Type II diabetes mellitus (T2D) is one of the biggest threats to human health. Previous studies have revealed the critical contribution of glucagon induced hepatic gluconeogenesis in the hyperglycemia of T2D patients. Inspired by previous researches and our well-integrated HP program, our project implemented a designer cell approach to control hyperglycemia by degrading hepatic Glucagon Receptors (GCGR) in a glycemic dependent manner. To be specific, the PR Predator system we proposed in iGEM 2018 is modified for intracellular GCGR degradation. A ChREBP activating hybrid promoter is also introduced to integrate endogenous hepatic glucose sensing signals to exogenous transgene expression.
To prove our concept and demonstrate our designs, we introduced the GCGR Predator to liver carcinoma cell line HepG2 and mouse primary hepatocytes. As expected, GCGR Predator mediated GCGR degradation and impaird hepatocyte glucagon sensitivity in vitro and ex vivo by affecting GCGR-downstream metabolic programs. We also demonstrated that the glucose sensing module is capable of activating transgene expression in a glucose dose-dependent manner. Furthermore, we showed that glucose sensor-GCGR Predator chimera restores glucose homeostasis of T2D patients in silico.
This year, by the hard-work of our entire team, we contributed 16 high quality, well documented biobrick parts, and characterized three previously existed biobricks in HepG2 cells, which is a rarely characterized chassis. We also expanded the function of previously reported TATA box promoter (BBa_M50098) by introducing ChREBP binding domains. The improved part is thus capable of sensing extracellular glucose level.
In general, our project in iGEM 2019 not only included a brand-new protein degradation system into the synthetic biology toolkit, but also developed a new synthetic glucose sensing approach that has not yet being reported. More importantly, we provided a promising close-loop, virus-based, protein-targeting, synthetic biology inspired approach for glycemic control in T2D
The whole journey is filled with suffering and interesting moments. With our tears and love, we hope that our efforts could help create a better world.