Abstract

Introduction Glucagon-like peptide-1 (GLP-1) is a prominent therapeutic agent capable of normalizing fasting blood glucose levels in diabetic patients. While GLP-1-expressing mRNA encapsulated in lipid nanoparticles (LNPs) has been evaluated for diabetes treatment in primate models, circular RNAs (circRNAs) represent a more stable alternative to linear mRNA, offering significant potential for the development of next-generation GLP-1-encoding RNA therapeutics. Objective This study aims to design and synthesize a safe, long-acting circRNA encoding a GLP-1 receptor agonist (GLP-1RA). Methods We developed a scarless PIE (SLPIE) system that embeds essential exonic sequences within a split internal ribosome entry site (IRES), thereby minimizing immunogenicity. Scarless circRNAs expressing GLP-1RA or its analogs were synthesized using this SLPIE platform, encapsulated in LNPs, and evaluated for antidiabetic efficacy. Results Our SLPIE system facilitated efficient RNA circularization. Systematic optimization identified the EV-B107_IRES as highly compatible with the SLPIE strategy and superior in translational efficiency. Incorporating 5–7% m⁶A modification during RNA synthesis enhanced translation via DOPE-based LNPs while maintaining minimal immunogenicity. Notably, scarless circRNAs encoding GLP-1 or its derivative, GLP-1_FcBO (fused with an IgG-Fc affinity peptide), demonstrated potent and sustained hypoglycemic and anti-obesity effects in diabetic mice, matching the efficacy of Semaglutide over a 7-day period. Additionally, we established a scalable production platform using an RNase III -deficient   E. coli   strain. Conclusion The SLPIE system thus offers a simple, flexible, and scalable advancement for circRNA therapeutics and opening new avenues for treating diabetes and obesity.