Improving the Safety of Genome Editing With Human Kidney Organoids

提高人肾组织基因组编辑的安全性 项目摘要 该项目的目标是在类器官培养中应用基因组编辑器，以建立一个预测模型， 人肾细胞类型的不良事件，包括危及生命的急性和慢性疾病 后果基因组编辑平台使得能够有效地操纵基因组中的特定DNA序列。 人类基因组，因此具有巨大治疗潜力。肾脏是重要靶点 器官，有机会在体内以及离体干预。然而，人们缺乏知识， 肾细胞对基因组编辑的反应 已知肾脏易受急性毒性损伤、长期肿瘤发生以及慢性毒性损伤的影响。 免疫介导的反应。预测这些影响的一个主要障碍是缺乏人类实验 重现体内反应的模型。啮齿动物模型固有地低通量，并且具有有限的 预测人类安全性的能力，而肾细胞系去分化太严重，无法准确模拟肾单位。 为了克服这一障碍，我们从多能干细胞中衍生出了人类肾脏类器官， 体外器官结构和功能替代物。类器官具有肾脏的许多关键特征 肾单位，包括不同的细胞类型，在远端到近端的安排，可以表达特定的表型 与肾损伤和遗传性疾病相关，并且适合于高通量筛选（HTS）。 基于我们的初步工作，我们假设基因编辑会对肾脏产生有害影响 它们是特异性的、可预测的，并且可以在类器官模型中重现，以优化它们的设计， 应用程序.这项工作具有重要意义，因为它将为治疗发展建立一个新的范式 在人类细胞中，其中在类器官中的多维HTS随后进行深度测序和详细的 分析确定最安全和最有前途的候选人。它具有高度的创新性，因为它将揭示 基因组编辑的不利后果，我们目前还不知道，通过尖端的测定， 从未被应用于类器官。类器官的关键发现将在人类肾脏中得到验证 组织样本和人体肾脏芯片 本研究将围绕三个目标展开。第一个目的是提高基因编辑的安全性 通过检测和改善对肾脏中关键细胞类型的生理损伤， 类器官目标2是通过分析致癌突变来降低无意致癌的风险， 在经历基因组编辑的人肾类器官中的转化事件。最后，在目标3中，我们建议 通过阐明免疫原性后果，确定编辑相关肾炎的潜在综合征， 肾单位区室中的CRISPR-Cas9活性。总的来说，这三个目标将建立一个强大的， 潜在的高通量框架，在其中改善候选化合物的安全性并增加药物的有效性。 FDA批准的肾脏和其他器官治疗方法的数量。

IMPROVING THE SAFETY OF GENOME EDITING WITH HUMAN KIDNEY ORGANOIDS PROJECT SUMMARY The goal of this project is to apply genome editors in organoid cultures to establish a predictive model for adverse events in human kidney cell types, including both acute and chronic disorders with life-threatening consequences. Genome editing platforms enable the efficient manipulation of specific DNA sequences in the human genome, and therefore have enormous potential as therapeutics. The kidneys are important target organs, with opportunities for interventions in vivo as well as ex vivo. However, there is a dearth of knowledge about how kidney cells respond to genome editing. Kidneys are known to be susceptible to acute toxic injury, long-term tumorigenesis, as well as chronic immune-mediated responses. A major barrier to predicting these effects is the lack of human experimental models that recapitulate in vivo responses. Rodent models are inherently low-throughput, and have limited ability to predict human safety, while kidney cell lines are too dedifferentiated to accurately model nephrons. To overcome this barrier, we have derived human kidney organoids from pluripotent stem cells as a surrogate for organ structure and function in vitro. Organoids possess many of the key features of kidney nephrons, including diverse cell types in distal-to-proximal arrangements, can express specific phenotypes associated with kidney injury and genetic disease, and are amenable to high throughput screening (HTS). Based on our preliminary work, we hypothesize that gene editing will have deleterious effects on the kidney that are specific, predictable, and can be recapitulated in an organoid model to optimize their design for safe application. This work is of great significance because it will establish a new paradigm for therapy development in human cells, in which multi-dimensional HTS in organoids followed by deep sequencing and detailed analysis identifies the safest and most promising candidates. It is highly innovative because it will bring to light adverse consequences of genome editing of which we are currently unaware, via cutting-edge assays that have never before been applied to organoids. Key findings in organoids will be validated in human kidney tissue samples and human kidneys-on-chips. The proposed research will be pursued as three Aims. The first Aim is to enhance the safety of gene editing for human nephrons by detecting and ameliorating physiological damage to critical cell types in kidney organoids. Aim 2 is to reduce the risk of inadvertent carcinogenesis by profiling oncogenic mutations and transformation events in human kidney organoids subjected to genome editing. Finally, in Aim 3 we propose to identify potential syndromes of editing-associated nephritis by elucidating the immunogenic consequences of CRISPR-Cas9 activity in nephron compartments. Collectively, these three Aims will establish a robust and potentially high throughput framework in which to improve the safety of candidate compounds and increase the number of FDA-approved treatments for kidneys and other organs.