Microphysiological Human Tissue Systems for Monitoring of Genome Editing Outcomes

摘要： 基因组编辑技术具有巨大的潜力来治疗各种毁灭性的人类疾病和 精神错乱。然而，基因组编辑疗法必须克服许多挑战才能达到 他们的全部承诺。具体地说，基因组编辑可能有许多独特的不良后果 如基因组完整性、免疫反应和由于细胞更替而导致的治疗效果丧失等工具 目前还没有用于严格评估的最佳系统。此外，这些后果是 人类生理学、基因组序列和免疫系统所独有的，因此典型的动物模型 并不能提供完全的信息。为了解决这一未得到满足的需求，我们组建了一个协作团队 研究人员开发了先进的基因组编辑策略和方法来改造人类 微生理组织系统，概括了人类的生理和功能，重点是骨骼 和心肌。我们将在这项提案中结合这些技术来系统地评估组织 不同基因组的生理、基因组改变、组织再生和免疫反应 编辑策略和交付方法。具体地说，这将包括全面和公正的地图绘制 对人类基因组序列的意外修改，包括在靶点上和靶点外。我们还将 确定驻留组织干细胞、细胞周转以及组织损伤和再生在稳定性中的作用 基因组编辑。最后，我们可以将免疫细胞结合到这些微生理组织中，以了解 对细菌衍生的基因组编辑组件免疫的后果。总体而言，这项提议将 开发一个平台，系统地应对实现变革性 基因组编辑疗法在人体组织中的潜力。

Abstract: Genome editing technologies have significant potential to treat a variety of devastating human diseases and disorders. However, there are a number of challenges that genome editing therapies must overcome to reach their full promise. Specifically, there are many possible adverse consequences that are unique to genome editing tools, such as genome integrity, immune responses, and loss of therapeutic efficacy due to cell turnover, for which there are currently are no optimal systems for rigorous assessment. Moreover, these consequences are unique to human physiology, genome sequence, and immune systems, and therefore typical animal models are not completely informative. To address this unmet need, we have assembled a team of collaborative investigators that have developed advanced genome editing strategies and methods for engineering human microphysiological tissue systems that recapitulate human physiology and function, with an emphasis on skeletal and cardiac muscle. We will combine these technologies in this proposal to systemically evaluate tissue physiology, genomic alterations, tissue regeneration, and immune response in response to various genome editing strategies and delivery methods. Specifically, this will include comprehensive and unbiased mapping of unintended modifications to human genome sequences, including at on-target and off-target sites. We will also determine the role of resident tissue stem cells, cell turnover, and tissue injury and regeneration in the stability of genome editing. Finally, we can incorporate immune cells into these microphysiological tissues to understand the consequences of immunity to bacteria-derived genome editing components. Collectively, this proposal will develop a platform to systematically address the most significant challenges to realizing the transformative potential of genome editing therapies in human tissues.