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，

基本信息

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
项目类别：
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

项目摘要

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）是癫痫死亡的主要原因。许多SUDEP案例具有与心律失常相关的遗传变异，特别是长 QT 综合征 (LQTS)。我们的实验室和其他研究表明，与基因型阴性家族相比，LQT2 患者患癫痫的风险高出 2 倍以上成员。 LQT2 是由编码 K+ 通道蛋白 (Kv11.1) 的 KCNH2 变体引起的，该蛋白可产生 K+ 电流（IKr）。它对于心脏复极至关重要，稳定神经元静息膜电位，并且抑制重复射击。患有 KCNH2 介导的癫痫和 LQT2 的患者需要安全有效的抗癫痫药物（ASM）。一个关键障碍是缺乏临床相关的 LQT2 动物模型癫痫。我们将开发第一个 Kcnh2 介导的癫痫转化模型、SUDEP 和 LQT2。作为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 孔结构域 (Kcnh2(+/mut)) 中具有杂合移码突变的兔子。该型号具有优越构建效度。由于啮齿类动物与人类的心电功能差异以及非生理性的 Kcnh2 表达模式，目前的模型不适合 Kcnh2 介导的神经元的转化研究心脏病理学。许多啮齿动物模型无法重现临床癫痫的自然进展，包括非癫痫相关的神经元损伤，需要与生理无关的触发因素，并且具有低 ASM 筛查的预测有效性。兔子是药物测试和癫痫研究的模型，心律失常和猝死。与啮齿类动物相反，人类和兔子的神经元细胞类型相似。 R61：外部面部验证：使用可量化和临床相关的终点，我们将测试 Kcnh2(+/mut) 是否有效兔子重现了 LQT2 癫痫患者的神经心脏病理。初步数据表明我们生成了 Kcnh2 介导的癫痫、SUDEP 和 LQT2 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发作介导的猝死。 R33：尽管 LQT2 患者有很高的风险癫痫和 SUDEP，目前还没有针对 LQT2 癫痫患者的既定 ASM。我们证明了与关闭 ASM 相比，LQT2 患者发生心律失常的风险增加，尤其是 Na+ 通道阻断 ASM（例如苯妥英）。外部人脸验证：（1）我们将证明肌细胞和皮质神经元来自 Kcnh2(+/mut) 的兔子 IKr 降低并且过度兴奋。 (2) 与LQT2患者类似，细胞和内体内试验将测试苯妥英是否对 Kcnh2(+/mut) 兔子有不良心脏影响。初步数据表明与 WT 兔子相比，苯妥英阻断 IKr 并导致 Kcnh2(+/mut) 的 QTc 更大程度增加，这表明可预测我们模型的有效性。影响：我们将开发 Kcnh2 介导的癫痫、SUDEP 和 LQT2 模型，再现人类 LQT2 神经心脏病理。它将提供一个平台来识别有效和安全的 ASM 可减少 LQT2 中的癫痫发作和 SUDEP，并补充我们使用 LQTS 患者登记的研究。