PROJECT SUMMARY Genome editing technologies are advancing rapidly toward clinical therapies, but robust methods for accurately assessing the potential adverse effects of genome editing activity and delivery vehicles on physiological tissue function have yet to be developed and broadly disseminated. Since animal models are often poor predictors of human biological responses, it is imperative to establish well-defined tissue systems composed of human cells as an intermediate testbed to assess the safety as well as the efficacy of genome editing and its effect(s) on tissue function. Human pluripotent and post-natal tissue-derived stem cells provide valuable sources of human differentiated cells, such as cardiomyocytes, neurons and hepatobiliary cells, that can be used to create 3D tissues to model diseases and test therapies ex vivo. Traditionally, the toxicity of novel therapies in the heart, nervous system and liver manifest with severe consequences that can often lead to organ failure and mortality. For this reason, a thorough preclinical characterization of potential toxicities and adverse events caused by genome editing in human microtissues that recapitulate critical physiologic functions will be essential for these therapies to be ultimately translated for clinical use. The primary objective of this proposal is to develop and validate human tissue platforms capable of sensitively and accurately detecting adverse effects of genome editing on physiologic tissue function. To achieve this objective, we have established a multi-disciplinary team of leading investigators with complementary expertise in tissue engineering, genome editing, stem cell biology, single cell genomics and technology development. We will pursue this overall goal through three projects in parallel that focus initially on the development of individual microtissue platforms in concert with a specific genome editing strategy before proceeding to testing each of the editing scenarios on all three of the tissue systems. In the first project, we will examine the effects of single-site editing on defined off-target sites and endogenous loci on cardiac microtissue electrical and mechanical function. In the second project, we will examine large scale genomic alterations following deletion of variable size fragments in neurons and the effects on physiologic parameters. In the third project, we will assess biomarker expression and secretion by hepatobiliary microtissues following the genomic insertion of exogenous genes. These human tissue models tested with various clinical genome editing strategies will be integrated within the Somatic Gene Editing Consortium, to test novel delivery vehicles and help inform the development of future genome editing therapies. Altogether, the outcomes of this work should significantly benefit the safety and predictability of curative genome therapies of the future.

PROJECT NARRATIVE Genome editing technologies are poised to revolutionize the practice of modern medicine for the treatment of various types of genetic diseases. However, reliable testbed systems using human cells and tissues are needed to accurately predict both intended and unintended consequences of therapeutic interventions. The primary objective of this proposal is to develop and validate human tissue models capable of sensitively and accurately detecting adverse effects of genome editing on physiologic tissue function.