Microphysiological Human Tissue Systems for Monitoring of Genome Editing Outcomes

摘要： 基因组编辑技术具有治疗各种毁灭性人类疾病的巨大潜力， 紊乱然而，基因组编辑疗法必须克服许多挑战， 他们的全部承诺。具体来说，有许多可能的不利后果是基因组编辑所独有的 工具，如基因组完整性，免疫反应，以及由于细胞更新而导致的治疗效果丧失， 目前还没有严格评估的最佳系统。此外，这些后果是 这是人类生理学、基因组序列和免疫系统所特有的，因此典型的动物模型是 不完全是信息。为了满足这一未满足的需求，我们组建了一个合作团队， 研究人员已经开发出先进的基因组编辑策略和方法， 微生理组织系统，概括人体生理和功能，重点是骨骼 和心肌。我们将联合收割机结合这些技术，在本提案中系统地评估组织 生理学、基因组改变、组织再生和响应于各种基因组的免疫应答 编辑策略和交付方法。具体而言，这将包括全面和公正的映射， 对人类基因组序列的非预期修饰，包括在靶位点和脱靶位点。我们还将 确定常驻组织干细胞的作用、细胞更新以及组织损伤和再生的稳定性 基因组编辑最后，我们可以将免疫细胞整合到这些微生理组织中， 对细菌衍生的基因组编辑组件的免疫力的后果。总的来说，这项建议将 建立一个平台，系统地应对实现变革性发展所面临的最重大挑战， 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.