Regulatory Assessment Gaps and Genotoxicity Considerations in mRNA-LNP Technologies

Current FDA and ICH genotoxicity requirements allow pharmaceutical developers to meet safety obligations using legacy assays designed for small, cell permeable chemicals, resulting in the use of bacterial reverse-mutation tests (Ames) and in vitro micronucleus assays in human peripheral blood mononuclear cells (PBMCs) to evaluate the presumed non genotoxicity of lipid nanoparticle (LNP) based platforms, including ionizable lipids used in mRNA vaccines. However, these assays have the potential to be structurally and mechanistically incapable of detecting relevant risks. Ionizable lipids and fully formulated LNPs exceed the size exclusion limits of bacterial porins in Escherichia coli and Salmonella typhimurium, preventing intracellular entry and interaction with genomic DNA; therefore, negative Ames results do not rule out mutagenic potential. Similarly, in vitro micronucleus assays reported as negative for COVID-19 mRNA vaccines used non activated PBMCs, which largely consist of lymphocytes that do not efficiently endocytose LNPs or free ionizable lipids due to size, charge, and absence of uptake pathways; as a result, the negative findings reflect absent intracellular exposure rather than absence of risk. Ionizable lipids can form covalent adducts with chemically modified RNA in vitro and may likewise bind residual linearized plasmid DNA contaminants present in mRNA vaccine preparations and potentially with human nucleic acids, a high-risk concern that has not been tested in vivo. If plasmids reach the nucleus, they could integrate into genomic DNA, while cytosolic plasmids may activate cGAS-STING signaling. Free or LNP associated ionizable lipids can electrostatically interact with nucleic acids once intracellular, further increasing the plausibility of mutagenic or immunomodulatory effects. Because existing genotoxicity assays do not deliver ionizable lipids or LNPs into relevant human cell compartments, their negative results cannot be considered evidence of safety, yet the FDA and other relevant agencies have not addressed this concern. Updated testing frameworks must use formulated LNPs in human cell models that support endocytosis, endosomal escape, nuclear access, and analytical detection of covalent adducts using mass spectrometry, reverse-phase ion-pair HPLC, additional genomics testing, and downstream microRNA and proteomic analyses to accurately evaluate genomic and immunological risk.