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

使用 CRISPR-Cas 酶靶向破坏癌症新基因组

• 批准号: 10678361
• 负责人: Mitchell L Leibowitz
• 金额: $ 6.91万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678361
• 关键词: Affect; Age; Apoptosis; Apoptotic; Biological Assay; Biological Markers; CRISPR/Cas technology; Cancer Patient; Cancer cell line; Cell Cycle; Cell Death; Cell Death Induction; Cell Line; Cell Survival; Cells; Cessation of life; Chromosomal Duplication; Chromosomes; Clonality; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Complex; Cytotoxic agent; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Sequence Alteration; DNA Sequence Rearrangement; Dana-Farber Cancer Institute; Data; Development; Double Minutes; Effectiveness; Elements; Encyclopedias; Enzymes; Evaluation; Event; Excision; Future; Gene Rearrangement; Gene Targeting; Genes; Genome; Genomics; Guide RNA; Hour; Human; Immunotherapy; Institution; Investigation; Journals; Knock-out; Malignant Neoplasms; Measures; Mediating; Medical; Mentors; Methods; Modality; Mutate; Mutation; Natural Killer Cells; Normal Cell; Nucleic Acids; Operative Surgical Procedures; Outcome; Pathway interactions; Patients; Peer Review; Predisposition; Proteins; Publications; Radiation therapy; Research; Resistance; Scientist; Series; Site; Stains; System; Therapeutic; Tissues; Toxic effect; Training; United States; Variant; cancer cell; cancer genome; cancer genomics; cancer therapy; cancer type; career; cell killing; cell type; chemotherapy; chromosome loss; chromosome missegregation; chromothripsis; computational pipelines; cytotoxic; cytotoxicity; data repository; design; experience; experimental study; genome database; genome editing; genomic locus; improved; new therapeutic target; next generation; novel; novel therapeutics; pan-genome; professor; repaired; research faculty; screening; small molecule; symposium; targeted nucleases; targeted treatment; tool; tumor; whole genome

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.

项目总结/摘要 在理解癌症基因组方面的进展已经导致了新疗法的发展， 靶向癌症DNA蛋白质产物的不同改变。由此产生的靶向治疗，以及 蛋白质靶向免疫疗法为癌症患者的生存带来了显着进步。但并非所有 患者受益于这些新疗法，而许多癌症继续使用DNA双- 链断裂（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生物学方法。 实验性癌症治疗的模式：直接靶向DNA而不是其蛋白质产物。这 该提案旨在对这一模式进行初步调查和评价。 假设：在多个重排连接处靶向诱导DNA DSB，特异于癌症 基因组，可以导致癌细胞特异性细胞毒性，并避免对健康组织的损害。 具体目标1.表征癌症特异性Cas9靶向基因组位点的景观， 癌症和癌细胞系。在这个目标中，将开发一个计算管道来表征光谱 Cas9靶向基因组改变在整个癌细胞系百科全书和泛癌症分析 超过300种癌细胞系和2500种癌症基因组的全基因组数据库。序列特征， 可能影响癌症治疗中靶点有效性的特征，包括靶点克隆性、靶点拷贝 数量和可靶向基因组改变在癌症类型中的分布。这条管道将成为 这是一个公开可用的工具，用于生成Cas9靶向重排断点的列表以用于未来的研究。 目标2.开发使用靶向癌症特异性靶向的Cas9在癌细胞中诱导细胞毒性的方法 DNA重排断裂点。在一系列的原理验证实验中，这一目标将评估Cas9是否 可以通过将DNA断裂靶向单个高度扩增位点或多个高度扩增位点来诱导靶向细胞死亡， 癌症新基因组中的独特位点以及这是否会在健康组织中产生毒性。 目标3.鉴定负责对Cas9介导的DNA损伤的抗性或敏感性的蛋白质因子： 这一目标将包括CRISPR敲除筛选，以发现影响细胞对多种Cas9- 诱导DNA断裂。 该提案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： 丹娜-法伯癌症研究所的马修·迈耶森教授，他在这两个领域都很有经验。除了 实验和计算研究，申请人将在会议上展示结果，培养未来的科学家， 并与癌症基因组学、基因组编辑和癌症治疗方面的同事和导师互动 领域的计划中的研究最终将在同行评审的期刊上发表。最终这 培训应使申请人为在美国研究机构从事研究工作做好准备。