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.

项目总结/摘要 必需肌节蛋白肌联蛋白（TTNtv）的杂合截短变体是最常见的遗传变异 扩张型心肌病（DCM），一个或两个心室的扩张和收缩功能障碍， 心DCM通常进展为心力衰竭（HF），这是一种与高发病率和高死亡率相关的破坏性疾病。 死亡率，包括首次HF住院后5年内50%的死亡。虽然一个确切的机制， TTNtv如何导致DCM的发病机制仍在研究中，TTN蛋白量不足引起的 TTNtv显著破坏心脏生理学并促进DCM的发展。为了定义治疗 针对由TTN中的显性截短变体（TTNtv DCM）引起的DCM的策略，Kim博士首先开发了一种 有效的模型系统：同基因野生型（WT）和突变体人诱导多能干细胞衍生 携带杂合TTNtv的心肌细胞（hiPSC-CM）。TTNtv/+ hiPSC-CM的TTN降低 表达和收缩性受损，类似于患有DCM的人类患者。此外，通过比较分析， 在hiPSC的心肌细胞分化期间序列保守和转录激活信号， 博士Kim鉴定了TTN的转录增强子，当从WT hiPSC-CM中删除时， 表达并破坏肌节形成和功能。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饰其序列来增加， 通过将成簇规则间隔短回文重复序列（CRISPR）介导的激活剂引导至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调节元件和通过引入外源性转录因子 活化剂将改善TTNtv DCM模型系统中的心肌细胞的功能。在这份提案中，金博士计划 以三个具体目标来验证她的假设在具体目标1中，Kim博士将确定调控遗传元件 TTN基因的表达，这是目前未知的。在具体目标2中，Kim博士将调节 通过基因组编辑TTN元件以增加TTN表达。在具体目标3中，金博士计划评估 在TTNtv DCM模型系统中增加TTN表达的作用。这项工作将在 布里格姆妇女医院（BWH）的心血管内科，该医院是哈佛医学院的核心教学医院 学校（HMS）。金博士将在克莉丝汀·塞德曼博士的指导下进行这项研究。 HMS的Smith医学教授和BWH心血管遗传学中心主任，以及Jonathan博士 塞德曼，亨里埃塔的B。和弗雷德里克·H. HMS的Bugher基金会遗传学教授。金博士的目标是 成为R 01资助的独立临床科学家，具有心肌病遗传学方面的专业知识。金医生 计划利用她的K 08奖来加强她在基因调控和基因组编辑方面的技能和知识， 将作为她R 01申请的基础，她将应用基因工程技术， 制定DCM的治疗策略。