Investigation of the impact of lipid nanoparticle compositions on the delivery and T cell response of circRNA vaccine

Circular RNA (circRNA) is an emerging class of vaccines for various diseases, such as cancer immunotherapy. For cancer therapeutic vaccines, it is critical to deliver circRNA to lymphoid tissues such as lymph nodes (LNs) and dendritic cells (DCs) and then elicit antigen-specific T cell responses. Lipid nanoparticles (LNPs) have shown great success for mRNA vaccines and may also have great potential as nanocarriers for circRNA vaccines. Here, we studied the impact of LNP composition on the efficiency of immune delivery, protein expression, and the T cell responses for circRNA vaccine. First, we used model mRNA and circRNA encoding firefly luciferase (mRNA-fLuc) to study protein expression and used two small circRNA vaccines to study T cell responses. We investigated a combination of six ionizable lipids, three helper lipids, and six different molar ratios of cholesterol and β-sitosterol for their impact on the physicochemical properties of RNA LNPs, in vitro DC transfection, in vivo protein expression in draining LNs, and antigen-specific T cell responses. Among these ionizable lipids, SM-102 was the most effective for DC transfection and enabling circRNA vaccines to elicit T cell responses. DOPE and β-sitosterol incorporation in LNPs resulted in efficient protein expression, albeit β-sitosterol incorporation appeared to be associated with reduced T cell response. Overall, circRNA was efficiently delivered to DCs and macrophages in mouse draining lymph nodes by LNPs of SM-102 (50 %), cholesterol (38.5 %), DOPE (10 %), and DMG-PEG2000 (1.5 %), resulting in the induction of potent antigen-specific CD8⁺ T cell response in mice. These findings may provide insights into designing the compositions of LNPs as the carrier for circRNA therapeutics and vaccines.