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

项目总结/摘要 体细胞基因组编辑（SCGE）具有显著的前景，可以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 修改人类致病突变，对疗效和安全性的突出关注已经减少， SCGE的临床应用前景广阔。我们目前对SCGE方法安全性的认识存在重大差距 包括：1）靶基因组编辑率和脱靶基因组编辑率，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对SCGE试剂作出反应。而 临床试验是确定有效性和安全性的有效工具，在详尽研究之后， 临床前研究优化了其他系统的有效性和安全性。该提案的目标是适应 仿生人类心脏微组织（CMT）--由来自人类诱导的心肌细胞工程化 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 系统在功能性人体组织上。因为CMT概括了体内心脏三维 结构、生物力学特性以及对心脏组织至关重要的复杂多细胞相互作用 由于它们具有体内平衡和功能，因此它们是体外测定心脏功能的理想选择。CMT已优化 对于定量一系列动态表型的功能测定， 预测体内心脏功能的组织收缩性、钙处理和电生理学。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，如扩张性和肥厚性心肌病，导致心脏 失败 在他们全面的初步数据的指导下，包括下一代DNA和RNA的应用， 在对CMT进行测序分析后，研究人员提出了两个特定目标来确定SCGE的功效 和安全性：1）通过对收缩性、钙处理和电性能的综合评估来询问CMT 功能与单细胞转录组学结合，并与脱靶基因组测序配对， 靶向肌联蛋白编码基因TTN的SCGE试剂的不良后果，和2）工程化自体 CMT与两种不同类型的巨噬细胞组装以研究心脏功能和SCGE试剂。 这些目标的实施将为SCGE试剂的安全性和有效性提供多尺度的见解 通过产生信息测试平台和相关方法的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基因组编辑的承诺的关键一步。 心血管和其他疾病的精准医疗。

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.