Utilizing Patient-Specific hiPSC-CMs to Investigate Pediatric Calmodulinopathy

项目总结/摘要 小儿钙调素病是一种危及生命的心律失常综合征， 三种钙调素（CaM）基因中任何一种的杂合突变。大多数（68%）患有致病性CaM的人 变体将经历重大的心脏事件，如心脏骤停或心脏猝死， 年龄五岁。钙调素病导致异质性的临床特征，具有两种主要的临床表现， 表型为长QT综合征（CALM-LQTS）和儿茶酚胺能多形性室性心动过速 （CALM-CPVT）。尽管目前的治疗，56%的钙调蛋白病患者发生突破性心脏 事件，强调需要改进的治疗方法。目前有效治疗的障碍是我们的 对钙调素基因变异如何影响病理表型的认识不完全。人类 诱导多能干细胞（hiPSC）可以分化成心肌细胞（hiPSC-CM）， 保持患者的遗传背景。在这种疾病的背景下应用hiPSC-CM使我们能够 以更好地理解细胞水平上的患者特异性异质性。在这项研究中，我们将建立一个冷静的- 使用hiPSC的LQTS hiPSC-CM模型，所述hiPSC产生自具有致病性CaM变体的五个人，所述人存在 关于CALM-LQTS我们假设，我们可以使用患者特异性hiPSC来模拟CALM-LQTS表型。 CM，确定临床上合适的药物，纠正它，并确定驱动这一点的潜在机制 表型。我们将首先使用盲法电生理学确定细胞CALM-LQTS表型， 收缩性和钙成像研究，然后筛选一组FDA批准的药物，以确定候选药物 来缓解这种表型。然后，我们将进行实验，以确定驱动 细胞CALM-LQTS表型。我们将首先确定钙循环失调的贡献， 钙调蛋白病的主要机制，在我们的CALM-LQTS hiPSC-CM模型中。然后我们将利用这个模型 研究CALM野生型和变体等位基因表达水平，以确定等位基因失衡是否有助于 细胞CALM-LQTS表型。我们还将评估每种变体对细胞的致病性。 通过使用基因组编辑的表型。对这些编辑过的细胞系进行表型分析将使我们能够确定这些变异是否 是细胞CALM-LQTS表型所必需和充分的。成功实现这些目标将 提供患者特异性方法，以确定CALM-LQTS的必要临床治疗，并阐明 这种罕见而严重的疾病的驱动因素。通过赞助商的支持和既定的培训 计划，博士前奖学金申请人将发展在该领域的专业知识和必要的技能， 实验设计，分析，沟通和技术技术，以完成这项研究战略， 过渡到他们的转化医学研究生涯的下一阶段。

PROJECT SUMMARY/ABSTRACT Pediatric calmodulinopathy is a life-threatening cardiac arrhythmia syndrome resulting from a single heterozygous mutation in any of three calmodulin (CaM) genes. Most (68%) persons with a pathogenic CaM variant will experience a major arrhythmic event such as aborted cardiac arrest or sudden cardiac death by the age of five years. Calmodulinopathies result in heterogenous clinical features with the two predominant clinical phenotypes being long QT syndrome (CALM-LQTS) and catecholaminergic polymorphic ventricular tachycardia (CALM-CPVT). Despite current treatments, 56% of calmodulinopathy patients experience breakthrough cardiac events, underscoring the need for improved therapeutics. A current barrier to effective treatment is our incomplete understanding of how calmodulin genetic variants contribute to pathologic phenotypes. Human induced pluripotent stem cells (hiPSCs) can be differentiated into cardiomyocytes (hiPSC-CMs) while maintaining the patient’s genetic background. Application of hiPSC-CMs in the context of this disease allows us to better understand patient-specific heterogeneity at the cellular level. In this study, we will establish a CALM- LQTS hiPSC-CM model using hiPSCs generated from five persons with a pathogenic CaM variant who present with CALM-LQTS. We hypothesize that we can model a CALM-LQTS phenotype using patient-specific hiPSC- CMs, identify clinically appropriate drugs that correct it, and determine underlying mechanisms that drive this phenotype. We will first determine the cellular CALM-LQTS phenotype using blinded electrophysiological, contractility, and calcium imaging studies and then screen a panel of FDA-approved drugs to identify candidates that alleviate this phenotype. We will then perform experiments to identify underlying mechanisms that drive the cellular CALM-LQTS phenotype. We will first determine the contribution of calcium cycling dysregulation, the primary mechanism of calmodulinopathy, in our CALM-LQTS hiPSC-CM model. We will then leverage this model to investigate CALM wildtype and variant allele expression levels to determine if allelic imbalance is contributing the cellular CALM-LQTS phenotype. We will also assess the pathogenicity of each variant on the cellular phenotype by using genome editing. Phenotyping of these edited lines will allow us to determine if these variants are necessary and sufficient for the cellular CALM-LQTS phenotype. Successful completion of these aims will provide a patient-specific methodology to identify necessary clinical treatments for CALM-LQTS and elucidate the drivers of this rare and severe disease. Through the support of their sponsor and their established training plan, the predoctoral fellowship applicant will develop expert knowledge in the field and the necessary skills in experimental design, analysis, communication, and technical techniques to complete this research strategy and transition to the next stage of their translational medicine research career.