Rapidly scalable platforms for direct in vivo screening of functional drivers in lethal cancers

可快速扩展的平台，用于直接体内筛选致命癌症的功能驱动因素

• 批准号: 9902374
• 负责人: Sidi Chen
• 金额: $ 40.7万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9902374
• 关键词: Advanced Development; Animals; Bar Codes; Brain Neoplasms; CRISPR screen; CTLA4 gene; Cancer Model; Cause of Death; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complement; Complex; Data; Development; FDA approved; Genes; Genetic; Genetic Models; Genetic Screening; Genetically Engineered Mouse; Genome; Glioblastoma; Goals; Heterogeneity; Histopathology; Human; Immunocompetent; Immunotherapy; Lentivirus Vector; Libraries; Life; Link; Liver; Malignant Neoplasms; Maps; Measurement; Mediating; Methods; Modeling; Molecular; Mutation; Names; Nature; Neurosciences; Normal Cell; Oncology; Organ; Outcome; PIK3CG gene; Pathology; Patients; Penetrance; Performance; Pharmaceutical Preparations; Phenotype; Preclinical Testing; Primary carcinoma of the liver cells; Protocols documentation; Resource Sharing; Savings; Science; Study models; System; Technology; Testing; Therapeutic; Tissue Engineering; Tissues; Validation; Variant; anticancer research; base; behavioral study; cancer genome; cancer type; causal variant; clinically relevant; cohort; combinatorial; comparative; flexibility; genetic variant; high throughput screening; human disease; immune checkpoint blockade; improved; in vivo; insight; mutant; new technology; novel; novel strategies; pre-clinical; preservation; profiles in patients; programmed cell death protein 1; promoter; response; screening; sequencing platform; standard of care; tool; translational cancer research; treatment response; trend; tumor; tumor heterogeneity; tumor immunology; tumor initiation; tumor microenvironment; tumor-immune system interactions; tumorigenesis

Project Summary: Cancer is a major cause of death worldwide with outstanding challenges for a cure. Such challenges are primarily due to the nature of tumor heterogeneity and evolvability. Thus, the ability to generate unbiased, quantitative and causal maps of functional drivers and their combinations in native tumor microenvironment is a key to accelerate therapeutic discovery. To date, little has been done to comprehensively and combinatorially test which of the mutations identified in human patients can indeed functionally drive tumorigenesis of normal cells in native organs. The major barriers include accurate delivery, precise genome manipulation, efficient massively parallel perturbation, and unbiased, high-sensitivity quantitative readout, all of which have to be achieved simultaneously in the native tissue microenvironment. We recently established a novel approach named Pooled AAV Screen with Targeted Amplicon Sequencing (PASTAS) for direct in vivo screening of causative cancer drivers and combinations. This method generates precision models of cancer that (1) spontaneously develop from tumor-originating cells in the native organ microenvironment, (2) develop in fully immunocompetent animals and preserve the immune microenvironment, (3) genetically mimic significant mutations found in patients, (4) closely mimic the histopathology of human disease and clinical features, (5) encompass high degree of genetic and cellular heterogeneity, (6) offer flexibility to target any choice of target genes and rapidly scalable as pooled mutant screens, and (7) is easy to use by the community. In this study, we will conduct advanced development, robust validation and full establishment of this screening system. We will first establish technical parameters for optimal performance of this technology by quantitative measurements using independent patient cohorts with two lethal cancer types: glioblastoma and liver hepatocellular carcinoma. Then, we will extend the utility for causative driver discovery in therapeutic settings. Finally, we will advance the development of a lentiviral vector-based orthogonal approach to open up larger screening capabilities. Such screening systems and models will enable rapid identification of causative factors that directly drive transformation of healthy cells, tumor initiation, progression and therapeutic responses to treatments. More importantly, compared to existing alternatives, the fully immunocompetent setting allows robust pre-clinical testing of immunotherapies, in genetically matched animal avatars, as well as screening for genes that modulate the response to these therapies. Outcome and impact: This R33 will deliver optimized and validated PASTAS / PLeSTASS systems to link causative genes to oncogenesis in native TME; to enable autochthonous immunotherapy screen in fully immunocompetent setting for identification of targets that modulate the response to these life-saving drugs; and to share resources and protocols for the community to collectively yield novel and far-reaching insights in oncology.

项目概述：癌症是全球主要的死亡原因，治疗面临着巨大的挑战。等 挑战主要是由于肿瘤异质性和可进化性的性质。因此， 天然肿瘤中功能驱动因子及其组合的无偏、定量和因果图 微环境是加速治疗发现的关键。迄今为止， 全面和组合地测试在人类患者中鉴定的哪些突变确实可以 在功能上驱动天然器官中正常细胞的肿瘤发生。主要障碍包括准确交付， 精确的基因组操作，高效的大规模并行扰动，以及无偏的高灵敏度 定量读出，所有这些都必须在天然组织微环境中同时实现。 我们最近建立了一种新的方法，称为Pooled AAV Screen with Targeted Amplicon Sequencing （PASTAS）用于直接体内筛选致病性癌症驱动因子和组合。该方法产生 精确的癌症模型，（1）从天然器官中的肿瘤起源细胞自发发展 （2）在完全免疫活性动物中发育并保护免疫微环境， (3)在基因上模拟在患者中发现的显著突变，（4）密切模拟人类的组织病理学， 疾病和临床特征，（5）包括高度的遗传和细胞异质性，（6）提供 靶向任何选择的靶基因的灵活性和作为合并的突变体筛选的快速可扩展性，以及（7）易于 由社区使用。在这项研究中，我们将进行先进的开发，强大的验证和全面的 建立这个筛选系统。我们将首先建立技术参数， 该技术通过使用具有两种致命癌症类型的独立患者队列进行定量测量： 胶质母细胞瘤和肝细胞癌。然后，我们将扩展该实用程序，用于发现因果驱动程序 在治疗环境中。最后，我们将推进基于慢病毒载体的正交试验的开发。 方法来打开更大的筛选能力。这种筛选系统和模型将使快速 鉴定直接驱动健康细胞转化、肿瘤起始、进展的致病因素 和对治疗的治疗反应。更重要的是，与现有的替代品相比， 免疫活性环境允许在基因匹配动物中进行免疫疗法的稳健的临床前测试 化身，以及筛选调节对这些疗法的反应的基因。成果和影响： 该R33将提供优化和验证的PASTAS / PLeSTASS系统，以将致病基因与 天然TME中的肿瘤发生;在完全免疫活性环境中进行自体免疫治疗筛选 确定调节对这些救命药物反应的靶点;分享资源， 为社区提供协议，共同产生肿瘤学方面新颖而深远的见解。