Novel molecular therapies for CPVT

该提案描述了一个为期五年的培训和职业发展计划， 研究员Vassilios Bezzerides博士将成为通道病领域的独立研究员， 遗传性心律失常该计划将建立在Bezzerides博士现有的细胞和 通过提供诱导多能干细胞（iPSC）、基因组 编辑和翻译研究技术。这个项目的主要导师将是威廉·普博士， 波士顿儿童医院和哈佛医学院的儿科和心脏病学教授。蒲博士是一位 国际公认的心脏发育和iPSC疾病建模专家。所附提案 概述了一个全面的培训计划，包括一个咨询委员会， 心血管研究和生物工程方面的专业知识，正式的课程，以及将 提供疾病建模和新疗法开发方面的严格培训。该提案的重点是 研究儿茶酚胺能多形性室性心动过速（CPVT）新治疗策略， 一种高度恶性的遗传性心律失常疾病，特征是在心脏病发作期间出现危及生命的室性心律失常。 压力或锻炼的时间。目前的CPVT治疗是不充分的，与治疗相关的毒性和频繁的 治疗失败。大多数CPVT病例是由兰尼碱受体2型（RYR 2）突变引起的， 编码主要的心肌细胞内Ca+2释放通道。使用iPSC-CM，我们开发了一种新的 CPVT的工程化人类心肌模型（“光芯片”），其在200 - 300 μ g/ml的浓度下再现了这种心律失常的关键特征。 组织水平。我们的初步数据表明，抑制Ca 2 +/钙调蛋白激酶II（CaMKII）可以阻止钙调蛋白的表达。 CPVT iPSC-CM的促排卵表型。在目标1中，使用药理学，基因组编辑和工程 组织，Bezzerides博士将通过评估致病性来确定CaMKII抑制是否广泛适用 CPVT基因型来自RYR 2基因内的四个典型致病区域中的每一个，并确定了RYR 2基因内的CPVT基因型。 心律失常抑制所需的抑制程度。在目标2中，Bezzerides博士将进一步开发CaMKII 抑制作为CPVT的临床适用治疗策略。为了完善这一策略， 靶向CaMKII在心脏传导系统细胞中的有效性， 结果。一个积极的结果将作为进一步研究的基础，作为迈向首次人体试验的一步。在 目标3，Bezzerides博士将使用基因组编辑，膜片钳和第二代光芯片检测， 探讨迟发性钠电流阻滞在CPVT治疗中的作用。虽然有争议，晚钠 电流阻断可能是氟卡尼治疗CPVT的机制基础。更好地理解 这一机制可能导致新的治疗选择，具有更高的疗效和更低的毒性。所有这些 这些研究将促进对CPVT中血栓形成的理解，为新的治疗开辟新的途径， 并为Bezzerides博士领导的独立研究项目提供基础。

This proposal describes a five-year training and career development program that will prepare its principle investigator, Dr. Vassilios Bezzerides, to be an independent investigator in the field of channelopathies and inherited arrhythmia disorders. This program will build on Dr. Bezzerides’ existing background in cellular and clinical electrophysiology by providing additional expertise in induced pluripotent stem cells (iPSCs), genome editing, and translational research techniques. The principal mentor for this program will be Dr. William Pu, Professor of Pediatrics and Cardiology at Boston Children’s Hospital and Harvard Medical School. Dr. Pu is an internationally recognized expert in cardiac development and iPSC disease modeling. The enclosed proposal outlines a comprehensive training program with structured mentorship including an advisory committee with expertise in cardiovascular research and bioengineering, formal coursework, and a research plan that will provide rigorous training in disease modeling and novel therapy development. This proposal is focused on the investigation of novel therapeutic strategies for catecholaminergic polymorphic ventricular tachycardia (CPVT), a highly malignant inherited arrhythmia disorder characterized by life-threatening ventricular arrhythmias during times of stress or exercise. Current CPVT therapy is inadequate, with both therapy related toxicity and frequent treatment failures. Most CPVT cases are caused by mutations in ryanodine receptor type 2 (RYR2), which encodes the major cardiomyocyte intracellular Ca+2 release channel. Using iPSC-CMs, we developed a novel engineered human myocardial model (“opto-chip”) of CPVT that reproduces key features of this arrhythmia at a tissue level. Our preliminary data demonstrates that inhibition of Ca2+/calmodulin kinase II (CaMKII) blocks the pro-arrhythmic phenotype of CPVT iPSC-CMs. In Aim 1, using pharmacology, genome editing, and engineered tissues, Dr. Bezzerides will determine if CaMKII inhibition is a broadly applicable by evaluating pathogenic CPVT genotypes from each of the four canonical pathogenic regions within the RYR2 gene and determine the degree of inhibition necessary for arrhythmia suppression. In Aim 2, Dr. Bezzerides will further develop CaMKII inhibition as a clinically applicable therapeutic strategy for CPVT. To refine this strategy Dr. Bezzerides will test the effectiveness of target CaMKII in the cells of the cardiac conduction system using clinically relevant outcomes. A positive result would serve as the basis for further study as a step towards a first-in-human trial. In Aim 3, Dr. Bezzerides will use genome editing, patch clamp, and a second generation opto-chip assay to dissect the role of late sodium current blockade in the treatment of CPVT. Although controversial, late sodium current blockade may be the mechanistic basis for flecainide’s efficacy in CPVT. Better understanding of the mechanism may lead to new therapeutic options with greater efficacy and lower toxicity. Together, these studies will advance the understanding of arrhythmogenesis in CPVT, open new avenues for novel therapies, and provide a foundation for an independent research program led by Dr. Bezzerides.