Human microtissues for in situ detection and functional measurement of adverse consequences caused by genome editing

项目总结 基因组编辑技术正在迅速地向临床治疗迈进，但准确地 评估基因组编辑活动和运送工具对生理组织的潜在不利影响 其功能还有待开发和广泛传播。因为动物模型往往不能很好地预测 人类的生物反应，必须建立由人类细胞组成的明确的组织系统 作为评估基因组编辑的安全性和有效性及其影响的中间试验台(S) 组织功能。人类多能性和出生后组织来源的干细胞提供了宝贵的人类 分化的细胞，如心肌细胞、神经元和肝胆细胞，可用于创建3D 用于模拟疾病和测试体外治疗方法的组织。传统上，新疗法对心脏的毒性， 神经系统和肝脏表现出严重的后果，常常会导致器官衰竭和死亡。 出于这个原因，彻底的临床前表征潜在的毒性和不良事件 人类微组织中概括关键生理功能的基因组编辑将是必不可少的 最终将转化为临床使用的治疗方法。这项提议的主要目标是发展和 验证能够灵敏和准确地检测基因组不良影响的人类组织平台 关于生理组织功能的编辑。为了达到这个目标，我们成立了一个由多个专业组成的团队， 在组织工程、基因组编辑、干细胞生物学方面拥有互补专业知识的领先研究人员， 单细胞基因组学与技术发展。我们将通过以下三个项目来实现这一总体目标： 平行，最初侧重于与特定的特定微组织平台的开发 基因组编辑策略，然后继续测试所有三个组织上的每个编辑方案 系统。在第一个项目中，我们将检查单站点编辑对定义的非目标站点和 内源性基因座对心脏微组织电和机械功能的影响。在第二个项目中，我们将 检测神经元中可变大小片段缺失后的大规模基因组改变及其影响 对生理参数的影响。在第三个项目中，我们将通过以下方式评估生物标志物的表达和分泌 外源基因基因组插入后的肝胆微组织。这些人体组织模型 通过各种临床基因组编辑策略的测试将集成到体细胞基因编辑中 测试新的递送工具，并帮助为未来基因组编辑疗法的发展提供信息。 总而言之，这项工作的结果应该大大有利于治疗性基因组的安全性和可预测性。 未来的疗法。

PROJECT SUMMARY Genome editing technologies are advancing rapidly toward clinical therapies, but robust methods for accurately assessing the potential adverse effects of genome editing activity and delivery vehicles on physiological tissue function have yet to be developed and broadly disseminated. Since animal models are often poor predictors of human biological responses, it is imperative to establish well-defined tissue systems composed of human cells as an intermediate testbed to assess the safety as well as the efficacy of genome editing and its effect(s) on tissue function. Human pluripotent and post-natal tissue-derived stem cells provide valuable sources of human differentiated cells, such as cardiomyocytes, neurons and hepatobiliary cells, that can be used to create 3D tissues to model diseases and test therapies ex vivo. Traditionally, the toxicity of novel therapies in the heart, nervous system and liver manifest with severe consequences that can often lead to organ failure and mortality. For this reason, a thorough preclinical characterization of potential toxicities and adverse events caused by genome editing in human microtissues that recapitulate critical physiologic functions will be essential for these therapies to be ultimately translated for clinical use. The primary objective of this proposal is to develop and validate human tissue platforms capable of sensitively and accurately detecting adverse effects of genome editing on physiologic tissue function. To achieve this objective, we have established a multi-disciplinary team of leading investigators with complementary expertise in tissue engineering, genome editing, stem cell biology, single cell genomics and technology development. We will pursue this overall goal through three projects in parallel that focus initially on the development of individual microtissue platforms in concert with a specific genome editing strategy before proceeding to testing each of the editing scenarios on all three of the tissue systems. In the first project, we will examine the effects of single-site editing on defined off-target sites and endogenous loci on cardiac microtissue electrical and mechanical function. In the second project, we will examine large scale genomic alterations following deletion of variable size fragments in neurons and the effects on physiologic parameters. In the third project, we will assess biomarker expression and secretion by hepatobiliary microtissues following the genomic insertion of exogenous genes. These human tissue models tested with various clinical genome editing strategies will be integrated within the Somatic Gene Editing Consortium, to test novel delivery vehicles and help inform the development of future genome editing therapies. Altogether, the outcomes of this work should significantly benefit the safety and predictability of curative genome therapies of the future.