PROJECT SUMMARY The studies proposed in this application will advance in vitro safety and efficiency testing for somatic cell genome editing in human cells in 3D organoid models. For genome editing, human organoids have the potential to be an ideal tool, as the therapeutic target is the human genome, which cannot be replicated in any other species. Organoids also have advantages for throughput and predictivity of human side effects. It is important, however, to test the utility and value of organoids in this context, for this to be demonstrated as an enabling technology for investigational new drugs. To achieve this, we will produce a set of diverse organoids representing human kidney, liver, brain, lung, retina, and/or heart as vital organ systems of great interest to gene editing applications. For each organ lineage in our 'body in a dish', we will demonstrate assays to measure editing rates as well as side effects. These assays will be optimized to establish reference standards with quantifiable measurements of assay stability, reproducibility, and analytical range. Organoid datasets will be compared with datasets produced in parallel efforts by collaborating teams using similar gene editing technologies. The objective is to demonstrate safety and efficiency assays in human organoid cultures in conjunction with complementary assessments in other systems as a tractable paradigm to support the advancement of genome editing therapeutics to human clinical trials. To maximize impact, we will focus on assays that will be broadly useful for a wide variety of genome editing therapeutics, in multiple organ systems. Organoids derived from human pluripotent stem cells possess many key features of tissues, including diverse cell types in sophisticated arrangements, and express specific disease phenotypes associated with rare populations. For regulatory consideration, there is a critical need to determine their fidelity and prediction capacity. In these studies, we will demonstrate concordance and synergy between human organoids and other preclinical models. Thus, the Specific Aim proposed is to de-risk therapeutic genome editing approaches by assessing dose-dependent efficiency with adverse events in human organoids. Collectively, these studies will produce models of genome editing in human organoids with outcomes that can be compared to orthologous models to establish a regulatory paradigm which can be applied to a range of tissues and diseases.

PROJECT NARRATIVE To enable safe and efficient advancement of genome editing therapeutics to the human clinical setting, it is essential to obtain preclinical data supporting safety and efficiency, but simple cell cultures and animal models have substantial limitations in predicting outcome in human tissues. Human organoids are 'mini-organ'-like structures grown in lab dishes that possess many key features of tissues, including diverse cell types in sophisticated arrangements specific to different organs, and disease phenotypes specific to individuals from whom they are derived. The objective of these studies is to blueprint a new predictive approach for genome editing efficiency and safety in human organoids, which can be used to de-risk strategies for regulatory consideration relevant to a wide variety of genome editing therapies.