Synthesis of scarless circular RNAs expressing long-acting GLP-1RAs for type 2 diabetes therapy

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-deficientE. colistrain. CONCLUSION: The SLPIE system thus offers a simple, flexible, and scalable advancement for circRNA therapeutics and opening new avenues for treating diabetes and obesity.