Development of microfluidic enabled CRISPR-Cas9 functional genetic screening technologies for target discovery in cancer immunotherapy

开发微流控 CRISPR-Cas9 功能基因筛选技术，用于癌症免疫治疗中靶点发现

基本信息

批准号：
10549221
负责人：
Stephane Angers
金额：
$ 26.8万
依托单位：
NORTHWESTERN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10549221
关键词：
3D Print Anger Antibodies Benchmarking Biological Biological Assay CD47 gene CRISPR screen CRISPR/Cas technology Cancer Biology Cell Line Cell Separation Cell Surface Proteins Cell Survival Cell surface Cells Communities Data Data Set Databases Development Device or Instrument Development Devices Disease Drug Targeting Eating Ensure Enzymes Exhibits Fluorescence-Activated Cell Sorting Functional disorder Genes Genetic Genetic Screening Genotype Hour Human Biology Human Genome Immune checkpoint inhibitor Immunoglobulin Domain Immunotherapy Label Magnetism Malignant Neoplasms Microfluidic Microchips Microfluidics Modification Molecular Monitor Mutation Myeloid Cells PTPNS1 gene Pathway interactions Performance Phagocytosis Phenotype Post-Translational Protein Processing Process Proteins Recovery Regulatory Element Research Personnel Resolution Role Series Signal Pathway Signal Transduction Small Interfering RNA Sorting - Cell Movement Specificity Stimulator of Interferon Genes Stress Suppressor-Effector T-Lymphocytes Surveys System T-Cell Activation Technology Testing Therapeutic Time TimeLine Validation base cancer immunotherapy cancer therapy cancer type cell fixation cell type checkpoint therapy cost drug discovery functional genomics genome editing genome wide screen genome-wide human disease immune checkpoint instrumentation knock-down macrophage microfluidic technology molecular drug target multidisciplinary new technology new therapeutic target next generation novel novel therapeutics protein expression scale up screening targeted treatment therapeutic development tool tumor validation studies whole genome

项目摘要

Project Summary: Genome-scale genetic screens performed using CRISPR-Cas9 editing can interrogate determinants of cell viability and are powerful tools for the identification of genetic regulators. Using this technology, hundreds of millions of cells - each targeted with a specific genetic alteration - can be surveyed, typically based on a live/dead survival profiling. Phenotype-based genetic screens - where protein expression alterations are detected - represent a next-generation approach and can facilitate the identification of regulators of therapeutically-relevant proteoforms. Due to challenges related to implementation, phenotype-based screens are less commonly used compared to proliferation-based screens. Thus, rapid and robust selection approaches for targeted capture of live cells are required to realize the potential of phenotypic genome-scale screens for functional discovery, further annotation of the human genome, and discovery of novel targets for the development of therapeutics. Recently, through the use of a newly developed high-throughput approach for phenotypic CRISPR- Cas9 screening (Microfluidic Immunomagnetic Cell Sorting (MICS)) we processed an entire genome- wide screen containing more than 108 cells in under one hour to study factors that modulate the display of CD47 on the cell surface. This highly scalable cell sorting technology maintained high levels of cell viability throughout the screening process. CD47 is a widely expressed cell surface protein that acts as a “don’t eat me” signal through inhibitory interactions with SIRPa, a protein expressed on macrophages and other myeloid cells that negatively regulates phagocytosis. CD47 is highly expressed on various tumour types and blocking the CD47-SIRPa interaction has been explored as a novel cancer immunotherapy strategy that has shown promising results for some cancer types. We robustly identified modulators of CD47 function including QPCTL, an enzyme required for formation of the pyroglutamyl modification at the N-terminus of CD47 and interaction with SIRPa. The proposed study will expand the utility of the platform, develop new classes of microfluidic chips for cytometric analysis, and produce a comprehensive database of geno/phenotypic relationships. This new high-performance phenotypic assessment system will greatly accelerate target discovery for cancer therapeutics. In this project we will specifically interrogate VISTA (V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation) and cGAS/STING signaling pathways using a series of related cell lines to identify novel targets for immunotherapies. Importantly, these data will demonstrate the utility of the technology for rapid assessment of regulatory networks for drug discovery.

项目概要：使用 CRISPR-Cas9 编辑进行基因组规模的遗传筛选可以询问决定因素细胞活力，是使用该技术识别遗传调节因子的强大工具，数以亿计的细胞——每个细胞都有特定的基因改变——可以被调查，通常基于基于表型的基因筛选——其中蛋白质。检测到表达改变 - 代表下一代方法，可以促进由于与治疗相关的蛋白质形式的调节剂的鉴定。实施中，与基于增殖的筛选相比，基于表型的筛选不太常用因此，需要快速、稳健的选择方法来靶向捕获活细胞。实现表型基因组规模筛选在功能发现和进一步注释方面的潜力人类基因组的研究，以及发现治疗药物开发的新靶点。最近，通过使用新开发的高通量方法进行表型 CRISPR- Cas9 筛选（微流控免疫磁性细胞分选 (MICS)）我们处理了整个基因组 - 一小时内包含超过 108 个细胞的宽屏，用于研究调节细胞因子的因素这种高度可扩展的细胞分选技术保持了高水平的 CD47 显示。 CD47 是一种广泛表达的细胞表面，可在整个筛选过程中检测细胞活力水平。通过与 SIRPa（一种蛋白质）的抑制性相互作用，发出“别吃我”信号的蛋白质 CD47 在巨噬细胞和其他骨髓细胞上表达，对吞噬作用产生负调节。在多种肿瘤类型中高表达并阻断 CD47-SIRPa 相互作用作为一种新型癌症免疫治疗策略进行了探索，该策略已对一些人显示出有希望的结果我们可靠地鉴定了 CD47 功能的调节剂，包括 QPCTL（一种酶）。 CD47 N 末端焦谷氨酰修饰的形成以及与 SIRPa。拟议的研究将扩展该平台的实用性，开发新型微流控芯片用于细胞分析，并生成基因/表型关系的综合数据库。这种新的高性能表型评估系统将大大加速目标发现在这个项目中，我们将专门研究 VISTA（V 域）。含免疫球蛋白 (Ig) 的 T 细胞激活抑制剂）和 cGAS/STING 信号通路使用一系列相关细胞系来识别免疫疗法的新靶标。数据将证明该技术在快速评估监管网络方面的实用性药物发现。