Pre-clinical evaluation of mRNA-lipid nanoparticles’ potency and toxicity: current practices and future directions

Abstract Over the last few years, the success of COVID-19 mRNA vaccines has resulted in the emergence of RNA lipid nanoparticles (LNPs) with promising prospects for the prevention and treatment of various diseases. The context of the SARS-CoV-2 pandemic has led to the rapid development of vaccines with abbreviated non-clinical programs. However, there are currently no official guidelines defining the required standards for global marketing of mRNA based therapeutic products. Nevertheless, to guarantee a well-controlled product, it is essential to characterize both the drug substance and the final product in terms of their structure, composition, formulation, physico-chemical features, potency, and safety. This lack of guidance has resulted in a wide variety of heterogeneous in vitro tests being used to assess the potency and cytotoxicity of RNA-LNP. This review discusses the commonly used in vitro assays, primarily 2D monolayer assays, employed to evaluate the biological properties of RNA-LNP. We then explore novel alternative methods to bridge the gap between in vitro and in vivo results. We summarize (i) co-culture models, (ii) multilayer 3D assays and (iii) in vivo replacement models, exploring their potential applications in assessing the potency and safety of RNA-LNPs. Finally, we discuss the use of in silico and machine learning as models for optimizing and predicting the biological behavior of RNA-LNPs. Graphical Abstract