A new bladder cancer model based on tissue reprogramming and gene targeting

项目总结： 随着细胞重新编程和基因编辑的最新进展，有可能设想出新的 正常和疾病环境下的组织建模方法。具体来说，我们建议使用 转分化和基因打靶技术构建一种新的基因工程模型系统 人类癌症。我们最近开发了一种高度创新的方法来产生完全功能的前列腺 基于识别协同规范基因的计算系统方法的肾移植中的组织 (Talos等人，NAT Commun，2017)。我们建议将这些方法应用并扩展到人体建模中 通过结合成纤维细胞的谱系转化和组织重组分析，晚期膀胱癌 计算系统生物学算法与CRIPSR/Cas9介导的临床相关基因打靶 突变。在我们的初步研究中，我们已经表明成纤维细胞可以直接转化为上皮细胞 在亲上皮培养条件下瞬时表达多能因子的细胞。此外， 这些被诱导的上皮细胞能够在组织中进一步向膀胱组织终末分化 在膀胱特异性间质诱导力作用下进行体内重组实验。基于这些 初步数据，我们假设可以利用易于获得的成纤维细胞的固有可塑性来 通过组合关键的膀胱规范基因、重新编程技术来生成膀胱上皮 和组织重组试验。此外，我们假设重新编程的膀胱组织是顺从的。 通过CRISPR介导的基因靶向向恶性转化。 为了检验这一假设并产生一个新的人类癌症模型，我们建议执行(1)直接 人成纤维细胞激活正常主调控基因向膀胱上皮细胞转化的研究 膀胱上皮，通过膀胱调节遗传网络的生物信息学分析或通过 候选基因方法和(2)CRISPR介导的基因打靶建立人膀胱癌模型 与人类疾病相关的肿瘤抑制基因和癌基因的重新编程组织。我们的研究将 为研究膀胱癌的发生机制提供了新的见解，并为药物开发提供了新的平台 筛选和发现患者特定的早期预后生物标记物。

Project Summary: With recent advances in cellular reprogramming and gene editing it became possible to envision new approaches for tissue modeling in normal and disease contexts. Specifically, we propose to use transdifferentiation and gene targeting to generate a novel genetically-engineered model system for studies of human cancer. We recently developed a highly innovative methodology for generating fully functional prostate tissue in renal grafts based on a computational system approach that identifies synergistic specification genes (Talos et al., Nat Commun, 2017). We propose here to apply and expand these methods for modeling human bladder cancer by combining lineage conversion of fibroblasts with tissue recombination assays, advanced computational systems biology algorithms and CRIPSR/Cas9-mediated gene targeting of clinically-relevant mutations. In our preliminary studies, we have shown that fibroblasts can be directly converted into epithelial cells following transient expression of the pluripotency factors in pro-epithelial culture conditions. Moreover, these induced epithelial cells are amenable to further terminal differentiation into bladder tissue in tissue recombination assays in vivo under the inductive force of bladder specific mesenchyme. Based on these preliminary data, we hypothesize that the inherent plasticity of readily-accessible fibroblasts can be exploited to generate bladder epithelia through a combination of key bladder specification genes, reprogramming techniques and tissue recombination assays. Moreover, we hypothesize that the reprogrammed bladder tissue is amenable to malignant transformation through CRISPR-mediated gene targeting. To test this hypothesis and generate a new model of human cancer, we propose to perform (1) Direct conversion of human fibroblasts into bladder epithelium by activation of master regulator genes of normal bladder epithelium, identified by bioinformatic analysis of regulatory genetic networks of bladder or by a candidate gene approach and (2) Modeling human bladder cancer by CRISPR-mediated gene targeting in the reprogrammed tissue of tumor suppressors and oncogenes relevant for human disease. Our studies will provide novel insights into the mechanisms underlying bladder tumorigenesis and a novel platform for drug screening and for discovery of patient-specific early prognostic biomarkers.