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

用于原位检测和功能测量基因组编辑引起的不良后果的人体微组织

• 批准号: 10455604
• 负责人: Bruce R Conklin
• 金额: $ 72.08万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-25 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10455604
• 关键词: 3-Dimensional; Adverse effects; Adverse event; Affect; Alleles; Animal Model; Biological; Biological Assay; Biological Markers; Cardiac; Cardiac Myocytes; Cell Death; Cell Differentiation process; Cells; Clinical; Clinical Medicine; Congenital Disorders; DNA Sequence Alteration; Detection; Development; Disease; Disease model; Dominant-Negative Mutation; Effectiveness; Engineering; Event; Excision; Exhibits; Exposure to; Failure; Future; Genes; Genetic Diseases; Genome; Genomic DNA; Genomics; Goals; Heart; Hepatobiliary; Hepatotoxicity; Human; Human Development; Human Engineering; In Situ; Individual; Lead; Liver; Liver diseases; Measurement; Measures; Mechanics; Metabolic; Methods; Mitotic; Modern Medicine; Mutate; Natural regeneration; Nervous system structure; Neurons; Organ; Organ failure; Organoids; Outcome; Patients; Pharmaceutical Preparations; Physiological; Pluripotent Stem Cells; Population; Reagent; Reporter; Research; Research Personnel; Safety; Site; Source; Specimen; System; Technology; Testing; Therapeutic Intervention; Tissue Engineering; Tissue Model; Tissues; Toxic effect; Transgenic Organisms; Translating; Work; adverse outcome; base editing; curative treatments; delivery vehicle; drug development; gene therapy; genome editing; human tissue; infancy; loss of function; mortality; multidisciplinary; neurotoxicity; novel; novel therapeutics; off-target site; postnatal; pre-clinical; relating to nervous system; response; somatic cell gene editing; stem cell biology; stem cell derived tissues; targeted nucleases; technology development; therapeutic gene; therapeutic genome editing; therapy development

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.

项目概要 基因组编辑技术正在迅速向临床治疗方向发展，但准确的方法还需要稳健的方法 评估基因组编辑活动和运载体对生理组织的潜在不利影响 功能尚待开发和广泛传播。由于动物模型通常不能很好地预测 人类的生物反应，必须建立由人体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对人类的影响的中间测试平台 组织功能。人类多能干细胞和产后组织干细胞为人类提供了宝贵的来源 可用于创建 3D 的分化细胞，例如心肌细胞、神经元和肝胆细胞 用于模型疾病和离体测试疗法的组织。传统上，新疗法对心脏的毒性， 神经系统和肝脏会产生严重后果，常常导致器官衰竭和死亡。 因此，需要对潜在毒性和不良事件进行彻底的临床前表征。 重现关键生理功能的人类微组织基因组编辑对于这些研究至关重要 最终转化为临床使用的疗法。该提案的主要目标是开发和 验证能够灵敏、准确地检测基因组不利影响的人体组织平台 编辑生理组织功能。为了实现这一目标，我们建立了一个多学科团队 领先的研究人员在组织工程、基因组编辑、干细胞生物学、 单细胞基因组学和技术开发。我们将通过三个项目来实现这一总体目标 并行，最初侧重于与特定的微组织平台相结合的开发 在继续测试所有三个组织上的每个编辑场景之前的基因组编辑策略 系统。在第一个项目中，我们将检查单站点编辑对定义的脱靶站点的影响，以及 内源性位点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 检查神经元中可变大小片段删除后的大规模基因组改变及其影响 关于生理参数。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： 外源基因插入基因组后的肝胆微组织。这些人体组织模型 经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 联盟，测试新型运载工具并帮助为未来基因组编辑疗法的开发提供信息。 总而言之，这项工作的结果应显着有利于治疗性基因组的安全性和可预测性 未来的疗法。