Novel molecular therapies for CPVT

这份提案描述了一项为期五年的培训和职业发展计划，该计划将为其原则做准备 研究员瓦西利奥斯·贝泽里德斯博士将成为通道病领域的独立研究员和 遗传性心律失常。该计划将建立在贝泽里德斯博士现有的细胞和 通过提供诱导多能干细胞(IPSCs)、基因组方面的额外专业知识，提供临床电生理学 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儿科和心脏病学教授。浦博士是一位 国际公认的心脏发育和IPSC疾病建模专家。随函附上的建议书 概述了具有结构化指导的全面培训计划，包括一个咨询委员会，该委员会 心血管研究和生物工程方面的专业知识，正规课程，以及将 在疾病建模和新疗法开发方面提供严格的培训。这项建议的重点是 儿茶酚胺能多形性室性心动过速(CPVT)治疗新策略探讨 一种高度恶性的遗传性心律失常，其特征是在 压力或锻炼的时间。目前的CPVT治疗是不够的，与治疗相关的毒性和频繁的 治疗失败。大多数CPVT病例是由兰诺定受体2型(RYR2)突变引起的，这种突变 编码主要的心肌细胞内钙释放通道。利用IPSC-CMS，我们开发了一种新颖的 CPVT的工程化人体心肌模型(“光电芯片”)，以一次 组织水平。我们的初步数据表明，抑制钙/钙调蛋白激酶II(CaMKII)可以阻断 CPVT IPSC-CMS的前心律失常表型。在目标1中，使用药理学、基因组编辑和工程 组织，贝泽里德斯博士将通过评估致病因子来确定CaMKII抑制是否广泛适用 RYR2基因内四个典型致病区域的CPVT基因型，并确定 抑制心律失常所必需的抑制程度。在目标2中，贝泽里德斯博士将进一步开发CaMKII 抑制作为CPVT的临床适用的治疗策略。为了改进这一策略，贝泽里德斯博士将测试 靶向CaMKII在心脏传导系统细胞中的作用 结果。积极的结果将作为进一步研究的基础，作为迈向首例人体试验的一步。在……里面 目标3，贝兹泽里德博士将使用基因组编辑、膜片钳和第二代光芯片分析来 剖析晚期钠电流阻断在CPVT治疗中的作用。虽然有争议，但晚期钠 目前的阻断作用可能是氟卡胺治疗CPVT有效的机制基础。更好地理解 机制可能导致新的治疗选择，更有效和更低的毒性。加在一起，这些 研究将促进对CPVT中心律失常发生的理解，为新的治疗方法开辟新的途径， 并为贝泽里德斯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.