Cancer-specific metabolites as cues to engineer and target anti-tumor therapeutic bacteria

摘要 随着合成生物学的不断进步，治疗性细菌在癌症治疗中的应用 现在更接近成为现实。然而，要实现这一目标，仍然需要更好地靶向治疗 细菌向肿瘤部位转移，以提高治疗的有效性和安全性。在这里，我们建议使用 一种关键的癌症代谢物，在结直肠癌和其他肿瘤中产生，作为激活基因电路的线索 在治疗性细菌中控制这些细菌，并将它们靶向癌症部位。要做到这一点 Goal，我们将把这种代谢物的天然转录调节因子克隆到 工程化治疗性大肠杆菌，并优化其性能。同时，我们将使用特定的RNA 我们已经为这种代谢物开发了适配子，以构建合成核糖开关来控制蛋白质 在工程菌中表达。随后，这两个工具；转录 调节剂和合成核糖开关将被结合使用，在体内建立遗传电路 工程化大肠杆菌菌株。然后这些遗传电路将1)引导经过改造的大肠杆菌(沿着这个键 代谢物浓度梯度)通过控制其运动性向肿瘤部位移动，2)诱导 这种大肠杆菌附着在肿瘤细胞上，以及3)在肿瘤细胞内诱导治疗分子的产生 肿瘤微环境对这种关键代谢物的反应。 在这笔赠款的时间范围内，我们将致力于开发这些大肠杆菌基因电路，它可以 在生理上相关的浓度下对这种代谢物做出反应，我们将测试这些 在癌细胞系上的基因工程大肠杆菌。今后的工作将通过随后的赠款进行，将 包括在结肠癌的小鼠模型上试验这些工程菌。

Abstract With the ongoing advances in synthetic biology, the application of therapeutic bacteria for cancer treatment is now closer to becoming a reality. Achieving this goal, however, still needs better targeting of the therapeutic bacteria towards the cancer sites to improve the efficacy and safety of the treatment. Here, we propose to use a key cancer metabolite produced in colorectal cancers and other tumors as a cue to activate genetic circuits inside the therapeutic bacteria to control those bacteria and target them to the cancer site. To achieve this goal, we are going to clone the naturally occurring transcriptional regulator for this metabolite into engineered therapeutic E. coli and optimize its performance. In parallel, we will employ specific RNA aptamers which we already developed for this metabolite, to build synthetic riboswitches to control protein expression in the engineered Escherichia coli strain. Subsequently, these two tools; the transcription regulator, and the synthetic riboswitches, will be used in combination to build genetic circuits inside the engineered E. coli strain. These genetic circuits will then 1) Direct the engineered E. coli (along this key metabolite concentration gradient) towards the tumor site through controlling its motility, 2) Induce the attachment of this E. coli to the tumor cells, and 3) Induce the production of a therapeutic molecule within the tumor microenvironment in response to this key metabolite. During the time frame of this grant, we will be working on developing these E. coli genetic circuits which can respond to this metabolite at the physiologically relevant concentrations and we are going to test these engineered E. coli on cancer cell lines. Future work, which will be pursued through subsequent grants, will involve trying these engineered bacteria in mice models for colon cancer.