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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：
10640157
负责人：
STUART G CAMPBELL
金额：
$ 52.91万
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/10640157
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 Functional disorder Future Genes Genetic Goals Guide RNA Heart Heart failure Heterozygote Human Individual Kinetics Knowledge 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.

项目总结/摘要心肌病发生在约1：200的个体中，通常由遗传基因变异引起，编码调节肌节的蛋白质，肌节是心脏细胞产生力量的细胞器。由于不完整的了解变异致病性和心肌病发病机制，医生目前在他们为心肌病患者提供诊断、预后和治疗选择的能力。中的变体编码肌节蛋白肌联蛋白的TTN基因是最常见的遗传性病变，扩张型心肌病（DCM），其特征在于心腔扩张，收缩功能降低，猝死和进行性心力衰竭的风险。DCM中最常见的TTN变异类型是一个截断突变，预计将缩短TTN蛋白长度和减少TTN蛋白量。值得注意的是，定位于远端TTN结构域的截短变体比那些截短变体更具致病性。定位于近端结构域，但这种关系的机制基础是不确定的。它仍然是不完全理解TTN截短变体通常如何导致DCM，这与我们缺乏了解TTN变异致病性的“长度依赖性”。这些知识差距限制了疾病扩张型心肌病患者的诊断、生物标志物鉴定和治疗开发。的中心目标我们的研究是为了确定TTN变异体如何破坏TTN长度和剂量导致DCM，并利用这一点。开发TTN变异携带者DCM治疗方法的知识。我们假设健康的心脏收缩功能和结构取决于TTN长度和剂量的调节， TTN截短的致病性可以通过与TTN截短相关的不同结构和功能后果来解释。与特定的截短位点。在目标1中，我们将确定TTN截短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，从诱导的多能干细胞分化，其中变体已经通过CRISPR介导的CRISPR介导的免疫调节被引入。基因组编辑我们将询问这些模型的组织力学表型（如被动张力和Frank-Starling行为），TTN蛋白长度和水平（使用专门的方法），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可变剪接（使用表达分析和转录组学）。在目标2中，我们将使用以下方法恢复TTN蛋白水平：最近开发的CRISPR激活方法应用于DCM工程化心脏组织模型，一般评价TTN同种型的功能和作为DCM概念验证治疗剂的功能。通过这些目的是，我们将获得对DCM相关TTN截短变体的病理生理学的重要新见解，揭示特征以解释DCM患者中鉴定的可变致病性，并开发治疗方法，直接瞄准TTN我们预计这一新知识将提高医生的诊断能力，并治疗因TTN变异而患DCM的患者。