Engineering Large Chromosomal Deletions in Mice to Advance Precision Oncology

项目总结/摘要 基因组缺失，包括局灶性和大臂水平染色体缺失，是常见的遗传事件 促进癌症的发生和发展。局灶性缺失对机制产生了重要的见解 通过帮助识别位于缺失位点的肿瘤抑制基因（TSG）来研究潜在的肿瘤发生。在 相比之下，对于癌症中大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是局灶性缺失的30倍。我们和其他研究人员已经证明， 这种缺失影响多个相邻的TSG的活动，并构成一种驱动力， 肿瘤发生此外，由大规模缺失引发的多个基因的伴随丢失可以产生 表型从根本上不同于由单一TSG丧失引起的表型，并可能提供 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 他们的分析应该集中在染色体缺失的综合影响上，而不是染色体缺失的影响。 一个关键的TSG基因工程小鼠（GEM）模型为研究基因工程小鼠的遗传学行为提供了理想的工具。 肿瘤生物学中遗传畸变的后果。然而，由于靶向克隆能力有限， 载体（~300 Kb）和传统基因打靶中靶向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高度挑战性， 因此，对这种病变在肿瘤发生中的作用的研究明显不足。为了填补这一关键空白，我们必须 开发了一种创新的方法，染色体工程在大的遗传距离，通过 CRISPR/Cas9技术在小鼠胚胎干细胞中的应用将所得到的携带所需的 基因组编辑使创造条件性敲除小鼠成为可能， 大的删除。考虑到前列腺癌中大染色体缺失的普遍性和重要性， 我们建议利用我们的创新方法来生成和充分表征两个新的GEM模型， 每个都有一个共同但不同的前列腺癌相关的大缺失，目的是验证和 将这些模型认证为人类前列腺癌的遗传学和生物学鲁棒代表。在 目标1，我们将使用CRISPR/Cas9技术开发条件小鼠系，以靶向常见缺失的大 人前列腺癌的染色体位点。 在目标2中，我们将开发前列腺癌的小鼠模型， 大的染色体缺失，以分析其对前列腺癌发展的生物学和表型影响。 总之，这些模型及其特征将满足此赠款机会的多个目标。 成功完成这些调查不仅将大大有助于实施精确的 肿瘤学研究，而且还提高了动物模型转化研究的适用性。

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.