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

基本信息

批准号：
10407081
负责人：
Benjamin Solomon Freedman
金额：
$ 65.28万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-16 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10407081
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 Models 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 Injury to Kidney 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

项目摘要

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.

提高人类肾脏类器官基因组编辑的安全性项目概要该项目的目标是在类器官培养中应用基因组编辑器，建立预测模型人类肾细胞类型的不良事件，包括危及生命的急性和慢性疾病结果。基因组编辑平台能够有效地操纵特定的 DNA 序列人类基因组，因此具有巨大的治疗潜力。肾脏是重要目标器官，有机会进行体内和离体干预。但知识还是有欠缺关于肾细胞如何响应基因组编辑。众所周知，肾脏容易受到急性毒性损伤、长期肿瘤发生以及慢性损伤的影响。免疫介导的反应。预测这些影响的一个主要障碍是缺乏人体实验重现体内反应的模型。啮齿动物模型本质上是低通量的，并且具有有限的预测人类安全的能力，而肾细胞系去分化程度太高，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提取了人肾类器官作为体外器官结构和功能的替代物。类器官具有肾脏的许多关键特征肾单位，包括从远端到近端排列的多种细胞类型，可以表达特定的表型与肾损伤和遗传疾病有关，并且适合高通量筛查（HTS）。根据我们的初步工作，我们假设基因编辑会对肾脏产生有害影响是具体的、可预测的，并且可以在类器官模型中重述，以优化其设计以实现安全应用。这项工作具有重要意义，因为它将为治疗发展建立新的范式在人类细胞中，其中对类器官进行多维 HTS，然后进行深度测序和详细分析确定最安全和最有前途的候选人。它具有高度创新性，因为它将揭示我们目前还没有意识到基因组编辑的不利后果，通过尖端检测以前从未应用于类器官。类器官的重要发现将在人类肾脏中得到验证组织样本和人类肾脏芯片。拟议的研究将作为三个目标进行。第一个目标是增强基因编辑的安全性通过检测和改善肾脏关键细胞类型的生理损伤来治疗人类肾单位类器官。目标 2 是通过分析致癌突变和降低无意致癌的风险进行基因组编辑的人类肾脏类器官的转化事件。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识别编辑相关性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有力的、潜在的高通量框架，可提高候选化合物的安全性并提高 FDA 批准的肾脏和其他器官治疗方法的数量。