In vivo regulators of TERT promoter mutant glioblastoma

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死亡而进行永生化的能力。 衰老和细胞凋亡是由细胞反复分裂引起的。为了实现这一目标，90%的癌症 重新激活端粒酶逆转录酶（TERT），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，端粒覆盖染色体的末端， 染色体DNA的末端复制问题。通常，TERT在干细胞中表达，但在干细胞中表达。 在体细胞中转录沉默。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，在TERT启动子中含有激活突变。 这些突变导致转录因子GA结合蛋白（GABP）的结合，以及转录因子GA结合蛋白（GABP）的重新激活。 通过不完全理解的机制。了解和靶向基因和途径 与这些现象相关的研究有可能为GBM开辟未来的主要治疗途径， 一种常见且最严重的成人脑癌。目前的建议将利用一个集中在体内 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GBM的因子 TERT启动子突变GBM细胞的永生化。目标屏幕将在控制和 GABP突变体细胞系，以阐明基因的功能，既协同和独立于 GABP。同时，该提案旨在设计靶向GBM的Cas9核糖核蛋白（RNP）复合物 细胞，特别是使用受体介导的摄取。Cas9 RNP复合物目前正在开发， 未来的治疗意图，考虑到与其他药物相比，毒性和脱靶效应的趋势降低 CRISPR编辑方法这些GBM特异性RNP将以编辑基因为最终目标而设计 逆转TERT启动子突变体GBM细胞中的细胞永生，在体外和体内。总的来说，这项工作 将开发基于CRISPR的技术，用于体内筛选和靶向GBM细胞，目标是 阐明和抑制GBM细胞永生的主要因素。

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.