High-throughput, all-optical assay in human cardiomyoctes for clinically relevant prediction of drug induced cardiotoxity.

 描述（由申请人提供）：毒性约占药物消耗的20%，其中近三分之一归因于心血管问题，特别是疟疾。1将新药推向市场的成本可能超过12亿美元，需要10年以上的研究。因此，在发育早期识别心脏毒性是至关重要的。目前，促心律失常药物作用的体外筛选试验侧重于测量hERG钾通道的抑制，这与潜在致死性尖端扭转型心律失常（TdP）有关。2然而，hERG试验缺乏高灵敏度和特异性：并非所有QT间期延长都是由于hERG单独阻滞所致，也并非所有hERG阻滞剂都会导致QT间期延长或诱导TdP。为了解决这些局限性，监管机构已经创建了综合性体外致心律失常试验（CiPA）倡议2，该倡议建议使用源自人类干细胞的心肌细胞作为致心律失常药物潜力的体外模型。建立这种毒性筛选模型需要详细表征 心肌细胞动作电位（AP）和钙瞬变（CT），AP/CT对药物的反应，以及与人类临床结果的相关性。然而，执行这些测量所需的高通量工具一直缺乏。在这里，我们建议开发一个高通量，全光学电生理平台，用于SEM细胞来源的心肌细胞的心脏毒性筛选。通过我们的第一阶段奖项，我们证明了Optopatch平台可用于检测急性和慢性药物治疗后人源性CM的电生理特征变化，尽管每次记录的通量为单个孔。为了高度并行化这些测量，我们建议构建一个96孔板Optopatch仪器，用于在起搏条件下从24个威尔斯孔同时记录电压和钙波形。这种几何形状将使我们的测定的通量提高近两个数量级。将优化光配位构建体以允许在每个细胞中掺入致动蛋白和报告蛋白。我们将利用这个平台筛选50种化合物，这些化合物在不同来源的人类心肌细胞中具有已知的尖端扭转型室性心动过速风险评分，并使用这些数据开发一种致心律失常性的预测算法。

 DESCRIPTION (provided by applicant): Toxicity accounts for approximately 20% of drug attrition, of which nearly one third is attributed to cardiovascular issues, in particular arrhythmias.1 The cost of bringing a new drug to market can exceed $1.2 billion and require more than 10 years of research. Thus, it is critical to identify cardiotoxicity early in developmen. Current in vitro screening assays for pro-­‐arrhythmic drug effects focus on measuring inhibition of the hERG potassium channel, which has been linked to potentially lethal Torsades de Pointes arrhythmias (TdP).2 The hERG assay, however, lacks high sensitivity and specificity: not all QT prolongation is due to block of hERG alone and not all hERG blockers result in QT prolongation or induce TdP. To address these limitations, regulatory bodies have created the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative2, which proposes use of cardiomyocytes derived from human stem cells as an in vitro model for arrhythmogenic drug potential. Establishment of this model for toxicity screening requires detailed characterization of the cardiomyocyte action potential (AP) and calcium transient (CT), the response of APs/CTs to drugs, and the correlation to clinical outcomes in humans. High-­‐throughput tools needed for performing these measurements, however, have been lacking. Here we propose to develop a high-­‐throughput, all-­‐optical electrophysiology platform for cardiotoxicity screening in sem cell-­‐derived cardiomyocytes. With our Phase I award, we demonstrated that the Optopatch platform could be used to detect changes in the electrophysiological characteristics of human derived CMs following both acute and chronic drug treatment, albeit with the throughput of a single well per recording. To highly parallelize these measurements, we propose building a 96-­‐well plate Optopatch instrument for simultaneous recording of voltage and calcium waveforms under paced conditions from 24 wells. This geometry will provide nearly two orders of magnitude improvement in throughput of our assay. Optopatch constructs will be optimized to allow for incorporation of the actuator and reporter proteins in each cell. We will utilize this platform to screen 50 compounds with known risk scores for Torsades de Pointes in different sources of human cardiomyocytes and use this data to develop a predictive algorithm of arrhythmogenicity.