Development of mechanogenetic tools for subcellular applications in cancer immunotherapy

Cancer has long ranked as #1 in the view of doctors and health professionals due to its high death rate. Recent development of molecular engineering and synthetic biology sheds lights on cancer diagnosis and treatment. For example, CAR-T based immunotherapy has become an emerging field in cancer therapeutics. However, major challenges still remain for cancer diagnosis and therapeutics, such as time-consuming detection in cancer diagnosis and uncontrolled cytotoxicity in CAR-T therapy. Therefore, there is a critical need to engineer molecular tools for more rapid and better control of live cells. Rapid and efficient measurement of cancer cells is a major challenge in early cancer diagnosis. Fluorescent proteins have revolutionized biology by allowing live cell visualization. Therefore, biosensors based on fluorescent proteins could potentially be developed as the point-of-care detection methods of live cancer cells. In chapter 2 we discussed the development of a FRET based EphA4 biosensor. EphA4 is involved in cellular mechanotransduction and is an engaging target for cancer research. We discovered that EphA4 has different activation at subcellular membrane locations in single live cells, which provides powerful tools to monitor the dynamic molecular activities at subcellular compartments.Various synthetic biology approaches have been adopted for targeted cancer therapeutics. For example, optogenetics integrating optical and genetics has enabled the sensing and control of molecular events in living systems. However, there is a critical need to remotely manipulate live cells with high precision deep in the body. Mechanical manipulation, in contrast to optogenetics, could allow deep and non-invasive control of these cells. Chapter 3 goes on to discuss the combination of developing a laser based mechanical stimulation tools and molecular biosensors to activate and study mechano-sensitive cells. Together, Chapter 2 and Chapter 3 built the basis for developing and integrating engineered molecular tools with novel clinical therapeutic stimulation devices. Chapter 4, as a major work of this thesis, presents the engineering of a remote controlled mechanogenetics system which integrates ultrasound mediated mechanical stimulation and gene activation of CD19CAR for controllable application in cancer immunotherapy. Collectively, our suite of engineered molecular tools shows potential in extrapolation to advance biomedical and clinical applications