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异的功能后果 使用从患者特异性诱导的多能干细胞(IPSCs)分化的人类神经元。我们最初会 重点关注SCN2A和KCNQ2基因，它们编码电压门控Na+(NaV1.2)和K+(KV7.2) 通道分别为。SCN2A和KCNQ2基因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性。总而言之，这两个版本的变体 基因约占遗传性癫痫所有突变的10%。分子致病机制 KCNQ2和SCN2A相关癫痫的临床表现的主要原因仍不清楚。 更重要的是，没有针对性的治疗方法能够减轻癫痫负担并改善 这些破坏性的神经疾病存在发育结果。在目标1中，我们将使用患者- 特定皮质神经元阐明癫痫相关的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- 含有致病变异体的特定IPSC品系和相应的等基因对照品系。我们将使用 转录图谱(单细胞RNA测序)与电生理方法的结合 (全细胞膜片钳记录和高通量光遗传记录)以确定 突变对神经元功能和兴奋性的影响。在目标2中，我们将评估患者的内在兴奋性 NAV通道阻滞剂和KV7激动剂治疗前后神经元的临床疗效 这些细胞是从患者身上衍生出来的。我们的目标将是对药物的体外有效性进行排名 恢复每个基因变体的正常神经元兴奋性，然后关联体外药物反应 并记录了这些患者对AEDs的临床反应。该项目需要战略协作 基斯基尼斯博士的实验室专注于使用基于干细胞的方法来建立 神经疾病和Q-State生物科学公司，该公司在麦克马纳斯博士的科学领导下 一直在开发光遗传技术，以实现高通量的人类神经元电记录 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(不同的优先顺序和管理核心)、项目1(高吞吐量功能 离子通道变种的评估)和项目3(小鼠模型的开发和研究 经络病相关的癫痫)。核心A正在构建工具，以便为实验评估确定变体的优先顺序 由三个中心项目。将项目2的结果与项目1和项目3的结果相关联将有所帮助 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 这些发现将通过证明经络病相关癫痫的机制效应来影响这一领域 通过为精确的药物选择提供对人类神经元平台的系统评估，以及通过提供对人类神经元平台的系统评估来实现精确的药物选择。

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.