A Metabolite Co-Delivery Strategy to Improve mRNA Lipid Nanoparticle Delivery

Lipid nanoparticles (LNPs) effectively protect mRNA and facilitate its entry into target cells for protein synthesis. Despite these successes, cellular entry alone may not be enough for optimal protein expression, as mRNA translation also depends on the availability of essential metabolites, including metabolic energy sources, coenzymes, and amino acids. Without adequate metabolites, mRNA translation may be less efficient, potentially leading to higher dosing requirements or poorer therapeutic outcomes for LNP therapies. To address this, we develop a metabolite co-delivery strategy by encapsulating essential metabolites within mRNA LNPs, hypothesizing that our approach can uniformly improve mRNA delivery. Instead of adding a fifth component to the organic phase, our strategy involves mixing the metabolite with the mRNA payload in the aqueous phase, while maintaining the molar ratio of the components in the organic phase during LNP formulation. We verify our approach <i>in vitro</i> and <i>in vivo</i>, highlighting the broad applicability of our strategy through mechanism and efficacy studies across multiple cell lines, and physiological conditions, such as normoxia (i.e., 21% oxygen), hypoxia (i.e., 1% oxygen), and in mice. Taken collectively, we anticipate that our metabolite co-delivery strategy may serve as a generalizable strategy to enhance <i>in vitro</i> and <i>in vivo</i> protein expression using mRNA LNPs, potentially offering broad applicability for the study and treatment of disease.