Engineering Large Chromosomal Deletions in Mice to Advance Precision Oncology

在小鼠中进行大量染色体缺失以推进精准肿瘤学

• 批准号: 10445187
• 负责人: MING CHEN
• 金额: $ 41.65万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445187
• 关键词: Affect; Animal Model; Biological; CRISPR/Cas technology; Cancer Biology; Characteristics; Chromosome Deletion; Chromosomes; Cloning; Code; Credentialing; Data Set; Development; Embryo; Engineering; Epithelial; Event; Gene Targeting; Genes; Genetic; Genetically Engineered Mouse; Genome; Genomic Segment; Genomics; Goals; Grant; Human; Human Chromosomes; Impairment; Investigation; Knockout Mice; Lead; Lesion; LoxP-flanked allele; Malignant Neoplasms; Malignant neoplasm of prostate; Modeling; Mus; Mutate; PTEN gene; Phenotype; Prevalence; Prostate; Proteins; Recurrence; Research; Research Personnel; Role; Sampling; Technology; Therapeutic; Time; Translational Research; Tumor Biology; Tumor Suppressor Genes; Work; arm; cancer genome; cancer initiation; cancer therapy; conditional knockout; driving force; embryonic stem cell; genome editing; homologous recombination; human disease; human model; in vivo; innovation; insight; mouse model; novel; precision oncology; pressure; probasin; prostate cancer model; prostate carcinogenesis; stem; tool; tumor progression; tumorigenesis; vector

Project Summary/Abstract Genomic deletions, including both focal and large arm-level chromosomal deletions, are frequent genetic events that promote cancer initiation and progression. Focal deletions have yielded significant insights into mechanisms underlying tumorigenesis by helping identify tumor suppressor genes (TSGs) residing in the deleted loci. In contrast, little is known about the biological and phenotypic impacts of large chromosomal deletions in cancer despite their occurrence 30 times more frequently than focal deletions. We and other researchers have shown that such deletions impact the activities of multiple neighboring TSGs and constitute a driving force in tumorigenesis. Additionally, concomitant loss of multiple genes triggered by broad-scale deletions can create phenotypes that differ fundamentally from those arising from the loss of a single TSG, and may offer unanticipated therapeutic opportunities. These losses should therefore be considered as distinct genetic events and their analysis should be focused on the integrated effects of chromosomal deletion rather than the impact of a single critical TSG. Genetically engineered mouse (GEM) models provide an ideal tool for investigating the consequences of genetic aberrations in tumor biology. However, due to the limited cloning capacity of targeting vectors (~300 Kb) and the rarity of on-target homologous recombination events in traditional gene-targeting technology, modelling large chromosomal deletions in mice has proven highly challenging, and the in vivo role of such lesions in tumorigenesis has therefore been significantly understudied. To fill this critical gap, we have developed an innovative approach to chromosomal engineering over large genetic distances through CRISPR/Cas9 technology in mouse embryonic stem (ES) cells. The resulting ES clones carrying the desired genome edits make possible the creation of conditional knockout mice that accurately mimic cancer-associated large deletions. Given the prevalence and significance of large chromosomal deletions in prostate cancer, here we propose to utilize our innovative approach to generate and fully characterize two novel GEM models that each harbor a common but distinct prostate cancer-associated large deletion, with a goal of validating and credentialing these models as genetically and biologically robust representations of human prostate cancer. In Aim 1, we will develop conditional mouse lines using CRISPR/Cas9 technology to target commonly deleted large chromosomal loci in human prostate cancer. In Aim 2, we will develop mouse models of prostate cancer harboring large chromosomal deletions to analyze their biological and phenotypic impacts on prostate cancer development. Taken together, these models and their characterization will meet multiple goals of this grant opportunity. Successful completion of these investigations will not only contribute greatly to the implementation of precision oncology research but also enhance the applicability of animal models to translational research.

项目概要/摘要 基因组缺失，包括局灶性和大臂水平的染色体缺失，是常见的遗传事件 促进癌症的发生和进展。焦点缺失对机制产生了重要的见解 通过帮助识别位于删除基因座中的肿瘤抑制基因（TSG）来揭示潜在的肿瘤发生。在 相比之下，人们对癌症中大量染色体缺失的生物学和表型影响知之甚少 尽管它们的发生频率比局部缺失高 30 倍。我们和其他研究人员已经证明 此类删除会影响多个相邻 TSG 的活动，并构成推动 肿瘤发生。此外，大规模缺失引发的多个基因的同时丢失可能会导致 与单个 TSG 丢失所产生的表型有根本不同，并且可能提供 意想不到的治疗机会。因此，这些损失应被视为不同的遗传事件 他们的分析应侧重于染色体缺失的综合影响，而不是染色体缺失的影响 单个关键 TSG。基因工程小鼠 (GEM) 模型为研究 肿瘤生物学中遗传畸变的后果。但由于靶向克隆能力有限 载体 (~300 Kb) 以及传统基因靶向中靶上同源重组事件的罕见性 技术，在小鼠中模拟大的染色体缺失已被证明具有很高的挑战性，并且体内作用 因此，此类病变在肿瘤发生中的作用尚未得到充分研究。为了填补这一关键空白，我们有 开发了一种在大遗传距离上进行染色体工程的创新方法 小鼠胚胎干 (ES) 细胞中的 CRISPR/Cas9 技术。由此产生的 ES 克隆携带所需的 基因组编辑使创造能够准确模仿癌症相关的条件基因敲除小鼠成为可能 大的删除。鉴于前列腺癌中大量染色体缺失的普遍性和重要性，这里 我们建议利用我们的创新方法来生成并充分表征两个新颖的 GEM 模型， 每个都包含一个共同但不同的前列腺癌相关大缺失，目的是验证和 证明这些模型是人类前列腺癌的遗传和生物学稳健代表。在 目标1，我们将利用CRISPR/Cas9技术开发条件小鼠品系，以针对常见缺失的大分子 人类前列腺癌的染色体位点。 在目标 2 中，我们将开发携带前列腺癌的小鼠模型 大量染色体缺失，以分析其对前列腺癌发展的生物学和表型影响。 总的来说，这些模型及其特征将满足本次资助机会的多个目标。 这些调查的顺利完成，不仅将为精准施策的实施做出巨大贡献。 肿瘤学研究还增强了动物模型在转化研究中的适用性。