causes a larger increase in QTc in Kcnh2(+/mut) vs. WT rabbits, which suggests predictive validity of our model. Impact: We will develop a model of Kcnh2-mediated epilepsy, SUDEP, and LQT2 that reproduces human LQT2 neuro-cardiac pathologies. It will provide a platform for identifying effective and safe ASMs to reduce seizures and SUDEP in LQT2, and complements our research using the LQTS patient registry.

癫痫猝死（Sudden Unexpected Death in Epilepsy，SUDEP）是癫痫患者死亡的主要原因。许多SUDEP案例 有与心律失常有关的遗传变异，特别是长QT综合征（LQTS）。我们的实验室和其他 结果显示，与基因型阴性家族相比，LQT 2患者患癫痫的风险高出2倍以上。 成员LQT 2是由编码K+通道蛋白（Kv11.1）的KCNH 2变体引起的，该蛋白产生K+ 电流（IKr）。它对心脏复极化至关重要，稳定神经元静息膜电位， 抑制重复性放电KCNH 2介导的癫痫和LQT 2患者需要安全有效的抗癫痫药物。 癫痫药物（ASMs）。一个关键的障碍是缺乏临床相关的LQT 2动物模型， 癫痫我们将开发Kcnh 2介导的癫痫，SUDEP和LQT 2的第一个翻译模型。作为LQT 2 具有KCNH 2孔结构域变体的患者癫痫发作的风险最高，我们使用CRISPR-Cas9来生成 在Kcnh 2孔结构域中具有杂合移码突变（Kcnh 2（+/mut））的兔。这种型号具有上级 结构效度由于啮齿动物与人类在心脏电功能方面的差异，以及非生理性Kcnh 2 表达模式，目前的模型不适合Kcnh 2介导的神经元的翻译研究。 心脏病。许多啮齿动物模型不能再现临床癫痫的自然进展，包括 非癫痫发作相关的神经元损伤，需要生理上不相关的触发，并且具有低的 ASM筛查的预测有效性。兔子是药物测试和研究癫痫发作的既定模型， 心律失常和猝死与啮齿动物相比，人类和兔子的神经元细胞类型相似。 R61：外部面验证：使用可量化和临床相关终点，我们将检验Kcnh 2（+/mut） 兔子再现了在患有癫痫的LQT 2患者中所见的神经心脏病理。初步数据表明 我们产生了Kcnh 2介导的癫痫、SUDEP和LQT 2的临床相关兔模型。有 大脑和心脏中Kv11.1表达减少、QTc延长、自发性非心源性癫痫 癫痫发作和自发性癫痫介导的猝死。R33：尽管LQT 2患者存在高风险， 癫痫和SUDEP，对于LQT 2癫痫患者没有确定的ASM。我们证明了 与关闭ASM相比，LQT 2患者的心律失常风险增加，特别是Na+通道阻滞 ASM（例如，苯妥英）。外部面验证：（1）我们将证明肌细胞和皮质神经元 来自Kcnh 2（+/mut）的兔具有降低的IKr并且是过度兴奋的。(2)与LQT 2患者相似，细胞和 体内试验将测试苯妥英在Kcnh 2（+/mut）兔中是否具有不利的心脏作用。初步数据表明 与WT兔相比，苯妥英钠阻断Kcnh 2（+/mut）的IKr并导致QTc增加更大，这表明预测性 我们模型的有效性。影响：我们将开发一种Kcnh 2介导的癫痫、SUDEP和LQT 2模型， 复制人类LQT 2神经心脏病理。它将提供一个平台， ASM减少LQT 2的癫痫发作和SUDEP，并补充我们使用LQTS患者登记的研究。