Project 2 - Investigation of human neuron models of channelopathy-associated epilepsy

项目 2 - 通道病相关癫痫的人类神经元模型的研究

• 批准号: 10477453
• 负责人: Evangelos Kiskinis
• 金额: $ 36.2万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10477453
• 关键词: Aftercare; Agonist; Antiepileptic Agents; Biological Models; Biological Sciences; Biophysics; CRISPR/Cas technology; Cell Line; Cells; Clinical; Collaborations; Collection; Coupled; Defect; Development; Disease; Drug Screening; Effectiveness; Electrophysiology (science); Epilepsy; Evaluation; Functional disorder; Gene Expression Profiling; Generations; Genes; Genetic; Glutamates; Goals; Human; In Vitro; Induced pluripotent stem cell derived neurons; Interneurons; Investigation; Ion Channel; Leadership; Link; Measures; Methods; Modeling; Molecular; Mutation; Neonatal; Neurologic; Neurological Models; Neuronal Differentiation; Neurons; Optics; Outcome; Pathogenicity; Patient Recruitments; Patient Selection; Patients; Persons; Pharmaceutical Preparations; Pharmacology; Pharmacology Study; Pharmacotherapy; Physiology; Positioning Attribute; Property; Refractory; Regimen; Research; Seizures; Severities; Syndrome; Technology; Variant; Work; channel blockers; clinical efficacy; early onset; epileptic encephalopathies; excitatory neuron; experimental study; genetic variant; genome editing; improved; in vivo; induced pluripotent stem cell; induced pluripotent stem cell technology; inhibitory neuron; innovation; mouse model; nervous system disorder; neurophysiology; optimal treatments; optogenetics; patch clamp; prevent; response; single-cell RNA sequencing; stem cell based approach; success; targeted treatment; tool; voltage; voltage clamp

In Project 2 we will determine the functional consequences of epilepsy-associated ion channel gene variants using human neurons differentiated from patient-specific induced pluripotent stem cells (iPSCs). We will initially focus on the SCN2A and KCNQ2 genes, which encode the voltage-gated Na+ (NaV1.2) and K+ (KV7.2) channels respectively. Mutations in SCN2A and KCNQ2 are responsible for monogenic early onset epileptic encephalopathy (EE) with overlapping clinical features and diverse severity. Collectively, variants in these two genes account for ~10% of all mutations identified in genetic epilepsy. The molecular pathogenic mechanisms responsible for the clinical manifestations of KCNQ2- and SCN2A-related epilepsies remain largely unknown. More importantly, no targeted therapeutic approach capable of diminishing seizure burden and improving developmental outcomes exists for these devastating neurological disorders. In Aim 1, we will use patient- specific cortical neurons to elucidate the functional consequences of epilepsy-associated KCNQ2 and SCN2A variants. We will specifically examine cortical excitatory and inhibitory neurons derived from existing patient- specific iPSC lines with pathogenic variants and corresponding isogenic control lines. We will use a combination of transcriptional profiling (single-cell RNA-sequencing) with electrophysiological approaches (whole cell patch clamp recording and high-throughput optogenetic recordings) to determine the impact of mutations on neuronal function and excitability. In Aim 2, we will assess the intrinsic excitability of patient neurons before and after treatment with NaV channel blockers and KV7 agonists that have clinical efficacy in the patients from whom the cells were derived. Our goal will be to rank the in vitro effectiveness of drugs in restoring normal neuron excitability for each genetic variant, and then to correlate the in vitro drug responses with the clinical responses to AEDs documented for these patients. This project entails a strategic collaboration between Dr. Kiskinis, whose lab focuses on using stem cell-based approaches to establish models of neurological disease, and Q-State Biosciences, Inc., which under the scientific leadership of Dr. McManus has been developing optogenetic technologies to enable high-throughput electrical recordings of human neurons and drug screening platforms for epilepsy syndromes. This project will work closely with the other Center teams, including Core A (Variant Prioritization and Curation Core), Project 1 (High-Throughput Functional Evaluation of Ion Channel Variants) and Project 3 (Development and Investigation of Murine Models of Channelopathy-associated Epilepsy). Core A is building tools to prioritize variants for experimental evaluation by the three Center projects. Correlation of findings from Project 2 with those of Projects 1 and 3 will help determine the reliability and accuracy of iPSC technology to predict in vivo physiology and pharmacology. Our findings will impact the field by demonstrating mechanistic effects of channelopathy-associated epilepsy variants, and by providing a systematic evaluation of human neuron platforms for precise drug selection.

在项目 2 中，我们将确定癫痫相关离子通道基因变异的功能后果 使用从患者特异性诱导多能干细胞（iPSC）分化而来的人类神经元。我们将首先 重点关注 SCN2A 和 KCNQ2 基因，它们编码电压门控 Na+ (NaV1.2) 和 K+ (KV7.2) 分别频道。 SCN2A 和 KCNQ2 突变导致单基因早发性癫痫 脑病（EE）具有重叠的临床特征和不同的严重程度。总的来说，这两个变体 基因约占遗传性癫痫中发现的所有突变的 10%。分子致病机制 导致 KCNQ2 和 SCN2A 相关癫痫临床表现的机制仍然很大程度上未知。 更重要的是，没有针对性的治疗方法能够减轻癫痫发作负担并改善癫痫发作 这些毁灭性的神经系统疾病存在发育结果。在目标 1 中，我们将使用病人- 特定皮质神经元阐明癫痫相关 KCNQ2 和 SCN2A 的功能后果 变种。我们将专门检查来自现有患者的皮质兴奋性和抑制性神经元 具有致病性变异的特定 iPSC 系和相应的等基因对照系。我们将使用一个 转录分析（单细胞 RNA 测序）与电生理学方法的结合 （全细胞膜片钳记录和高通量光遗传学记录）以确定 神经元功能和兴奋性的突变。在目标 2 中，我们将评估患者的内在兴奋性 使用具有临床疗效的 NaV 通道阻滞剂和 KV7 激动剂治疗前后的神经元 细胞来源的患者。我们的目标是对药物的体外有效性进行排名 恢复每个遗传变异的正常神经元兴奋性，然后关联体外药物反应 记录了这些患者对 AED 的临床反应。该项目需要战略合作 Kiskinis 博士的实验室专注于使用基于干细胞的方法来建立模型 神经系统疾病和 Q-State Biosciences, Inc. 在 McManus 博士的科学领导下， 一直在开发光遗传学技术以实现人类神经元的高通量电记录 以及癫痫综合征药物筛选平台。该项目将与其他中心密切合作 团队，包括核心 A（变体优先级划分和管理核心）、项目 1（高通量功能 离子通道变异体的评估）和项目 3（小鼠模型的开发和研究） 通道病相关性癫痫）。核心 A 正在构建工具来确定实验评估变体的优先级 由三个中心项目组成。项目 2 的研究结果与项目 1 和 3 的研究结果的关联将有助于 确定 iPSC 技术预测体内生理学和药理学的可靠性和准确性。我们的 研究结果将通过证明通道病相关癫痫的机制效应来影响该领域 变体，并通过对人类神经元平台进行系统评估以进行精确的药物选择。