Development of strategies to enhance titin (TTN) expression and treat dilated cardiomyopathy caused by TTN haploinsufficiency

项目摘要/摘要 基本肌节蛋白titin(TTNtv)的杂合截断变体是最常见的遗传 扩张型心肌病(DCM)的原因，一侧或双侧脑室的扩张和收缩功能障碍 心。DCM通常进展为心力衰竭(HF)，这是一种破坏性的疾病，与高发病率和 死亡率，包括首次入院治疗后5年内死亡率为50%。而一个确切的机制是 TTNTV如何导致DCM的发病机制仍在研究中，导致TTN蛋白含量不足 TTNtv严重扰乱心脏生理，促进扩张型心肌病的发展。为了定义治疗性 TTN显性截断变异引起DCM的策略(TTNtv DCM)，Kim博士首先提出了一种 高效的模型系统：同基因野生型(WT)和突变型人诱导多能干细胞来源 携带杂合子TTNtv的心肌细胞(hiPSC-CMS)。TTNtv/+hiPSC-CMS降低了TTN 表达和收缩能力受损，类似于人类扩张型心肌病患者。此外，通过比较分析， HPSCs在心肌细胞分化过程中的序列保守和转录激活信号 Kim博士发现了一种TTN的转录增强子，当从WT hiPSC-CMS中删除时，TTN显著减少 表达并破坏肌节的形成和功能。此外，金博士的初步工作 证明了TTN增强子的转录活性可以通过修改ITS序列和 通过将簇状规则间隔短回文重复序列(CRISPR)介导的激活子定向到TTN 人类细胞中的基因座。基于这些初步数据，金博士形成了一个假说，即增加TTN基因 调节内源性TTN调控元件和外源转录的表达 激活剂可改善TTNtv DCM模型系统的心肌细胞功能。在这份提案中，金博士计划 在三个具体目标上检验她的假设。在具体目标1中，金博士将确定受调控的遗传因素 TTN基因的表达，目前尚不清楚。在具体目标2中，金博士将调整监管 TTN元件通过基因组编辑来增强TTN的表达。在具体目标3中，金博士计划评估 TTNtv DCM模型系统中TTN表达增加的影响。这项工作将在 哈佛医学院核心教学医院布里格姆妇女医院的心血管内科 学校(HMS)。金博士将在克里斯汀·塞德曼博士的指导下进行这项研究，托马斯·W·W。 HMS医学教授、BWH心血管遗传学中心主任Smith博士和Jonathan博士 塞德曼，亨丽埃塔·B和弗雷德里克·H·布格尔基金会的遗传学教授。金博士的目标是 成为R01资助的独立临床医生兼科学家，拥有心肌病遗传学方面的专业知识。金博士 计划利用她的K08奖来加强她在基因调控和基因组编辑方面的技能和知识，这 将作为她R01应用程序的基础，在那里她将把基因工程技术应用到 制定扩张型心肌病的治疗策略。

Project Summary/Abstract Heterozygous truncating variants in the essential sarcomere protein titin (TTNtv) are the most common genetic cause of dilated cardiomyopathy (DCM), dilatation and contractile dysfunction of one or both ventricles of the heart. DCM often progresses to heart failure (HF), a devastating disorder associated with high morbidity and mortality including death in 50% within 5 years after the first HF hospitalization. While an exact mechanism of how TTNtv leads to pathogenesis of DCM is still under investigation, insufficient amount of TTN protein caused by TTNtv significantly disrupts cardiac physiology and contributes to development of DCM. To define therapeutic strategies for DCM caused by dominant truncating variants in TTN (TTNtv DCM), Dr. Kim first developed an efficient model system: isogenic wild-type (WT) and mutant human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying heterozygous TTNtv. TTNtv/+ hiPSC-CMs have decreased TTN expression and impaired contractility, similar to human patients with DCM. Additionally, by comparative analyses of sequence conservation and transcriptional activation signals during cardiomyocyte differentiation of hiPSCs, Dr. Kim identified a transcriptional enhancer of TTN, when deleted from WT hiPSC-CMs, markedly reduced TTN expression and disrupted sarcomere formation and function. Furthermore, Dr. Kim’s preliminary work demonstrated that transcriptional activity of the TTN enhancer can be increased by modifying its sequences and by directing clustered regularly interspaced short palindromic repeats (CRISPR)-mediated activator to the TTN locus in human cells. Based on these preliminary data, Dr. Kim formed the hypothesis that increasing TTN gene expression by modulating endogenous TTN regulatory elements and by introducing exogenous transcriptional activators will improve function of cardiomyocytes in TTNtv DCM model systems. In this proposal, Dr. Kim plans to test her hypothesis in three specific aims. In Specific Aim 1, Dr. Kim will identify regulatory genetic elements of TTN gene expression, which are currently unknown. In Specific Aim 2, Dr. Kim will modulate regulatory elements of TTN via genome editing to augment TTN expression. In Specific Aim 3, Dr. Kim plans to assess effects of increased TTN expression in TTNtv DCM model systems. This work will take place in the Division of Cardiovascular Medicine at Brigham and Women’s Hospital (BWH), a core teaching hospital of Harvard Medical School (HMS). Dr. Kim will perform the research under the mentorship of Dr. Christine Seidman, the Thomas W. Smith Professor of Medicine at HMS and director of Cardiovascular Genetics Center at BWH, and Dr. Jonathan Seidman, the Henrietta B. and Frederick H. Bugher Foundation Professor of Genetics at HMS. Dr. Kim’s goal is to become an R01-funded independent clinician-scientist with expertise in genetics of cardiomyopathy. Dr. Kim plans to use her K08 award to strengthen her skills and knowledge in gene regulation and genome editing, which will serve as a foundation for her R01 application where she will apply genetic engineering technologies to develop therapeutic strategies for DCM.