Targeting the cancer neo-genome for destruction with CRISPR-Cas enzymes

项目摘要/摘要 在了解癌症基因组方面的进展导致了新疗法的发展，这种疗法 针对癌症DNA的蛋白质产物的明显变化。由此产生的靶向治疗，连同 蛋白质靶向免疫疗法已经显著提高了癌症患者的存活率。然而，并不是所有的 患者从这些新的疗法中受益，相反，许多癌症继续接受DNA双倍治疗- 断链(DSB)-产生放射和化学疗法。 核酸靶向方法，如CRISPR-Cas9系统，现在能够研究一种新的 实验性癌症治疗方法：直接针对DNA，而不是其蛋白质产物。这 建议的目的是对这一模式进行初步调查和评估。 假设：针对癌症，在多个重排连接处靶向诱导DNA双链断裂 基因组，可导致癌细胞特异性的细胞毒性和对健康组织的损害。 具体目标1.描述癌症特异性Cas9靶向基因组位置的全景 癌症和癌细胞系。在这一目标中，将开发一种计算管道来表征光谱 肿瘤细胞系百科全书中Cas9靶向基因组改变的研究和泛癌分析 全基因组数据库，包括300多个癌细胞系和2500个癌症基因组。序列特征： 在肿瘤治疗中可能影响靶点有效性的特征包括靶点克隆性、靶点复制 癌症类型中靶向基因组改变的分布情况。这条管道将成为 可公开使用的工具，用于生成CAS9目标重排断点列表，以供未来研究使用。 目的2.开发针对癌症特异性的Cas9诱导癌细胞细胞毒作用的方法 DNA重排断点。在一系列原则证明实验中，这一目标将评估Cas9 可以通过将DNA断裂定向到单个高度扩增的位置或几个 癌症新基因组中的独特位置以及这是否会对健康组织产生毒性。 目的3.确定对Cas9介导的DNA损伤具有抗性或敏感性的蛋白质因素： 这一目标将包括CRISPR基因敲除筛查，以发现影响细胞对多个Cas9- 诱导DNA断裂。 这项提议将在以下指导下提供癌症基因组学和靶向治疗方面的培训 Dana-Farber癌症研究所的Matthew Meyerson教授在这两个领域都很有经验。除了……之外 在实验和计算研究方面，申请者将在会议上展示结果，培训未来的科学家， 并与癌症基因组学、基因组编辑和癌症治疗学的同事和导师互动 菲尔兹。这项计划中的研究计划最终发表在同行评议的期刊上。归根结底，这 培训应该为申请者在美国研究机构的研究人员职业生涯做好准备。

Project Summary/Abstract Advances in understanding the cancer genome have led to the development of novel therapeutics that target distinct alterations in protein products of cancer DNA. The resulting targeted therapies, together with protein-targeting immunotherapies, have led to significant advances in cancer patient survival. However, not all patients benefit from these new therapies and instead many cancers continue to be treated with DNA double- strand break (DSB)-generating radio- and chemo- therapies. Nucleic acid targeting approaches such as the CRISPR-Cas9 system now enable investigation of a new modality for experimental cancer therapeutics: targeting DNA directly rather than its protein products. This proposal is aimed at the initial investigation and evaluation of this modality. Hypothesis: Targeted induction of DNA DSBs at multiple rearrangement junctions, specific to the cancer genome, can lead to cancer-cell specific cytotoxicity and spare damage to healthy tissue. Specific Aim 1. Characterize the landscape of cancer-specific Cas9-targetable genomic sites across cancers and cancer cell lines. In this aim, a computational pipeline will be developed to characterize the spectrum of Cas9-targetable genome alterations across the Cancer Cell Line Encyclopedia and Pan-Cancer Analysis of Whole Genomes databases of over 300 cancer cell lines and 2500 cancer genomes. Sequence features that may affect target effectiveness in cancer therapy will be characterized, including target clonality, target copy number, and the distribution of targetable genomic alterations across cancer types. This pipeline will become a publicly available tool to generate lists of Cas9-targetable rearrangement breakpoints for use in future studies. Aim 2. Develop methods to induce cytotoxicity in cancer cells using Cas9 targeted to cancer-specific DNA rearrangement breakpoints. In a series of proof-of-principle experiments, this aim will assess whether Cas9 can induce targeted cell death through targeting DNA breaks to either single highly amplified sites or several unique sites in the cancer neo-genome and whether this generates toxicity in healthy tissue. Aim 3. Identify protein factors responsible for resistance or sensitivity to Cas9-mediated DNA damage: This aim will comprise a CRISPR knockout screen to discover proteins that affect cell sensitivity to multiple Cas9- induced DNA breaks. This proposal will provide training in cancer genomics and targeted therapeutics under the guidance of Professor Matthew Meyerson at Dana-Farber Cancer Institute, who is experienced in both fields. In addition to experimental and computational research, the applicant will present results at conferences, train future scientists, and interact with colleagues and mentors in the cancer genomics, genome editing, and cancer therapeutics fields. The planned research is intended to culminate in publication in peer-reviewed journals. Ultimately, this training should prepare the applicant for a career as research faculty at a United States research institution.