(PQ4) Novel tools for in vivo study of genetic interactions in cancer progression

(PQ4) 用于体内研究癌症进展中遗传相互作用的新工具

基本信息

批准号：
10599597
负责人：
Sidi Chen
金额：
$ 12.27万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10599597
关键词：
Address Alleles Award Brain Cancer Hospital Cancer Model Cancer Patient Cell Line Cells Companions Complex Data Set Development Disease Progression Drug resistance Ensure Environment Event Evolution Genes Genetic Genetic study Genetically Engineered Mouse Genomics Goals Guide RNA Health Heterogeneity Human Joints Knock-out Liver Malignant Neoplasms Malignant neoplasm of lung Mammalian Cell Mediating Mentors Mentorship Methodology Modeling Molecular Mouse Strains Mus Mutagenesis Neoplasm Metastasis Oncogenes Oncogenic Organ Orthologous Gene Outcome Parents Pathologic Phenotype Physiological Play Process Regulatory Element Relapse Research Research Personnel Research Project Grants Research Training Retroviral Vector Role Sampling Scientist Specificity System Testing Training Transgenic Founder Mouse Transgenic Mice Tumor Suppressor Proteins Underrepresented Minority Validation Viral Vector Work Yale Cancer Center base career development causal variant clinically relevant computational pipelines design drug sensitivity endonuclease flexibility genome editing high throughput screening human disease human model in vivo in vivo Model lung tumorigenesis minority trainee mouse model novel parent project preclinical study screening success tool transplant model tumor tumor progression tumorigenesis vector

项目摘要

PROJECT SUMMARY: The evolution of human cancer is a complex process driven by multiple molecular and cellular events. We endeavored to harness single-effector RNA-guided endonucleases (RGNs) for genome editing, parallel screening and in vivo modeling of human cancer. Recently, we generated a platform to systematically interrogate several hundred loci directly in vivo. To overcome current limitations in multigene editing and achieve more accurate control of simultaneity and sequentiality of multi-allelic tumor modeling, we utilized Cpf1, an RGN that can edit its target simply with crRNAs independent of tracrRNA thus allowing simultaneous editing of multiple genes with a single crRNA array. We developed a preliminary Cpf1-based crRNA array screening (CCAS) system in mammalian cells, and applied it in mouse models of progression and metastasis. In our first aim, we will perform validation and optimization of CCAS for in vivo double-knockout phenotyping of cancer co-drivers. We will establish its technical rigor, efficiency and specificity for simultaneous editing, as well as developing a set of computational pipelines for accurate calling of statistically significant gene pairs. We will apply this approach to study the genetic interactions of tumor suppressors found in lung cancer patients at Yale Cancer Center and Hospital, and identify potential co-drivers of metastasis to vital organs. In the second aim, we will carry out validation and optimization of a Cpf1-Flip system for sequential mutagenesis of cancer targets. We will demonstrate its broader applicability by testing clinically relevant gene sets identified from public studies of the genomics of metastasis as well as a large multi-sample metastasis dataset gathered on Yale cancer patients. We will then apply this methodology as an unbiased depletion screen to identify targets that are essential for survival in specific oncogenic backgrounds. We will develop novel versatile transgenic mouse strains and companion viral vectors for direct modeling of multigenic tumorigenesis in mice. We will combine these tools to enable high-throughput genetic interaction screening in healthy cells directly in the native organ to identify causative mutation pairs that drive tumorigenesis. We anticipate that developing and establishing these tools will transform multigenic tumor modeling and pre-clinical studies of human cancer, directly addressing NCI Provocative Question 4. These powerful toolkits will enable scientists to target any gene pairs or combinations simultaneously or sequentially, assessing the phenotypic outcome of their in vivo interactions in tumor progression, metastasis, synthetic lethality, drug sensitivity or other processes in cancer evolution. This supplement’s goal is to (1) extend and strengthen the existing research described in Aim 3; and to (2) promote diversity in health-related research by involving an under-represented minority trainee in the project. This will mostly involve Aim 3 of the parent award due to the stage of this project.

项目摘要：人类癌症的进化是由多个分子和细胞事件驱动的复杂过程。我们努力对线束单作用RNA引导的内切核酸酶（RGN）进行基因组编辑，平行筛查和体内人类癌症模型。最近，我们生成了一个系统的平台直接在体内询问数百个基因座。要克服当前的多基因编辑和实现对多行肿瘤建模的同时性和顺序性的更准确控制，我们利用了 CPF1，可以简单地用独立于tracrrna的CRRNA编辑目标的RGN，从而允许简单具有单个CRRNA阵列的多个基因的编辑。我们开发了基于CPF1的初步CRRNA阵列哺乳动物细胞中的筛选（CCAS）系统，并将其应用于进展和转移的小鼠模型中。在我们的第一个目标中，我们将对体内双敲击表型进行验证和优化癌症共同司机。我们将建立其技术的严格，效率和特殊性，以简单编辑，作为以及开发一组计算管道，以准确调用统计学上的重要基因对。我们将采用这种方法研究在肺癌患者中发现的肿瘤补充剂的遗传相互作用耶鲁大学癌症中心和医院，并确定转移的潜在共同驾驶到重要器官。在第二个目的，我们将对癌症的顺序诱变进行验证和优化CPF1-FLIP系统目标。我们将通过测试从临床相关的基因集来证明其更广泛的适用性关于转移基因组学以及大型多样本转移数据集的公共研究耶鲁癌症患者。然后，我们将将这种方法应用于公正的部署屏幕以识别目标这对于在特定的致癌背景中生存至关重要。我们将开发新颖的多功能转基因小鼠菌株和伴随病毒载体，用于直接建模小鼠多基因肿瘤。我们将结合这些工具以使健康细胞中的高通量遗传相互作用筛查直接在天然器官以识别驱动肿瘤发生的致病突变对。我们预计这是发展的建立这些工具将改变人类癌症的多基因肿瘤建模和临床前研究，直接解决NCI挑衅性问题4。这些强大的工具包将使科学家能够针对任何基因对或组合简单或依次，评估其体内的表型结果肿瘤进展，转移，合成致死性，药物敏感性或癌症的其他过程中的相互作用进化。该补充的目标是（1）扩展和加强AIM 3中描述的现有研究；和（2）通过参与代表性不足的少数学员，促进与健康相关的研究的多样性项目。由于该项目的阶段，这主要涉及父母奖的目标3。