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

多细胞型人肝芯片微生理平台用于检查基于 CRISPR 的基因调节

• 批准号: 10229418
• 负责人: Samira Kiani
• 金额: $ 52.63万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-05 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10229418
• 关键词: 3-Dimensional; Adopted; Adverse effects; Animal Model; Animals; Attention; Biochemical; Biological; Biological Models; CRISPR/Cas technology; Cell Line; Cells; Cellular Stress; Cellular Stress Response; Chromatin; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Communities; Complex; Cues; DNA; Detection; Disease model; Effectiveness; Engineering; Face; Future; Genes; Genetic Transcription; Genome; Genome engineering; Goals; Guide RNA; Hepatic; Hepatocyte; Hereditary Disease; Human; Human Cell Line; Hydrogels; In Vitro; Inflammation; Inflammatory; Innate Immune Response; Kupffer Cells; Lead; Libraries; Liquid substance; Liver; Maintenance; Mechanics; Medical; Metabolic Diseases; Microfabrication; Microfluidics; Modality; Mus; Oxygen; Physiological; Proteins; Reporting; Role; Safety; Specificity; Streptococcus pyogenes; System; Technology; Testing; Tissues; Toxic effect; Translations; adaptive immune response; base; biological adaptation to stress; biomarker evaluation; capillary bed; cell type; clinically relevant; cost; design; detection method; dosage; falls; gene therapy; genome editing; genome-wide; genomic locus; human model; human tissue; mechanical force; microphysiology system; organ on a chip; predicting response; premature; programs; response; safety and feasibility; scaffold; screening; shear stress; three dimensional structure; tool; transcriptomics; vector

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.

基因组工程技术能够解决基因治疗领域许多未满足的需求。 在新开发的基因组工程工具中，簇状规则间隔短回文 由于易于工程化和可编程性，重复 (CRISPR) 系统彻底改变了基因组编辑。 尽管潜力巨大，用于基因治疗的 CRISPR 仍面临着一些挑战。要想成功， 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。像这样， 精确检测基因组位点的脱靶效应是已收到的另一个重要因素 迄今为止受到相当大的关注。将 CRISPR 有效转化为人体试验需要以人为基础 可以提供有关基因组编辑生物学后果的直接信息的平台。最多 迄今为止，研究都是在细胞系或动物模型中进行的。审查工作进展甚微 平台中的基因编辑器可以产生与人类相关的读数，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利影响。人体器官芯片 平台可以促进基因组编辑技术和传递系统的安全性研究 人类。我们假设芯片上的人类肝脏可以用于准确评估毒性和 基于 CRISPR 的基因编辑离体脱靶活性。在目标 1 中，我们将研究细胞先天的 芯片平台上复杂人类肝脏中 Cas9 蛋白或 gRNA 的响应：我们假设 芯片平台上的人类肝脏可用于准确预测 Cas9 蛋白的细胞应激反应， 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 脱靶活性 芯片上肝脏内的原代人类肝细胞：我们假设芯片平台上的人类肝脏 可用于离体检查原代肝细胞中的 CRISPR 脱靶切割。使用临床图书馆 相关肝脏基因并设计具有不同特异性的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 芯片中 CRISPR 对人类肝细胞的不利影响。我们将测试机械非刚性的影响 基于水凝胶的支架和低度炎症条件对人类 CRISPR 基因编辑效率的影响 离体形成的组织。