Divergent Delivery and Expression Kinetics of Lipid and Polymeric Nanoparticles across mRNA Modalities.

Messenger ribonucleic acid (mRNA)-based therapies, including conventional linear mRNA (linRNA), circular RNA (circRNA), and self-amplifying RNA (saRNA), are being developed not only for vaccination but also for protein replacement, gene editing, and regenerative medicine. However, these mRNA modalities differ in structure and function, and their interactions with current non-viral delivery systems influence their therapeutic efficacy. Here, the in vivo expression kinetics of linRNA, circRNA, and saRNA delivered via lipid nanoparticles (LNPs) or bioreducible poly(cystamine bisacrylamide-co-4-amino-1-butanol) (pABOL) polymer are systematically evaluated. At 0.5 µg, Venezuelan equine encephalitis virus (VEEV)-based saRNA resulted in higher total luciferase expression than 5 µg of linRNA or circRNA highlighting its superior potency. LNPs significantly enhanced expression of non-amplifying mRNAs compared to pABOL, whereas pABOL delivery of saRNA yielded a ∼2-fold improvement over LNPs. Furthermore, saRNAs derived from New World alphaviruses expressed 2-6 times more protein than Old World saRNAs when delivered with LNPs; these differences are not observed with pABOL. These findings demonstrate that mRNA modality, saRNA genotype, and delivery platform interact to determine therapeutic protein output. This study provides actionable insights for optimizing mRNA-based therapeutics across diverse clinical applications.