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: 10459430
• 负责人: Samira Kiani
• 金额: $ 52.19万
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10459430
• 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; delivery vehicle; 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

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.

基因组工程技术具有解决基因治疗领域许多未满足需求的能力。 在新开发的基因组工程工具中，群集定期插入了空格短的palindromic 重复（CRISPR）系统由于易于工程性和可编程性而改变了基因组编辑。 尽管具有巨大的潜力，但基因疗法的CRISPR仍面临一些挑战。要成功， CRISPR必须是特定的，并且在基因组和细胞/组织中具有最小的脱靶效应。像这样， 对基因组基因概念的脱靶影响的精确检测是接受的另一个重要因素 到目前为止，非常关注。将CRISPR的有效翻译为人类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 到目前为止，已经在细胞系或动物模型中进行了研究。几乎没有进步来检查 平台中的基因编辑器可以产生人类相关的读数并使我们能够快速评估和 预测CRISPR在生理相关的人类模型中的不利影响。人体器官片 平台可以促进有关基因组编辑技术和输送系统安全性的研究 人类。我们假设可以采用芯片上的人肝脏来准确评估毒性和 基于CRISPR的基因编辑的非目标活动。在AIM 1中，我们将研究蜂窝先天 在芯片平台上对复杂人肝脏中Cas9蛋白或GRNA的反应：我们假设 可以使用芯片平台上的人肝脏来准确预测Cas9蛋白的细胞应力反应， 原代人肝细胞中的GRNA或递送车。在AIM 2中，我们将检查CRISPR非目标活动 肝脏中肝内的原发性人肝细胞：我们假设我们的人类肝脏在芯片平台上 可用于检查原发性肝细胞的靶裂解。使用临床上的库 相关的肝脏基因并设计具有不同特异性的GRNA，我们将研究CRISPR脱离目标 活动并验证肝组织中的一组转录组或分泌因素，这些因素可以作为未来标记 评估CRISPR关闭目标活动。在AIM 3中，我们将了解微环境提示的作用 在CRISPR对芯片中人肝细胞的不利影响中。我们将测试机械非刚性的影响 人类CRISPR基因编辑效率的基于水凝胶的脚手架和低度炎症条件 组织形成的离体。