Designing a toolkit of environmentally responsive synthetic cells

合成生物学在过去二十年里呈指数级增长，推动了许多行业的进步。从这些创新中获益最多的领域之一是医学，特别是癌症的治疗。癌症在历史上一直是难以治疗的，其挑战来自遗传和生理的异质性以及由于全身细胞毒性治疗而产生的有害副作用。这产生了寻找更精确的治疗靶点和更具体的应用方法的动力，这些方法可以针对单个癌症亚型，甚至单个患者量身定制。基因测序的改进和DNA合成成本的降低为实现这些目标的工程解决方案铺平了道路。研究人员正在开发靶向药物传递机制的方法之一是利用致病菌，致病菌自然聚集在肿瘤部位，擅长浸润肿瘤肿块，并且有现有的途径使宿主细胞能够摄取。然而，虽然这些细菌经过改造，不再具有毒性，只是用作合成基因电路的基础，但使用活生物体仍然存在一些安全问题，这可能会对监管部门的批准构成障碍。这些担忧也反映在经常用于其他疾病基因治疗的病毒载体上。由于没有生命，合成细胞具有广泛的益处，非常适合于治疗应用。因此，我们提出了一种利用合成细胞靶向递送能够原位生产治疗性蛋白质的遗传回路的机制。将环境响应性表达调控整合到这些细胞的遗传载荷中，将通过确保我们选择的治疗分子在更精确的限定位置表达，降低靶外肿瘤效应的风险。通过选择肿瘤微环境特有的环境特征，我们可以增加药物应用的特异性，同时减少副作用的可能性，并允许在肿瘤部位施用更大的药物剂量。到目前为止，我们已经开发了一种功能性热反应表达盒，并验证了在低氧条件下使用的缺氧功能荧光报告的活性。我们还演示了基本的巨型单层囊泡的形成，它将形成我们合成细胞系统的基础。这个项目的下一步将包括结合这些元素来生产对环境敏感的合成细胞，我们可以将治疗分子整合到细胞中进行原位合成和释放。

Synthetic biology has grown exponentially over the last two decades, fuelling advances in a wide range of industries. One of the fields which stands to benefit the most from these innovations is medicine and specifically the treatment of cancer. Cancer has always been historically difficult to treat, with challenges arising from genetic and physiological heterogeneities and harmful side effects as a result of systemic cytotoxic treatment. This has generated a drive to source more precise therapeutic targets and more specific methods of application that can be tailored to individual cancer subtypes, or even individual patients. Improvements in genetic sequencing and the reduced cost of DNA synthesis have paved the way for engineered solutions to these goals. One of the ways that researchers are developing a targeted drug delivery mechanism is through the use of pathogenic bacteria, which naturally aggregate at tumour sites, are adept at infiltrating the tumour mass and have existing pathways to enable uptake by host cells. However, while these bacteria are engineered such that they are no longer virulent and are employed simply as a chassis for synthetic gene circuits, there are still several safety concerns over the use of living organisms, which can pose a barrier for regulatory approval. These concerns are also echoed for the viral vectors that are often used in gene therapies for other diseases. By being non-living, synthetic cells confer a wide range of benefits that are well suited to therapeutic application. We therefore propose a mechanism using synthetic cells for targeted delivery of genetic circuitry capable of in-situ production of therapeutic proteins. The integration of environmentally responsive expression regulation into the genetic payload of these cells will reduce the risk of on-target off-tumour effects by ensuring expression of our chosen therapeutic molecule in more precisely delimited locations. By selecting environmental characteristics that are unique to the tumour microenvironment, we can increase the specificity of drug application, simultaneously reducing the likelihood of side effects and allowing administration of a larger drug dose at the tumour site. So far, we have developed a functional thermo-responsive expression cassette and have verified the activity of a hypoxia-functional fluorescent reporter for use in low-oxygen conditions. We have also demonstrated formation of basic Giant Unilamellar Vesicles which will form the chassis of our synthetic cell system. The next steps of this project will involve combining these elements to produce environmentally responsive synthetic cells into which we can integrate a therapeutic molecule for in-situ synthesis and release.