Designing a toolkit of environmentally responsive synthetic cells

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.