Establishing and reversing the functional consequences of Titin truncation mutations

建立并逆转肌联蛋白截断突变的功能后果

基本信息

批准号：
10510011
负责人：
STUART G CAMPBELL
金额：
$ 56.61万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10510011
关键词：
3-Dimensional Adrenergic Agents Agar Gel Electrophoresis Alternative Splicing Base Pairing Behavior Biological Assay Biology Biomimetics CRISPR-mediated transcriptional activation CRISPR/Cas technology Cardiac Cardiac Myocytes Cardiomyopathies Cells Clustered Regularly Interspaced Short Palindromic Repeats Communities Computing Methodologies DNA Data Dependence Development Diagnosis Dilated Cardiomyopathy Disease Distal Dose Extracellular Matrix Fibroblasts Foundations Functional disorder Future Genes Genetic Goals Guide RNA Heart Heart failure Human Individual Kinetics Knowledge Lead Learning Length Lesion Link Location Measures Mechanics Mediating Methods Modeling Molecular Mus Mutation Myocardial Contraction Myocardium Nonmuscle Myosin Type IIA Organelles Pathogenesis Pathogenicity Pathologic Pathway interactions Patients Phenotype Physicians Physiological Prognosis Protein Isoforms Proteins Publications RNA Binding Reagent Regulation Regulatory Element Research Resources Risk Sarcomeres Series Signal Transduction Site Stress Structure Sturnus vulgaris Sudden Death Technology Therapeutic Tissue Engineering Tissue Model Tissue-Specific Gene Expression Transcription Coactivator Variant Western Blotting Work adverse outcome biological adaptation to stress biomarker development biomarker identification cardiac tissue engineering cohort confocal imaging connectin disease prognosis dosage genome editing heart cell heart dimension/size heart function high risk human model improved induced pluripotent stem cell induced pluripotent stem cell derived cardiomyocytes inherited cardiomyopathy insight mechanotransduction mutation carrier prevent promoter proteostasis response scaffold sudden cardiac death therapeutic development therapeutic target transcriptomics

项目摘要

PROJECT SUMMARY/ABSTRACT Cardiomyopathies occur in ~1:200 individuals and are commonly caused by inheritance of variants in genes that encode proteins that regulate the sarcomere, the force-producing organelle of heart cells. Due to an incomplete understanding of variant pathogenicity and cardiomyopathy pathogenesis, physicians are currently limited in their ability to provide diagnoses, prognoses, and therapeutic options for cardiomyopathy patients. Variants in the TTN gene, which encodes the sarcomere protein titin, are the most frequently identified genetic lesion in dilated cardiomyopathy (DCM), which is characterized by heart chamber dilation, reduced contractile function, risk of sudden death, and progressive heart failure. The most frequent type of TTN variant identified in DCM is a truncation mutation that would be predicted to shorten TTN protein length and to reduce TTN protein quantities. Significantly, truncation variants localized to distal TTN structural domains are more pathogenic than those localized to proximal structural domains, but the mechanistic basis for this relationship is uncertain. It remains incompletely understood how TTN truncation variants cause DCM generally, which is compounded by our lack of understanding of the ‘length dependence’ of TTN variant pathogenicity. These knowledge gaps limit disease prognostication, biomarker identification, and therapeutic development for DCM patients. The central goal of our study is to define how disruptions in TTN length and dosage by TTN variants cause DCM, and exploit this knowledge to develop DCM therapeutics for TTN variant carriers. We hypothesize that healthy cardiac contractile function and structure depends on the regulation of TTN length and dosage, and that varying pathogenicity of TTN truncation can be explained by distinct structural and functional consequences associated with the specific site of truncation. In Aim 1, we will determine the functional consequences of TTN truncations across structural domains by harnessing 3-dimensional heart tissue models composed of human cardiomyocytes differentiated from induced pluripotent stem cells in which variants have been introduced by CRISPR-mediated genome editing. We will interrogate these models for tissue mechanical phenotypes (such as passive tension and Frank-Starling behavior), TTN protein length and levels (using specialized methods), proteostasis stress pathway responses (using immunoblotting), and mechanotransduction signaling and alternative splicing (using expression analysis and transcriptomics, respectively). In Aim 2, we will restore TTN protein levels using the recently developed method of CRISPR activation applied to DCM engineered heart tissue models for both evaluating the function of TTN isoforms generally and as a DCM proof-of-concept therapeutic. Through these Aims, we will gain critical new insights into the pathophysiology of DCM-associated TTN truncation variants, uncover features to explain the variable pathogenicity identified in DCM patients, and develop a therapeutic to target TTN directly. We anticipate this new knowledge will improve physicians’ capacity to diagnose, prognose, and treat patients with DCM due to TTN variants.

项目概要/摘要心肌病发生在大约 1:200 的个体中，通常是由基因变异遗传引起的，这些变异编码调节肌节的蛋白质，肌节是心脏细胞的力产生细胞器，由于不完整。目前，医生对变异致病性和心肌病发病机制的了解还有限他们为变异型心肌病患者提供诊断、预后和治疗选择的能力。 TTN 基因编码肌节蛋白肌联蛋白，是最常见的遗传性病变扩张型心肌病（DCM），其特征是心室扩张、收缩功能降低、 DCM 中最常见的 TTN 变异类型是猝死和进行性心力衰竭的风险。预计会缩短 TTN 蛋白长度并减少 TTN 蛋白数量的截断突变。值得注意的是，位于远端 TTN 结构域的截短变异比那些本地化的截短变异更具致病性。局限于近端结构域，但这种关系的机制基础仍然不确定。不完全了解 TTN 截断变异通常如何导致 DCM，而我们的缺乏使情况变得更加复杂这些知识差距限制了对 TTN 变异致病性“长度依赖性”的理解。 DCM 患者的预测、生物标志物识别和治疗开发。我们的研究旨在确定 TTN 变体对 TTN 长度和剂量的破坏如何导致 DCM，并利用这一点为 TTN 变异携带者开发 DCM 疗法的知识。收缩功能和结构取决于TTN长度和剂量的调节，并且不同 TTN 截短的致病性可以通过相关的不同结构和功能后果来解释在目标 1 中，我们将确定 TTN 截断的功能后果。通过利用由人类心肌细胞组成的 3 维心脏组织模型来跨结构域与诱导多能干细胞不同，诱导多能干细胞中通过 CRISPR 介导引入了变异体我们将询问这些模型的组织机械表型（例如被动张力）。和 Frank-Starling 行为）、TTN 蛋白质长度和水平（使用专门方法）、蛋白质稳态应激途径反应（使用免疫印迹），以及机械转导信号和选择性剪接（使用分别是表达分析和转录组学）。在目标 2 中，我们将使用最近开发的 CRISPR 激活方法应用于 DCM 工程心脏组织模型通过这些总体评估 TTN 亚型的功能以及作为 DCM 概念验证治疗的功能。目标是，我们将对 DCM 相关 TTN 截短变异的病理生理学获得重要的新见解，揭示特征来解释 DCM 患者中发现的可变致病性，并开发一种治疗方法我们预计这一新知识将提高医生诊断、预测、并治疗因 TTN 变异而导致的扩张型心肌病 (DCM) 患者。