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

在项目2中，我们将确定癫痫相关离子通道基因变体的功能后果 使用从患者特异性诱导多能干细胞（iPSC）分化的人类神经元。我们将初步 集中于SCN 2A和KCNQ 2基因，其编码电压门控Na+（NaV1.2）和K+（KV7.2）， 渠道分别。SCN 2A和KCNQ 2突变与单基因早发性癫痫相关 脑病（EE）具有重叠的临床特征和不同的严重程度。总的来说，这两个变量 基因占遗传性癫痫中所有突变的约10%。致病的分子机制 KCNQ 2和SCN 2A相关癫痫的临床表现的主要原因仍不清楚。 更重要的是，没有靶向治疗方法能够减少癫痫发作负担并改善 这些毁灭性的神经系统疾病的发展结果存在。在目标1中，我们将使用患者- 特异性皮质神经元，以阐明癫痫相关的KCNQ 2和SCN 2A的功能后果 变体。我们将专门研究来自现有患者的皮层兴奋性和抑制性神经元- 具有致病性变体的特异性iPSC系和相应的同基因对照系。我们将使用一个 转录谱分析（单细胞RNA测序）与电生理学方法的组合 （全细胞膜片钳记录和高通量光遗传学记录）来确定 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， 在用NaV通道阻断剂和KV 7激动剂治疗之前和之后的神经元中， 细胞来源的患者。我们的目标将是对药物的体外有效性进行排名， 恢复每种遗传变体的正常神经元兴奋性，然后将体外药物反应与 这些患者对AED的临床反应记录在案。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建立模型， 神经系统疾病和Q-State Biosciences，Inc.，在麦克马纳斯博士的科学领导下， 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 以及癫痫综合征的药物筛选平台。该项目将与其他中心密切合作 团队，包括核心A（变体优先级和策展核心）、项目1（高吞吐量功能 离子通道变异体的评价）和项目3（ 癫痫病相关的癫痫）。核心A是构建工具来优先考虑实验评估的变体 三个中心项目。将项目2的调查结果与项目1和项目3的调查结果联系起来， 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 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，并通过提供人类神经元平台的系统评估，以进行精确的药物选择。

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.