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

开发增强肌联蛋白 (TTN) 表达并治疗 TTN 单倍体不足引起的扩张型心肌病的策略

• 批准号: 10662742
• 负责人: Yuri Kim
• 金额: $ 16.89万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10662742
• 关键词: 5' Untranslated Regions; Applied Genetic Engineering; Award; Biological Assay; Biological Models; Cardiac; Cardiac Myocytes; Cardiovascular system; Cells; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Conserved Sequence; Data; Development; Dilatation - action; Dilated Cardiomyopathy; Disease; Elements; Engineering; Enhancers; Etiology; Evolution; Foundations; Functional disorder; Funding; Gene Expression; Gene Expression Regulation; Genetic; Genetic Engineering; Genetic Transcription; Genome; Genomics; Goals; Heart; Heart Abnormalities; Heart Ventricle; Heart failure; Heterozygote; Hospitalization; Hospitals; Human; Human Engineering; Impairment; In Vitro; Individual; Introns; Investigation; Knowledge; Laboratories; Left; Mediating; Medicine; Mentorship; Morbidity - disease rate; Mus; Mutagenesis; Mutate; Mutation; Myocardial dysfunction; Nucleic Acid Regulatory Sequences; Open Reading Frames; Pathogenesis; Pathogenicity; Patients; Physician Executives; Physiology; Population; Prevalence; Proteins; Regulator Genes; Regulatory Element; Reporter; Research; Role; Sarcomeres; Scientist; Sequence Analysis; Signal Transduction; Site-Directed Mutagenesis; Structure; System; Teaching Hospitals; Technology; Testing; Therapeutic; Transcription Coactivator; Transcriptional Activation; Translations; Variant; Ventricular; Woman; Work; comparative; connectin; functional improvement; gain of function mutation; genetic variant; genome editing; improved; in vivo; induced pluripotent stem cell derived cardiomyocytes; inherited cardiomyopathy; medical schools; mortality; mouse genome; mouse model; mutant; prevent; professor; promoter; protein expression; recruit; repaired; screening; skills; stem

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.

项目摘要/摘要 基本肌节蛋白滴定（TTNTV）中的杂合截断变体是最常见的通用元素 膨胀心肌病（DCM）的原因，一个或两个心室的扩张和收缩功能障碍 心。 DCM通常会发展为心力衰竭（HF），这是一种与高发病率相关的毁灭性疾病 死亡率包括在第一次HF住院后5年内50％的死亡。而确切的机制 TTNTV如何导致DCM发病机理仍在研究中，TTN蛋白的数量不足 TTNTV通过TTNTV显着破坏心脏生理，并有助于DCM的发展。定义治疗 DCM策略是由TTN（TTNTV DCM）中主要截断变体引起的，Kim博士首先开发了 有效的模型系统：等源性野生型（WT）和突变的人类诱导的多能干细胞衍生 携带杂合TTNTV的心肌细胞（HIPSC-CMS）。 TTNTV/+ HIPSC-CM已改进了TTN 表达和收缩性受损，类似于人类DCM患者。另外，通过比较分析 HIPSC的心肌细胞分化期间的序列保守和转录激活信号， Kim博士从WT HIPSC-CMS删除时确定了TTN的转录增强子，显着降低了TTN 表达和破坏的肌节形成和功能。此外，金博士的初步工作 证明TTN增强子的转录活性可以通过修改其序列和 通过将群集的定期间隔短的短质体重复（CRISPR）介导的激活剂引导到TTN 人类细胞中的基因座。基于这些初步数据，Kim博士形成了一个假设，即增加TTN基因 通过调节内源性TTN调节元件并引入外源转录来表达 激活剂将改善TTNTV DCM模型系统中心肌细胞的功能。在此提案中，金博士计划 以三个具体目标来检验她的假设。在特定目标1中，金博士将确定监管通用元素 目前未知的TTN基因表达。在特定的目标2中，金博士将调节监管 TTN的元素通过基因组编辑以增强TTN表达。在特定目标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 哈佛医学核心教学医院的杨百翰和妇女医院（BWH）的心血管医学 学校（HMS）。 Kim博士将根据Christine Seidman博士Thomas W.进行研究。 HMS的史密斯医学教授和BWH的心血管遗传学中心主任和乔纳森博士 Seidman，Henrietta B.和Frederick H. Bugher基金会在HMS的遗传学教授。金博士的目标是 成为R01资助的独立临床科学家，具有心肌病遗传学专业知识。金博士 计划使用她的K08奖来增强她在基因调节和基因组编辑方面的技能和知识，这 将作为她的R01应用程序的基础，她将在其中应用基因工程技术 开发DCM的理论策略。