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(SUDEP)是癫痫患者的主要死亡原因。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人 结果显示，LQT2患者患癫痫的风险是基因阴性家系的2倍 会员。LQT2是由编码K+通道蛋白(Kv11.1)的KCNH2变体引起的，Kv11.1产生K+ 当前(Ikr)。它对心脏复极、稳定神经元静息膜电位和 抑制重复发射。KCNH2介导的癫痫和LQT2患者需要安全有效的抗癫痫药物 癫痫药物(ASM)。一个关键的障碍是缺乏临床上相关的LQT2动物模型 癫痫。我们将开发第一个KCNH2介导的癫痫的翻译模型SUDEP和LQT2。AS LQT2 具有KCNH2孔结构域变异的患者癫痫发作的风险最高，我们使用CRISPR-Cas9生成 KCNH2多孔区杂合性移码突变(KCNH2(+/mut))兔。这种款式有很好的性能 构建效度。由于啮齿动物和人类心脏电功能的差异，以及非生理性的KCNH2 表达模式，目前的模型不适合KCNH2介导的神经细胞的翻译研究。 心脏病理学。许多啮齿动物模型无法再现临床癫痫的自然发展，包括 与癫痫无关的神经元损伤，需要与生理无关的触发因素，而且 ASM筛查的预测效度。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。与啮齿动物不同，人类和兔子的神经细胞类型相似。 R61：外部脸验证：使用可量化和临床相关的端点，我们将测试KCNH2(+/mut) 兔子复制了LQT2癫痫患者的神经心脏病理。初步数据显示 我们建立了KCNH2介导的癫痫、SUDEP和LQT2的临床相关兔模型。的确有 Kv11.1在脑和心脏的表达减少，QT间期延长，自发性非心源性癫痫 癫痫发作和自发性癫痫导致的猝死。R33：尽管LQT2患者有很高的风险 对于癫痫和SUDEP，目前还没有针对LQT2癫痫患者的ASM。我们证明了这一点 LQT2患者在开启ASM时与关闭ASM时相比，发生心律失常的风险增加，尤其是Na+通道阻断 ASM(如苯妥英)。外部面孔验证：(1)我们将证明肌细胞和皮质神经元 KCNH_2(+/MUT)使兔的IKR降低，并具有高度兴奋性。(2)与LQT2患者相似，细胞和在 体内试验将测试苯妥英对KCNH2(+/mut)兔是否有不良心脏作用。初步数据显示 苯妥英阻断IKR并导致KCNH2(+/MUT)兔较WT兔更大的QTc值增加，这表明 验证了我们模型的有效性。影响：我们将开发一种KCNH2介导的癫痫模型，SUDEP和LQT2 复制人类LQT2神经心脏病理。它将提供一个平台，以确定有效和安全 ASMS可以减少LQT2中的癫痫发作和SUDEP，并补充了我们使用LQTS患者登记的研究。