Human cardiac microtissues with innate immune sensing to study adverse consequences of genome editing

具有先天免疫传感的人类心脏微组织用于研究基因组编辑的不良后果

• 批准号: 10463658
• 负责人: John Travis Hinson
• 金额: $ 73.7万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10463658
• 关键词: 3-Dimensional; Adaptive Immune System; Address; Adverse effects; Affect; Alleles; Architecture; Arrhythmia; Autologous; Automobile Driving; Biological Assay; Biological Process; Biomechanics; Biomimetics; CRISPR/Cas technology; Calcium; Cardiac; Cardiac Function Study; Cardiac Myocytes; Cardiotoxicity; Cardiovascular Diseases; Cardiovascular system; Cell Line; Cell model; Cells; Cessation of life; Clinical Trials; Complex; Computational Biology; Contracts; DNA sequencing; Data; Dilated Cardiomyopathy; Disease; Disease model; Electrophysiology (science); Engineering; Fibroblasts; Gene Targeting; Genes; Genetic; Genetic Diseases; Genome; Genomics; Goals; Guide RNA; Heart; Heart failure; Homeostasis; Human; Human Engineering; Human Genetics; Hypertrophic Cardiomyopathy; Immunology; In Vitro; Inherited; Innate Immune System; Interferon Type I; Kinetics; Knowledge; Mendelian disorder; Methods; Modeling; Modification; Molecular; Morbidity - disease rate; Mutate; Other Genetics; Outcome; Patients; Phenotype; Physiology; Property; Public Health; Reagent; Research Personnel; Resolution; Resources; Risk; Risk Assessment; Safety; Site; Structure; System; Testing; Therapeutic; Tissue Model; Tissues; adverse outcome; clinical application; clinical development; connectin; disease-causing mutation; exhaustion; experience; genetic variant; genome editing; genome sequencing; genotoxicity; heart dimension/size; heart function; human disease; human tissue; immunogenicity; improved; in vivo; induced pluripotent stem cell; innate immune sensing; insight; macrophage; mortality; next generation; pre-clinical; precision medicine; preclinical study; prevent; reagent testing; response; single-cell RNA sequencing; somatic cell gene editing; technology development; therapeutic development; tool; transcriptome sequencing; transcriptomics; tumorigenesis

PROJECT SUMMARY/ABSTRACT Somatic cell genome editing (SCGE) has remarkable promise to transform our therapeutic toolbox for the treatment of human genetic disorders. However, despite having the tools available for identification and modification of human disease-causing mutations, outstanding concerns over efficacy and safety have curtailed the clinical application of SCGE broadly. Critical gaps in our current knowledge of the safety of SCGE approaches include: 1) what are the on- and off-target genome editing rates, 2) how do SCGE reagents affect human cellular and tissue function, and 3) how will the innate and adaptive immune system respond to SCGE reagents. While clinical trials are effective tools to determine efficacy and safety, they are most efficiently applied after exhaustive pre-clinical studies have optimized efficacy and safety in other systems. The goal of this proposal is to adapt biomimetic human cardiac microtissues (CMTs)--engineered from cardiomyocytes derived from human induced pluripotent stem cells (iPSCs), fibroblasts, and macrophages--to study the impact of SCGE reagents and delivery systems on a functional human tissue. Because CMTs recapitulate in vivo cardiac three-dimensional architecture, biomechanical properties, and complex multicellular interactions that are critical to cardiac tissue homeostasis and function, they are ideal for assaying cardiac functions in vitro. The CMTs have been optimized for functional assays that quantify a range of dynamic phenotypes that include orders-of-magnitude changes in tissue contractility, calcium handling, and electrophysiology that predict in vivo cardiac function. Importantly, CRISPR/Cas9 genome editors have been effectively applied to CMTs to generate monogenic disease models of common cardiovascular disorders such as dilated and hypertrophic cardiomyopathies that result in heart failure. Guided by their comprehensive preliminary data including application of next-generation DNA- and RNA- sequencing assays to CMTs, the researchers propose to pursue two Specific Aims to determine SCGE efficacy and safety: 1) interrogate CMTs by comprehensive assessment of contractility, calcium handling and electrical function in combination with single-cell transcriptomics paired with off-target genome sequencing to identify adverse consequences of SCGE reagents that target the titin-encoding gene TTN, and 2) engineer autologous CMTs assembled with two distinct classes of macrophages to study cardiac function and SCGE reagents. Execution of these Aims will provide multi-scale insights into the safety and efficacy of SCGE reagents by producing an informative testing platform and system of associated methods to identify adverse outcomes. Establishing these resources will be a pivotal step toward realizing the promise of genome editing and human precision medicine of cardiovascular and other disorders.

项目概要/摘要 体细胞基因组编辑（SCGE）有望改变我们的治疗工具箱 人类遗传疾病的治疗。然而，尽管有可用于识别和识别的工具 人类致病突变的修饰，对功效和安全性的突出担忧已经减少 SCGE的临床应用十分广泛。我们目前对 SCGE 方法安全性的了解存在重大差距 包括：1) 基因组编辑的脱靶率和脱靶率是多少，2) SCGE 试剂如何影响人类细胞 和组织功能，以及 3) 先天性和适应性免疫系统将如何响应 SCGE 试剂。尽管 临床试验是确定疗效和安全性的有效工具，在详尽无遗之后应用它们是最有效的 临床前研究优化了其他系统的功效和安全性。该提案的目标是适应 仿生人类心脏微组织（CMT）——由源自人类诱导的心肌细胞设计而成 多能干细胞 (iPSC)、成纤维细胞和巨噬细胞——研究 SCGE 试剂和递送的影响 功能性人体组织上的系统。因为 CMT 再现了体内心脏三维 对心脏组织至关重要的结构、生物力学特性和复杂的多细胞相互作用 体内平衡和功能，它们是体外测定心脏功能的理想选择。 CMT 已优化 用于量化一系列动态表型的功能测定，包括数量级的变化 组织收缩性、钙处理和预测体内心脏功能的电生理学。重要的是， CRISPR/Cas9基因组编辑器已有效应用于CMT，以生成单基因疾病模型 常见的心血管疾病，例如扩张型和肥厚型心肌病，导致心脏 失败。 以他们全面的初步数据为指导，包括下一代 DNA 和 RNA 的应用 针对 CMT 的测序分析，研究人员建议追求两个具体目标来确定 SCGE 功效 和安全性：1) 通过综合评估收缩性、钙处理和电学来询问 CMT 结合单细胞转录组学和脱靶基因组测序来识别 针对 titin 编码基因 TTN 的 SCGE 试剂的不良后果，以及 2) 工程自体 CMT 与两种不同类别的巨噬细胞组装在一起，用于研究心脏功能和 SCGE 试剂。 这些目标的执行将为 SCGE 试剂的安全性和有效性提供多尺度的见解 通过创建信息丰富的测试平台和相关方法系统来识别不良结果。 建立这些资源将是实现基因组编辑和人类前景的关键一步。 心血管和其他疾病的精准医学。