Engineering Native E. coli to Detect, Report, and Treat Colorectal Cancer

项目摘要/摘要 尽管结直肠癌(CRC)的发病率总体上有所下降，但它仍然是第四大常见原因 美国癌症死亡人数的最大比例。不幸的是，流行病学研究表明，癌症的发病率有惊人的增长。 登革热在50岁以下的人群中存在，他们没有进行常规筛查。此外，CRC检测也很困难 在高危人群中，包括那些有遗传倾向的人(例如家族性腺瘤性息肉病)、疾病 例如炎症性肠病)，或来自某些人口统计学特征(例如美国黑人)。因此，有一个 迫切需要制定创新的解决办法，以及早发现CRC和预防 从腺瘤到结直肠癌的过渡。为了满足这一需求，我们的跨学科研究团队将开发 使用合成生物学方法识别早期结直肠癌发展的基因工程细菌，监测 并报告腺瘤和结直肠癌微环境的变化，防止癌症进展。要实现 以上目标，工程菌都要嫁接和定植在恶劣的管腔环境中，感觉 并识别异常环境信号，计算该信号，并表示报告者或治疗者 探员。然而，具有这些特征的合适载体仍然缺乏，限制了合成生物学的应用。 癌症研究的专利。重要的是，CRC与大肠杆菌高度相关，我们有许多合成的 生物工具。此外，我们的初步概念验证研究表明，天然的大肠杆菌可以 被设计成永久定植完全常规(即非微生物组耗尽)宿主并执行功能- 感兴趣的治疗，例如，管腔胆汁酸的解结合。去结合胆汁酸和生成的法尼醇X-Re- 受体(FXR)激动剂可抑制结直肠癌的发展，提示工程化治疗的潜在用途 原生细菌。基于我们强有力的支持性初步结果，我们将区分本地的大肠杆菌和健康的， 腺瘤和结直肠癌组织的一种遗传模型，并对它们进行改造以检测和治疗结直肠癌 癌症的微环境。此外，我们还将描述不同肿瘤环境的影响。 工程菌在结肠有机物模型中定植及性能的影响因素 在数学建模的支持下的片上器官，从而识别特定的CRC信号以用于编程- 作为CRC报告和治疗药物的工程化天然大肠杆菌的反应。最后，我们将设计 通过定量报告CRC的水平来检测和减缓CRC的进展的本地细菌- 相关的半胱氨酸蛋白酶，并选择性地抑制其活性。本提案中描述的研究将 产生新的、急需的合成生物载体，可以开发为生物传感器和治疗 腺瘤和结直肠癌，以及许多其他疾病。此外，这个项目将丰富我们的基础 了解结直肠癌与微生物组的关系，阐明半胱氨酸蛋白酶在结直肠癌中的作用。 消退与治疗。

PROJECT SUMMARY/ABSTRACT Despite its overall decreasing occurrence, colorectal cancer (CRC) remains the fourth most common cause of cancer deaths in the US. Unfortunately, epidemiological studies demonstrate an alarming increase in inci- dence in populations below the age of 50, who are not routinely screened. Furthermore, CRC detection is difficult in high-risk groups, including those with a genetic predisposition (e.g. familial adenomatous polyposis), disease traits (e.g. inflammatory bowel disease), or from certain demographics (e.g. Black-Americans). Thus, there is a significant need for the development of innovative solutions for the early detection of CRC and the prevention of the transition from adenoma to CRC. To address this need, our interdisciplinary research team will develop genetically engineered bacteria using synthetic biology approaches to identify early CRC development, monitor and report changes in the adenoma and CRC microenvironment, and prevent cancer progression. To achieve the above objectives, engineered bacteria have to engraft and colonize the hostile luminal environment, sense and distinguish an abnormal environmental signal, compute this signal, and express a reporter or a therapeutic agent. However, appropriate vectors with these features remain lacking, constraining synthetic biology applica- tions for cancer research. Importantly, CRC is highly associated with E. coli, for which we have many synthetic biology tools. Furthermore, our preliminary proof-of-concept studies have revealed that native E. coli can be engineered to perpetually colonize fully conventional (i.e. non-microbiome depleted) hosts and to execute func- tions of interest, e.g., deconjugation of luminal bile acids. Deconjugated bile acid and resultant farnesoid X re- ceptor (FXR) agonism can suppress CRC development, indicating a potential therapeutic use of engineered native bacteria. Building on our strong supportive preliminary results, we will identify native E. coli from healthy, adenoma, and CRC tissues of a genetic model of CRC and engineer them to detect and treat CRC in response to the cancer microenvironment. Furthermore, we will characterize the effects of different tumor environment factors on the colonization and performances of engineered native E. coli in the colon organoid model in an organ-on-chip with the support of mathematical modeling, thereby identifying specific CRC signals for program- ming the responses of engineered native E. coli as CRC reporters and therapeutics. Finally, we will engineer native bacteria to detect and attenuate the progression of CRC by quantitatively reporting the level of CRC- related cysteine proteases and selectively inhibiting their activity. The research described in this proposal will generate new, much-needed synthetic biology vectors that can be developed as biosensors and therapeutics of adenoma and CRC, as well as many other diseases. Furthermore, this project will enrich our fundamental knowledge about the CRC-microbiome relationship and elucidate the roles of cysteine proteases in CRC pro- gression and treatment.