Improving the Safety of Genome Editing With Human Kidney Organoids

提高人肾类有机化合物基因组编辑的安全性 项目总结 该项目的目标是将基因组编辑应用于器官培养，以建立一个预测模型 人类肾脏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。基因组编辑平台能够有效地操作特定的DNA序列 人类基因组，因此具有巨大的治疗潜力。肾是重要靶点 器官，有机会进行体内和体外干预。然而，知识的匮乏。 关于肾脏细胞对基因组编辑的反应。 众所周知，肾脏易受急性毒性损伤、长期肿瘤形成以及慢性毒性损伤的影响。 免疫调节的反应。预测这些影响的一个主要障碍是缺乏人体实验 概括体内反应的模型。啮齿动物模型天生就是低吞吐量的，而且 预测人类安全性的能力，而肾细胞系去分化程度太高，无法准确地模拟肾单位。 为了克服这一障碍，我们从多能干细胞中提取了人类肾脏有机化合物作为 体外器官结构和功能的替代物。有机化合物具有肾脏的许多关键特征。 肾单位，包括远近端排列的不同类型的细胞，可以表达特定的表型。 与肾脏损伤和遗传疾病有关，并适用于高通量筛查(HTS)。 根据我们的初步工作，我们假设基因编辑将对肾脏产生有害影响。 它们是特定的、可预测的，并且可以在有机模型中重现，以优化其设计以确保安全 申请。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，其中多维HTS在有机物中随后进行深度测序和详细 分析确定了最安全、最有前途的候选人。它具有很高的创新性，因为它将揭示 我们目前还不知道的基因组编辑的不利后果，通过尖端的分析 以前从未被应用于有机化合物。有机类化合物的关键发现将在人类肾脏中得到验证 组织样本和芯片上的人体肾脏。 拟议的研究将以三个目标进行。第一个目标是提高基因编辑的安全性 通过检测和改善对肾脏关键细胞类型的生理性损伤来治疗人肾单位 有机化合物。目标2是通过分析致癌基因突变和 人类肾脏器官中基因组编辑后的转化事件。最后，在目标3中，我们建议 通过阐明编辑相关性肾炎的免疫原性后果来确定潜在的综合征 肾单位室的CRISPR-Cas9活性。这三个目标加在一起，将建立一个强有力的 潜在的高通量框架，在该框架中，提高候选化合物的安全性并增加 FDA批准的肾脏和其他器官治疗的数量。

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.