Lipid nanoparticles (LNPs) are clinically established carriers for nucleic acid therapeutics, most notably messenger RNA (mRNA). Beyond mRNA, LNPs can be used to deliver alternative gene-expressing nucleic acids with distinct biological properties, offering opportunities to modulate expression kinetics. Here, we compared LNPs formulated with firefly luciferase (FLuc)-encoding mRNA, self-amplifying RNA (saRNA), and plasmid DNA (pDNA), as well as defined combinations of these payloads, to assess how payload identity and composition influence expression in vitro and in vivo. Using a constant ALC-0315-based lipid composition and microfluidic formulation process, single-payload LNPs displayed distinct expression profiles following intramuscular and intravenous administration in mice: mRNA produced high, transient expression levels, saRNA produced delayed and sustained expression, and pDNA enabled prolonged low-level expression. Combined-payload formulations, prepared either by mixing the payloads before LNP formulation or by mixing separately formulated LNPs, exhibited expression profiles consistent with the proportional contributions of their constituent payloads, providing proof-of-concept evidence of additive behaviour under the conditions tested, with no clear evidence of synergistic or antagonistic interactions. Characterisation of LNP physicochemical properties, including mean hydrodynamic diameter, polydispersity, zeta potential, and encapsulation efficiency, showed minimal differences between formulations. These findings indicate that, within the common formulation system investigated, differences in expression profiles were associated with payload identity and composition. Overall, this study shows that distinct expression kinetics can be observed when different nucleic acid payloads are delivered using a common LNP formulation and that combined-payload preparations can exhibit additive expression behaviour under defined experimental conditions.
