Expediting in vitro characterization of mRNA-based gene therapies via high-content fluorescent imaging

mRNA-based therapeutics have progressed significantly over the past decade and hold great potential to treat several diseases, including but not limited to cancer, infectious diseases, and genetic disorders. Many mRNA therapeutics are encapsulated in lipid nanoparticles (LNPs), which stabilize the mRNA and, following patient administration, transfect target cells to deliver the therapeutic mRNA. Developing reliable and robust in vitro bioassays is critical for expediting early screening of LNP-mRNA formulations, as well as identifying and developing lead candidate drug products. In this study, high-content fluorescent imaging was used to monitor LNP-mRNA transfections in both two- and three-dimensional HepG2 cell cultures. This technique allowed for continuous and simultaneous measurement of multiple LNP-mRNA transfection parameters, including cellular cytotoxicity, mRNA delivery, and mRNA translation/protein expression. Moreover, high-content imaging demonstrated acceptable accuracy and precision when quantifying the potency of mRNA delivery and subsequent translation and protein expression, thereby establishing the method as fit-for-purpose. Furthermore, concurrent evaluation of two different LNP formulations showed high-content imaging is sensitive at detecting pharmacological differences among varying LNP formulations. This study details the efficiency and reliability of LNP-mRNA characterizations that can be accomplished with high-content imaging.