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.

项目摘要/摘要心肌病发生在〜1：200个个体中，通常是由于基因中变异的遗传而引起的编码调节肌膜的蛋白质，即心脏细胞的产生力细胞器。由于不完整了解变异的致病性和心肌病发病机理，医师目前受到限制他们为心肌病患者提供诊断，预后和治疗选择的能力。变体编码肌节蛋白滴定的TTN基因是最常见的遗传病变扩张的心肌病（DCM），其特征是心脏腔室分裂，收缩功能降低，突然死亡和进行性心力衰竭的风险。 DCM中确定的最常类型的TTN变体是截断突变将预测，将缩短TTN蛋白长度并减少TTN蛋白质量。值得注意的是，与远端TTN结构结构域的截短变体相比本地化为代理结构领域，但是这种关系的机械基础尚不确定。它仍然存在不完全理解TTN截断变体通常会导致DCM，这是由于我们缺乏的复杂理解TTN变异致病性的“长度依赖性”。这些知识差距限制了疾病 DCM患者的预后，生物标志物鉴定和热发育。中心目标我们的研究是定义TTN变体的TTN长度和剂量的破坏是如何引起DCM的，并利用这一点为TTN变体载体开发DCM疗法的知识。我们假设健康心脏收缩功能和结构取决于TTN长度和剂量的调节，并且变化 TTN截断的致病性可以通过相关的不同结构和功能后果来解释与特定的截断部位。在AIM 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三维心脏组织模型，跨结构域与诱导的多能干细胞区别基因组编辑。我们将询问这些模型的组织机械表型（例如被动张力和坦率的宣传行为），TTN蛋白长度和水平（使用专业方法），蛋白抗压力途径响应（使用免疫印迹）和机械转导信号传导和替代剪接（使用表达分析和转录组学分别）。在AIM 2中，我们将使用最近开发的CRISPR激活方法应用于DCM工程心组织模型通常评估TTN同工型的功能，并作为DCM概念验证疗法。通过这些目的，我们将获得有关DCM相关TTN截断变体的病理生理学的关键新见解，发现特征以解释在DCM患者中鉴定出的可变致病性，并发展为治疗性直接靶向TTN。我们预计这一新知识将提高医生诊断，预测的能力并因TTN变体而治疗DCM患者。