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满足的需求。 在新开发的基因组工程工具中，规则间隔短回文 重复（CRISPR）系统由于易于工程和可编程性而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仍面临着一些挑战。为了成功， CRISPR必须是特异性的，并且在基因组和细胞/组织中具有最小的脱靶效应。因此，在本发明中， 精确检测基因组基因座上的脱靶效应是另一个重要因素， 到目前为止相当关注。将CRISPR有效转化为人体试验需要基于人类的 这些平台可以提供有关基因组编辑的生物学后果的直接信息。最 迄今为止，研究是在细胞系或动物模型中进行的。在检查 平台中的基因编辑器可以产生人类相关的读数，使我们能够快速评估和 预测CRISPR在生理相关人类模型中的不良影响。人体器官芯片 平台可以促进对基因组编辑技术和递送系统安全性的研究， 人类我们假设芯片上的人类肝脏可以用于准确评估毒性， 基于CRISPR的基因编辑的离体脱靶活性。在aim 1中，我们将研究细胞先天性 在芯片平台上的复杂人类肝脏中对Cas9蛋白或gRNA的反应：我们假设， 芯片平台上的人肝脏可用于准确预测对Cas9蛋白的细胞应激反应， 在原代人肝细胞中的gRNA或递送载体。在目标2中，我们将检查CRISPR的脱靶活性， 芯片上的肝脏内的原代人类肝细胞：我们假设我们的芯片平台上的人类肝脏 可以用于离体检查原代肝细胞中的CRISPR脱靶切割。使用临床上 相关的肝脏基因和设计的具有不同特异性的gRNA，我们将研究CRISPR脱靶 活性，并验证肝组织中的一组转录组或分泌因子，这些因子可以作为未来的标记物， CRISPR脱靶活性的评估。在目标3中，我们将了解微环境线索的作用 在CRISPR对人类肝细胞的不利影响的芯片。我们将测试机械非刚性 基于水凝胶的支架和人类CRISPR基因编辑效率的低度炎症条件 离体形成的组织。

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.