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

项目摘要 使用CRISPR/Cas9系统的基因组编辑允许我们产生特定的突变或纠正突变 所需的站点。在动物模型中，系统地传递CRISPR/Cas9元件提供了概念验证 基因组编辑可能被用于治疗患者的遗传病。自从一个突变基因在它的 特定的基因座可能会在组织中不加区别地进行，非靶点效应可能会导致严重的后果 比如患者的致癌作用。由于基因组的差异，给定的gRNA的非靶向效应可能是 不同物种之间差异很大，需要对人体组织进行质量控制测试。肾是 可能是最容易受到体细胞基因组编辑影响的器官之一，因为它有大量的血液流动。肾 来源于人类多能干细胞(HPSCs)的有机化合物显示出许多天然的结构特征 肾组织，包括肾小球和肾小管结构，在体外提供基于人类细胞的肾脏平台。 为了在人体细胞中开发肾组织平台，以评估体细胞基因组编辑的不利影响， 在具体目标1中，我们将确定最佳的分化和CRISPR/Cas9转导方案。然后 我们将评估通过腺病毒载体传递CRISPR/Cas9元件的编辑效果和不良反应。 相关病毒(AAV)。为了进行概念验证，我们将针对Duchenne肌营养不良症(DMD)基因， 一个流行的体细胞基因组编辑目标，因为简单地去除患病的外显子就可以纠正读数 对大多数患者来说都是相框。我们将在适合筛选的96孔和384孔培养板中产生肾脏有机化合物 优化AAV转导的实验。我们将确定每个车厢的送货效率 肾组织和评价CRISRP/Cas9基因组编辑的靶上和靶外效应 基因组测序和循环测序。此外，我们将评估AAVs和CRISPR/Cas9的毒性反应 利用我们的肾脏损伤和DNA损伤生物标记物来研究肾脏有机物中的元素。为了实现更好的模拟 利用肾脏有机化合物的药代动力学和药效学，在具体目标2中，我们将联合专业知识 在肾脏器官和微生理系统中发展可灌流的体外血管肾组织。 我们最近的合作工作表明，流体切应力有助于血管的形成 肾脏器官中的内源性祖细胞。我们将优化有机化合物的鉴别条件 利用内皮前体，设计和构建定制的生物打印芯片，用于血管形成和 肾器质受控灌流。我们将测定肾脏的血管化和功能成熟度 芯片上的有机物作为芯片上的机械提示、介质成分和潜在的细胞外信号的函数 矩阵。我们将评估血管肾有机化合物的基因编辑效率、脱靶事件和毒性。 模特们。我们提出的工作，具有良好的里程碑，将在人类细胞中提供新的体外平台 测试体细胞基因组编辑的疗效和不良反应。

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.