Multicell type human liver on chip microphysiological platform to examine CRISPR based gene modulation

基因组工程技术有能力解决基因治疗领域中许多未得到满足的需求。 在新开发的基因组工程工具中，成簇的规则间隔短回文 由于易于设计和可编程，重复(CRISPR)系统已经彻底改变了基因组编辑。 尽管CRISPR具有巨大的潜力，但它在基因治疗方面面临着几个挑战。要想成功， CRISPR必须是特定的，并且在基因组和细胞/组织中都具有最小的偏离目标的影响。因此， 对基因组基因座上的非靶点效应的精确检测是另一个重要的因素 到目前为止，受到了相当大的关注。CRISPR向人体试验的高效转换需要以人为基础 可以提供有关基因组编辑的生物学后果的直接信息的平台。多数 到目前为止，研究都是在细胞系或动物模型中进行的。在检查方面进展甚微 平台中的基因编辑器可以产生与人类相关的读数，并使我们能够快速评估和 在生理相关的人体模型中预测CRISPR的不良影响。片上人体器官 平台可以促进对基因组编辑技术和传递系统的安全性的研究 人类。我们假设，可以采用芯片上的人体肝脏来准确评估毒性和 基于CRISPR的基因编辑的体外靶外活性。在目标1中，我们将研究细胞先天 在芯片平台上对复杂人类肝脏中Cas9蛋白或gRNA的响应：我们假设 芯片平台上的人肝可以用来准确地预测细胞对Cas9蛋白的应激反应， 原代人肝细胞中的gRNA或递送载体。在目标2中，我们将检查CRISPR的非目标活动 芯片上的肝脏中的原代人类肝细胞：我们假设我们的人体肝脏在芯片平台上 可用于检测CRISPR在体外对原代肝细胞的靶向切割。使用临床资料库 相关的肝脏基因和设计的具有不同特异性的gRNAs，我们将研究CRISPR脱靶 并验证肝组织中一组转录或分泌因子，这些因子可以作为未来的标志物 对CRISPR脱离目标活动的评估。在目标3中，我们将了解微环境线索的作用 在CRISPR中对人体肝细胞产生不良影响的一种芯片。我们将测试机械非刚性的冲击力 水凝胶支架和低度炎症条件对人CRISPR基因编辑效率的影响 组织在体外形成。

Genome engineering technology has the capability of resolving many unmet needs in the field of gene therapy. Among the newly developed genome engineering tools, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) System has revolutionized genome editing due to ease of engineering and programmability. Despite its tremendous potential, CRISPR for gene therapies faces several challenges. To be successful, CRISPR must be specific and have minimal off-target effects both in genome and cells/tissues. As such, precise detection of off-target effects on the genomic loci is another important factor that has received considerable attention so far. Efficient translation of CRISPR to human trials requires human-based platforms that can provide direct information about the biological consequences of genome editing. Most studies so far have been conducted in cell lines or animal models. Little progress has been made to examine the gene editors in platforms that can yield human relevant readouts and enable us to rapidly assess and predict adverse effects of CRISPR in physiologically relevant human models. Human organ on-a-chip platforms can facilitate studies on the safety of genome editing technologies and delivery systems in human. We hypothesize that human liver on a chip can be adopted for accurate assessment of toxicity and off-target activity of CRISPR-based gene editing ex vivo. In aim 1 we will investigate cellular innate response to Cas9 protein or gRNA in a complex human liver on a chip platform: We hypothesize that human liver on a chip platform can be employed to accurately predict cellular stress response to Cas9 protein, gRNA or delivery vehicles in primary human liver cells. In aim 2 we will examine CRISPR off-target activity in primary human liver cells within liver on a chip: We hypothesize that our human liver on a chip platform can be used to examine CRISPR off target cleavage in primary liver cells ex-vivo. Using a library of clinically relevant liver genes and designed gRNAs with different specificities, we will investigate CRISPR off-target activity and verify a set of transcriptomic or secretory factors in liver tissue that can serve as future markers for evaluation of CRISPR off target activity. In aim 3, we will understand the role of micro-environmental cues in CRISPR adverse effect on human liver cells in a chip. We will test the impact of mechanically non-rigid hydrogel-based scaffolds and low-grade inflammatory conditions in CRISPR gene editing efficiency in human tissue formed ex vivo.