Glucose-Responsive Promoter Engineering for Dynamic Regulation of Insulin Transgene Expression Delivered by Non-Viral Gene Therapy

Diabetes mellitus treatment currently relies on externally administered insulin, which lacks the precise glucose-responsive regulation of pancreatic β-cells. This study develops a synthetic glucose-responsive gene expression system using repeated glucose-responsive elements (R3G) to regulate insulin production in non-pancreatic cells. The R3G element, derived from carbohydrate response elements (ChoREs) of the liver pyruvate kinase promoter, was incorporated upstream of minimal promoters to create glucose-inducible constructs with low basal activity. Plasmid (pDNA) constructs were generated using the pGM206 backbone containing either reporter genes or a furin-cleavable human insulin cDNA (hsoINSfur). pDNA was delivered into HepG2 cells using the non-viral Glycosaminoglycan-binding Enhanced Transduction (GET) nanoparticle system. Glucose responsiveness was evaluated using Gaussia luciferase (GLuc) assays and a dual secreted reporter system incorporating secreted embryonic alkaline phosphatase (SEAP) as an internal normalisation control. Among the promoters tested, R3G-miniCMV and R3G-SCP1 demonstrated strong glucose-dependent activation while maintaining low basal expression. R3G-miniCMV produced the greatest dynamic range, showing up to ∼24-fold induction under hyperglycaemic conditions (25 mM glucose) relative to hypoglycaemic/glucose-free conditions. Dual-reporter assays confirmed that increased GLuc activity was due to glucose-responsive transcription rather than variations in transfection efficiency or cell viability/metabolism. Replacement of reporter genes with hsoINSfur demonstrated functional insulin secretion, with R3G-containing constructs producing significantly higher C-peptide levels under elevated glucose conditions compared with non-R3G controls. Overall, this study demonstrates that synthetic R3G promoters can confer robust glucose-responsive insulin expression in non-pancreatic cells using a safe non-viral delivery platform, providing a proof-of-concept system for future glucose-regulated insulin gene therapy applications.