Improving the Safety of Genome Editing With Human Kidney Organoids

提高人肾类器官基因组编辑的安全性

• 批准号: 9810503
• 负责人: Benjamin Solomon Freedman
• 金额: $ 71.33万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-16 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9810503
• 关键词: AIDS-Associated Nephropathy; Acute; Acute Disease; Acute Renal Failure with Renal Papillary Necrosis; Adverse effects; Adverse event; Antigen Presentation; Architecture; Autoimmune Diseases; Biological Assay; CRISPR/Cas technology; Cell Line; Cells; Chronic; Chronic Disease; Clinical Trials; Computer Simulation; DNA Damage; DNA Sequence; Distal; Early Diagnosis; Endothelial Cells; Epithelial Cells; Event; Experimental Models; FDA approved; Fibrosis; Frequencies; Genes; Genetic Diseases; Genome; Goals; Human; Human Genome; Immune; Impairment; In Vitro; Inflammation; Inflammatory; Injury; Intervention; Kidney; Kidney Diseases; Knowledge; Lead; Life; Lupus Nephritis; Malignant Neoplasms; Mediating; Metabolism; Methods; Modeling; Modification; Monitor; Mutation; Myocardial Infarction; Nephritis; Nephrons; Oncogenic; Organ; Organoids; Outcome; Phenotype; Physiological; Pluripotent Stem Cells; Pre-Clinical Model; Production; Proliferating; Renal carcinoma; Research; Risk; Rodent Model; Safety; Structure; Syndrome; Therapeutic; Time; Tissue Model; Tissue Sample; Tubular formation; Work; adaptive immune response; adverse outcome; base; carcinogenesis; cell type; clinical application; clinical effect; cytokine; deep sequencing; design; genome editing; high throughput screening; human tissue; immunogenic; improved; in vivo; innovation; kidney cell; podocyte; preconditioning; predictive modeling; replication stress; response; side effect; specific biomarkers; therapeutic genome editing; therapy development; tumorigenesis

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.

通过人类肾脏器官提高基因组编辑的安全性 项目摘要 该项目的目的是将基因组编辑在器官培养物中应用，以建立一个预测模型 人类肾细胞类型的不良事件，包括威胁生命的急性和慢性疾病 结果。基因组编辑平台可以有效操纵特定的DNA序列 人类基因组，因此具有巨大的治疗潜力。肾脏是重要的目标 器官，有体内干预的机会以及体内。但是，缺乏知识 关于肾细胞如何对基因组编辑的反应。 已知肾脏容易受到急性毒性损伤，长期肿瘤发生以及慢性 免疫介导的反应。预测这些影响的主要障碍是缺乏人类实验 概括体内反应的模型。啮齿动物模型本质上是低通量的，并且有限 能够预测人体安全的能力，而肾细胞系过于推导，无法准确地对肾单位进行建模。 为了克服这一障碍，我们从多能干细胞中衍生出人类肾脏器官 体外的器官结构和功能替代。类器官具有肾脏的许多关键特征 肾单位，包括远端到远端排列的各种细胞类型，可以表达特定的表型 与肾脏损伤和遗传疾病有关，并且可以接受高吞吐量筛查（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 特定的，可预测的，可以在器官模型中概括，以优化其设计以确保 应用。这项工作具有重要意义，因为它将为治疗开发建立新的范式 在人类细胞中，其中类器官中的多维HTS随后进行了深度测序和详细的测序 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，因为它将揭露 我们目前不知道的基因组编辑的不利后果是通过尖端测定的 从来没有应用于类器官。类器官中的主要发现将在人肾脏中得到验证 组织样品和片上的人类肾脏。 拟议的研究将作为三个目标进行。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关键细胞类型的生理损害，用于人类肾脏 器官。 AIM 2是通过分析致癌突变和 经过基因组编辑的人类肾脏器官的转化事件。最后，在目标3中，我们建议 通过阐明了与编辑相关肾炎的潜在综合征 肾室室中的CRISPR-Cas9活性。总的来说，这三个目标将建立强大的和 潜在的高吞吐量框架，以提高候选化合物的安全性并增加 肾脏和其他器官的FDA批准治疗次数。