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.

项目概要/摘要 必需肌节蛋白肌联 (TTNtv) 的杂合截短变异是最常见的遗传变异 扩张型心肌病（DCM）的病因，一侧或双侧心室扩张和收缩功能障碍 DCM 经常进展为心力衰竭 (HF)，这是一种与高发病率和相关性相关的破坏性疾病。 首次心力衰竭住院后 5 年内死亡率包括 50% 的死亡。 TTNtv如何导致DCM发病机制仍在研究中，TTN蛋白量不足导致 TTNtv 显着扰乱心脏生理并有助于 DCM 的发展。 针对TTN（TTNtv DCM）中显性截断变异引起的DCM的策略，Kim博士首先开发了一种 高效模型系统：同基因野生型（WT）和突变型人类诱导多能干细胞来源 携带杂合 TTNtv/+ hiPSC-CM 的心肌细胞 (hiPSC-CM) TTN 降低。 通过比较分析，表达和收缩力受损与 DCM 患者相似。 hiPSC 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 TTN 的转录增强子，当从 WT hiPSC-CM 中删除时，TTN 显着降低 此外，Kim 博士的前期工作。 TTN增强子的转录活性可以通过修改其序列来增加 通过将成簇规则间隔的短回文重复序列 (CRISPR) 介导的激活子引导至 TTN 基于这些初步数据，Kim 博士提出了增加 TTN 基因的假设。 通过调节内源 TTN 调控元件和引入外源转录因子来表达 Kim 博士计划在这项提案中使用激活剂来改善 TTNtv DCM 模型系统中的心肌细胞功能。 为了在三个具体目标中检验她的假设，Kim 博士将在具体目标 1 中识别调控遗传元件。 TTN 基因表达的影响目前尚不清楚，在具体目标 2 中，Kim 博士将调节监管。 Kim 博士计划在特定目标 3 中评估通过基因组编辑来增强 TTN 表达的 TTN 元素。 TTNtv DCM 模型系统中 TTN 表达增加的影响这项工作将在该部门进行。 哈佛医学院核心教学医院布莱根妇女医院 (BWH) 心血管内科 Kim 博士将在 Thomas W. School 的 Christine Seidman 博士的指导下进行这项研究。 HMS 史密斯医学教授兼 BWH 心血管遗传学中心主任，以及 Jonathan 博士 Seidman 是 HMS 的 Henrietta B. 和 Frederick H. Bugher 基金会遗传学教授 Kim 博士的目标是。 成为 R01 资助的独立临床医生科学家，拥有心肌病遗传学方面的专业知识。 计划利用她的 K08 奖项来加强她在基因调控和基因组编辑方面的技能和知识，这 将作为她的 R01 申请的基础，她将把基因工程技术应用于 制定 DCM 的治疗策略。