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

项目摘要/摘要 体细胞基因组编辑(SCGE)极有希望改变我们的治疗工具箱 人类遗传性疾病的治疗。然而，尽管有工具可用来识别和 对人类致病突变的修饰，对有效性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严重差距 包括：1)目标上和目标外的基因组编辑率是多少；2)单细胞凝胶电泳法试剂对人类细胞有何影响 和组织功能，以及3)先天和获得性免疫系统将如何对SCGE试剂做出反应。而当 临床试验是确定有效性和安全性的有效工具，它们在穷尽后最有效地应用 临床前研究已经优化了其他系统的有效性和安全性。这项提议的目标是适应 仿生人心脏微组织(CMTs)--由人诱导的心肌细胞构建而成 多能干细胞(IPSCs)、成纤维细胞和巨噬细胞--研究SCGE试剂和释放的影响 人体功能组织上的系统。因为CMTs概括了活体心脏的三维 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 动态平衡和功能，它们是在体外测试心脏功能的理想选择。已对CMTS进行优化 对于量化包括数量级变化的一系列动态表型的功能分析 可预测体内心脏功能的组织收缩能力、钙处理和电生理学。重要的是 CRISPR/Cas9基因组编辑器已有效地应用于CMTs以建立单基因疾病模型 常见的心血管疾病，如导致心脏的扩张性和肥厚性心肌病 失败了。 在他们全面的初步数据的指导下，包括应用下一代DNA和RNA- 对CMTs进行测序，研究人员建议追求两个特定的目标来确定SCGE的有效性 和安全性：1)通过综合评估收缩能力、钙处理和电学来询问CMTs 结合单细胞转录和脱靶基因组测序进行鉴定的功能 靶向Titin编码基因TTN的SCGE试剂的不良后果，以及2)工程自体 CMTs与两种不同类型的巨噬细胞组装在一起，研究心脏功能和SCGE试剂。 这些目标的实施将提供对SCGE试剂的安全性和有效性的多尺度洞察 通过建立一个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 心血管疾病等疾病的精准医学。

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.