Vascularized kidney organoids on chip for efficacy and toxicity testing of somatic genome editing

项目摘要 使用CRISPR/Cas9系统的基因组编辑使我们能够在特定的时间产生特定的突变或纠正突变。 理想的网站。在动物模型中，CRISPR/Cas9元件的全身递送提供了概念验证， 基因组编辑可用于治疗患者的遗传疾病。自从在其第一阶段对突变基因进行修正以来， 特定位点可能在组织间不加区别地进行，脱靶效应可能导致严重后果 例如患者的致癌作用。由于基因组差异，给定gRNA的脱靶效应可能是不一致的。 在不同物种之间存在广泛差异，需要在人体组织中进行质量控制测试。肾脏是 据推测，由于其大量的血液流动，它是最容易受到体细胞基因组编辑影响的器官之一。肾 来源于人多能干细胞（hPSC）的类器官表现出在天然干细胞中发现的许多结构特征。 肾组织，包括肾小球和肾小管结构，提供了一个基于人细胞的体外肾脏平台。 为了在人类细胞中开发肾脏组织平台，以评估体细胞基因组编辑的不良影响， 在具体目标1中，我们将确定最佳分化和CRISPR/Cas9转导方案。然后 我们将评估通过腺病毒介导的CRISPR/Cas9元件的编辑效果和不良反应。 相关病毒（AAV）。为了验证概念，我们将靶向杜氏肌营养不良症（DMD）基因， 这是体细胞基因组编辑的一个流行目标，因为简单地去除患病外显子就可以纠正阅读 对于大多数患者来说。我们将在适合筛选的96孔和384孔培养板中产生肾类器官 实验以优化AAV转导。我们将确定每个隔室的输送效率， 肾组织，并通过deep-seq、全基因组测序评估CRISRP/Cas9基因组编辑的靶向和脱靶效应。 基因组测序和CIRCLE-seq.此外，我们将评估对AAV和CRISPR/Cas9的毒性反应。 通过利用我们的肾损伤和DNA损伤生物标志物，为了更好的模拟 使用肾脏类器官的药代动力学和药效学，在具体目标2中，我们将结合专业知识， 在肾类器官和微生理系统中，以在体外发育可灌注的血管化肾组织。 我们最近的合作工作表明，流体剪切应力促进血管形成， 肾类器官中的内源性祖细胞。我们将优化类器官的分化条件 与内皮前体，并设计和构建定制的生物打印芯片的血管化， 肾类器官的受控灌注。我们将确定肾脏的血管化和功能成熟 芯片上的类器官作为芯片上的机械线索、培养基组成和底层细胞外基质的函数， 矩阵我们将评估基因编辑效率，脱靶事件和血管化肾类器官的毒性 模型我们提出的工作，具有良好的里程碑，将提供新的体外人类细胞平台 来测试体细胞基因组编辑的功效和副作用。

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.