In vivo regulators of TERT promoter mutant glioblastoma

TERT启动子突变胶质母细胞瘤的体内调节因子

基本信息

批准号：
9918280
负责人：
ALEXANDRA M AMEN
金额：
$ 4.89万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
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：
2018-06-01 至 2021-02-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9918280
关键词：
Adult Affect Apoptosis Binding Binding Proteins Biological Brain CRISPR interference CRISPR/Cas technology Cancer Cell Growth Cell Line Cell Survival Cell division Cell model Cells Chromosomes Clustered Regularly Interspaced Short Palindromic Repeats Complex DNA Data Development Disease Engineering Future Gene Expression Gene Expression Profiling Gene Silencing Gene Targeting Genes Genetic Genomics Glioblastoma Glioma Goals Growth Human In Vitro Individual Knock-out Libraries Malignant Neoplasms Malignant neoplasm of brain Mediating Methods Modeling Molecular Abnormality Mus Mutation Normal Cell Pathway interactions Patients Phenotype Plasmid Cloning Vector Process Promoter Regions Protein Isoforms RNA-Directed DNA Polymerase Regulation Regulator Genes Research Ribonucleoproteins Sampling Somatic Cell Specificity System Technology Telomerase Telomere Shortening Therapeutic Time Toxic effect Tumor Cell Line Viral Vector Work Xenograft Model Xenograft procedure base cancer cell cancer subtypes cancer type cell growth design druggable target gene function genome editing high throughput screening in vivo interest knock-down mutant neoplastic cell new therapeutic target promoter receptor screening senescence small molecule small molecule inhibitor stem cells telomere therapeutic development therapeutic target transcription factor tumor tumor growth uptake

项目摘要

Project Summary/Abstract A crucial step in the development of human cancer is the ability of cells to undergo immortalization by avoiding senescence and apoptosis that result from repeated cell divisions. To accomplish this, 90% of cancers reactivate telomerase reverse transcriptase (TERT), the catalytically active and rate-limiting subunit of telomerase. Telomerase functions to maintain telomeres, which cap the ends of chromosomes, protecting chromosomal DNA from the end replication problem. Normally, TERT is expressed in stem cells, but is transcriptionally silenced in somatic cells. However, recent work has demonstrated that many cancer subtypes, including 83% of primary glioblastoma (GBM) tumors, contain activating mutations in the TERT promoter. These mutations result in binding of the transcription factor GA binding protein (GABP), and reactivation of TERT, through incompletely understood mechanisms. Understanding and targeting genes and pathways related to these phenomena has the potential to open major future therapeutic avenues for GBM, the most common and most severe form of adult brain cancer. The current proposal will make use of a focused in vivo CRISPRi growth-based screen using an intracranial xenograft model of GBM to uncover factors that regulate immortality of TERT promoter mutant GBM cells. The targeted screen will be performed in both control and GABP mutant cell lines, in order to elucidate genes that function both synergistically with and independently of GABP. In parallel, the proposal aims to engineer Cas9 ribonucleoprotein (RNP) complexes that target GBM cells specifically, using receptor-mediated uptake. Cas9 RNP complexes are currently being developed with future therapeutic intent, given the tendency for reduced toxicity and off-target effects as compared to other methods of CRISPR editing. These GBM-specific RNPs will be designed with the ultimate goal of editing genes to reverse cellular immortality in TERT promoter mutant GBM cells, both in vitro and in vivo. Overall, this work will develop CRISPR-based technologies for both in vivo screening and targeting of GBM cells, with the goal of elucidating and inhibiting major factors that underlie GBM cellular immortality.

项目概要/摘要人类癌症发展的一个关键步骤是细胞通过避免永生化的能力细胞反复分裂导致的衰老和凋亡。为了实现这一目标，90%的癌症重新激活端粒酶逆转录酶 (TERT)，这是端粒酶的催化活性和限速亚基端粒酶。端粒酶的功能是维持端粒，端粒覆盖染色体末端，保护染色体DNA的末端复制问题。正常情况下，TERT 在干细胞中表达，但体细胞中的转录沉默。然而，最近的研究表明，许多癌症亚型，包括 83% 的原发性胶质母细胞瘤 (GBM) 肿瘤，含有 TERT 启动子的激活突变。这些突变导致转录因子 GA 结合蛋白 (GABP) 的结合，并重新激活 TERT，通过不完全了解的机制。了解和靶向基因和通路与这些现象相关的研究有可能为 GBM 开辟主要的未来治疗途径，成人脑癌的常见且最严重的形式。当前的提案将利用集中的体内使用 GBM 颅内异种移植模型进行基于 CRISPRi 生长的筛选，以揭示调节因子 TERT启动子突变的GBM细胞的永生性。目标屏幕将在控制和 GABP突变细胞系，以阐明与协同或独立发挥作用的基因 GABP。与此同时，该提案旨在设计针对 GBM 的 Cas9 核糖核蛋白 (RNP) 复合物特别是细胞，利用受体介导的摄取。 Cas9 RNP 复合物目前正在开发中考虑到与其他药物相比，毒性和脱靶效应降低的趋势，未来的治疗意图 CRISPR 编辑方法。这些 GBM 特异性 RNP 的设计最终目标是编辑基因在体外和体内逆转 TERT 启动子突变 GBM 细胞的细胞永生性。总的来说，这部作品将开发基于 CRISPR 的技术，用于 GBM 细胞的体内筛选和靶向，目标是阐明和抑制 GBM 细胞永生的主要因素。