Design And Construction Of Precision-contolled Designer Cells For Diabetes Therapy

Diabetes mellitus is so far one of the most prevalent chronic diseases worldside,accompanying with a pathophysiology involving metabolic impairments that can lead to many clinical complications.Hepatogenous diabetes is a complex disease that is typified by the simultaneous presence of type 2 diabetes and many forms of liver disease.The chief pathogenic determinant in this pathophysiological network is insulin resistance(IR),which is characterized by insensitivity of blood insulin level in liver,muscle and fat cells that impede the recruitment of glucose by tissue,strengthen the symptom in hyperinsulinemia and hyperglycemia.However,pharmacotherapies targeting IR remain limited and are generally inapplicable for liver disease patients,and conventional drug therapy for diabetes mellitus often fails to considerate the complex situations of complications with diabetes mellitus.Synthetic biology medicine provide a prospective road to achieve the spatiotemporal gene regulation in comprehensive and diversified gene networks-circuits which pursued by life science,and produce a development to clinical medical research in realizing informatization,precision and intelligent diagnosis and treatment,supplying a strong technology for revolutionary and rapid development in future medical service.We based on a variety of physiological indexes related to corresponding pathogenesis from different types of diabetes,such as hyperglycemia,hyperinsulinemia and hyperbileacidemia,to design and synthesize intelligent gene circuits inspired by the method of synthetic biology which include self-recognition to pathological indexes and feedback loop,to realize collaborative treatment of diabetes and complications.This paper focuses on artificial controlled engineering cells for diabetes diagnosis and treatment,exploring synthetic oleanolic acid(OA)regulation gene circuits contained in engineering cells for treatment of hepatogenous diabetes and synthetic intelligent close-loop engineering cell for insulin resistance syndrome respectively to investigate corresponding gene network in order to specialize designment for related physiology indexes and application of therapies.1.Design synthetic OA regulation gene circuit contained engineering cell for hepatogenous diabetes.The oleanolic acid(OA),widely distributed in plants,have been authorized using for protecting liver clinically in OTC medicine which was approved by medical field because of its safety and protecting efficiency.In this work,we designed gene circuit inspired by synthetic biology that combined the pharmacological activity of OA with Glucagon like peptide-1(GLP-1)to weaken insulin resistance,restore liver and pancreas function.The results indicated that our synthetic gene circuit in engineering cells could be activated by OA to express GLP-1,improving syndromes of glucose-tolerance,insulin resistance,hyperglycemia,dyslipidemia and liver dysfunction with other metabolism disorders in hepatogenous diabetes mice model.We didn't see any notable improvement in mice model with OA administration only(without our synthetic gene circuit).2.Design synthetic intelligent close-loop engineering cells for treatment of insulin resistance syndrome.By functionally rewiring the mitogen-activated protein kinase(MAPK)signalling pathway to produce MAPK-mediated activation of a hybrid transcription factor consisting of the tetracycline repressor,TetR,fused to the human ELK1-derived transactivation domain(TetR-Elk1),we assembled a synthetic insulin-sensitive transcription-control device that self-sufficiently distinguished between physiological and increased blood insulin levels and correspondingly fine-tuned the reversible expression of therapeutic transgenes from synthetic TetR-ELK1-specific promoters.Through the in vitro/vivo assay,we demonstrated that a self-adjusting synthetic gene circuit can be designed to sense and reverse the insulin-resistance syndrome in different mouse models.According to the results,when hyperinsulinemia occurs,insulin dose-dependent adiponectin was expressed and secreted into blood,intensify the sensitivity of insulin in liver,muscle and fat tissues,reducing the secretion of insulin by islet cells,which reversed insulin resistance syndrome;when blood insulin level decreased,the close-loop gene circuit could turn down the expression of adiponectin autonomously.We also took a long term therapeutic application assay in ob/ob and DIO animal models,the consequence of assay confirmed that our therapeutic method effectively improved animal model syndromes in hyperinsulinemia and insulin resistance.The therapeutic synthetic methods of OA regulation gene circuit and intelligent self-adjustable insulin-related close-loop gene circuit presented by this thesis performed satisfactory effects.This will open new possibilities for employing synthetic biology to advance personalized medicine by converting cells into living therapeutics to combat hitherto intractable diseases.