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

 说明(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，特别是心律失常。1将一种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。目前对促心律失常药物作用的体外筛选试验主要集中于测量HERG钾通道的抑制程度，HERG钾通道与潜在的致命性尖端心律失常(TDP)有关。然而，HERG试验缺乏高度的敏感性和特异性：并不是所有的QT延长都是由于阻断HERG本身，也不是所有的HERG阻滞剂都会导致QT延长或诱导TDP。为了解决这些局限性，监管机构创建了全面的体外前心律失常分析(CIPA)倡议2，该倡议建议使用来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描述 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、全光学电生理学平台，用于在扫描电子显微镜细胞来源的心肌细胞中进行心脏毒性筛选。在我们的第一阶段奖中，我们展示了Optopatch平台可以用于检测急性和慢性药物治疗后人源性CMS的电生理特征的变化，尽管每次记录的吞吐量只有一个井。为了使这些测量高度并行化，我们建议建造一台96孔板Optopatch仪器，用于同时记录24口井在起搏条件下的电压和钙波形。这种几何构型将使我们的分析的吞吐量提高近两个数量级。Optopatch结构将进行优化，以允许在每个细胞中整合致动器和报告蛋白。我们将利用这一平台在不同来源的人心肌细胞中筛选出50种风险分数已知的化合物，并使用这些数据来开发心律失常的预测算法。

 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.