Introduction
Type two diabetic mellitus (T2D) is a common and serious chronic metabolic syndrome comprises a cluster of diseases associated with excess nutrition and insufficient physical activity. To date, it is estimated that more than 352 million people around the globe are at risk of developing such a chronic and painful disease, particularly in low – and middle-income countries1 (Figure 1a). In China, around 10% of its population have diabetes, with nearly 60% exposed to the T2D related risk factors2. Due to its undiminished importance, diabetic mellitus has been widely studied in the past two decades.
Though pathological mechanism of T2D is still yet to be further understood, peripheral insulin resistance, impaired regulation of hepatic glucose production, and islet β-cell dysfunction are thought to be related to the development of T2D3. Current treatment also mainly focused on improving peripheral insulin resistance or increasing insulin secretion by lifestyle intervention or pharmaceutical approaches4. Notably, synthetic biology approaches have also been developed to provide “smart” glycemic control over the T2D patients by inducing insulin or GLP-1 production5-7. However, it is worth noticing that although the hyperglucagonemia syndrome has been discovered on T2D patients for years, research focusing on glucagon pathophysiology and related therapeutic approaches has historically been dwarfed by research on beta cells and insulin. Glucagon is a 29-amino acid peptide hormone secreted from the pancreatic alpha cells with a powerful stimulatory effect on hepatic glucose production acting to increase plasma glucose levels as a counter-regulatory hormone to insulin8. Though the mechanisms underlying hyperglucagonemia in patients with type 2 diabetes are not clear. It seemed quite reasonable to propose a brand-new glycemic control approach targeting glucagon pathway (Figure 2a).
Glucagon stimulates hepatic glucose production mainly via hepatic glucagon receptor (GCGR), a 62 kDa G-protein coupled receptor9. Liver specific disruption of GCGR has also shown reductions in fasting blood glucose and improvements in insulin sensitivity and glucose tolerance compared with wild-type controls under normal chow diet and high fat diet condition10-12. These results further implied that GCGR might serve as a decent drug target for glycemic control. However, as is suggested by our HP interview and several literatures, uncontrolled depletion of GCGR may result in lethal hypoglycemia and disrupted islet alpha cell function11,13. An improved approach for “smart” GCGR blockage is thus needed.
For such matter, inspired by our project in iGEM 2018, we further expanded the application of previously reported Trim-away system14 by shifting the antibody-antigen interaction into ligand-receptor interaction, thus providing us a new approach to degrade endogenous receptors possibly within ER or Trans-Golgi Network (Figure 2b). Our results suggested that our new protein degradation approach can effectively degrade GCGR in HepG2 cells. Impaired cellular glucagon response and glucose production were also observed. Furthermore, to achieve the feedback control of the GCGR degradation system, we also incorporated Carbohydrate-response element-binding protein (CHREBP) pathway, the endogenous hepatic glucose sensing pathway15, into our system by in-silico-assisted16,17 designing of a CHREBP-activating hybrid promoter (Figure 2c). As expected, fluorescent reporter assay showed a dose-dependent increase of fluorescent intensity under the stimulation of different amount of glucose. Taking one step forward, we also successfully demonstrated that the GCGR degradation system we designed can be easily packaged into adenovirus-based delivery system and remain functional in primary hepatocytes after virus transfection. Our modeling also showed that by conjugating the glucose sensing module and the GCGR degrading module, our circuit can reestablish glycemic homeostasis of simulated T2D patient into a much healthier level without inducing the risk of hypoglycemia.
By the meantime, our HP members also set foot into the society and hospitals, investigating their understanding on diabetes prevention and treatment, as well as their opinions on virus delivered gene therapy. The feedbacks from local society, medical doctors and experts not only help us to reshape our project, but also pushed us forward to launch a few public awareness programs related to T2D and gene therapy.
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.
Reference
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