Project Summary Genome editing using CRISPR/Cas9 systems allows us to generate specific mutations or correct mutations at desired sites. In an animal model, systemic delivery of CRISPR/Cas9 elements provided proof-of-concept that genome editing may be used to treat genetic diseases in patients. Since the correction of a mutant gene at its specific loci may be done indiscriminately across tissues, off-target effects could lead to serious consequences such as carcinogenesis in patients. Owing to genomic differences, the off-target effects of a given gRNA may be widely discrepant across species and necessitates quality control testing in human tissue. The kidney is presumably one of the most susceptible organs to somatic genome editing due to its mass blood flow. Kidney organoids derived from human pluripotent stem cells (hPSCs), exhibit many architectural features found in native kidney tissue, including glomerular and tubular structures, providing a human cell-based kidney platform in vitro. To develop kidney tissue platforms in human cells for assessment of adverse effects of somatic genome editing, in Specific Aim 1, we will determine the optimal differentiation and CRISPR/Cas9 transduction protocols. Then we will evaluate the efficacy of editing and adverse effects of delivering CRISPR/Cas9 elements via adeno- associated viruses (AAVs). For proof-of-concept, we will target the Duchenne Muscular Dystrophy (DMD) gene, a popular target for somatic genome editing since simple removal of the diseased exons can correct the reading frame for most patients. We will generate kidney organoids in 96- and 384-well culture plates suited for screening experiments to optimize AAV transduction. We will determine the delivery efficiency to each compartment of kidney tissue and evaluate on-target and off-target effects of CRISRP/Cas9 genome editing by deep-seq, whole genome sequencing, and CIRCLE-seq. Further, we will evaluate toxicity responses to AAVs and CRISPR/Cas9 elements in kidney organoids by utilizing our kidney injury and DNA damage biomarkers. For better simulation of pharmacokinetics and pharmacodynamics using kidney organoids, in Specific Aim 2, we will unite expertise in kidney organoids and microphysiological systems to develop perfusable vascularized kidney tissues in vitro. Our recent collaborative work demonstrated that fluidic shear stress facilitates vascular formation from endogenous progenitor cells in kidney organoids. We will optimize the differentiation conditions for organoids with endothelial precursors, and design and construct customized bioprinted chips for vascularization and controlled perfusion of kidney organoids. We will determine vascularization and functional maturation of kidney organoids-on-chip as a function of mechanical cues on chip, media composition, and the underlying extracellular matrix. We will evaluate gene editing efficiency, off-target events, and toxicity in vascularized kidney organoid models. Our proposed work, with well-established milestones, will provide novel in vitro platforms in human cells to test efficacy and adverse effects of somatic genome editing.

Narrative The proposed work will take advantage of the state-of-the-art technology of kidney organoids that we recently generated from human pluripotent stem cells (hPSCs) and further advance this technology toward the goal of establishing kidney tissue platforms for assessment of somatic genome editing. We will optimize kidney organoid generation and AAV transduction for assessment of adverse effects of CRISR genome editing. Further, we will incorporate the 3D-printed vascular system into kidney organoids to simulate in vivo pharmacokinetics and pharmacodynamics on chips.