Improving the Safety of Genome Editing With Human Kidney Organoids

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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.