Resolving the architecture of mRNA lipid nanoparticles through advanced NMR and fluorescence techniques – LNP-HiRes

Lipid nanoparticles (LNPs) are a newly emerging type of drug formulation that encapsulates biological molecules such as nucleic acids and proteins. They have recently emerged as effective vehicles for mRNA vaccines. The performance, stability and delivery properties of these particles crucially depend on their architecture. However, due their complexity, their molecular-scale organization has so far escaped from full characterization. Several important structural features, such as the interactions between their many components, the environment of the nucleic acid cargo, its hydration or the lipid distribution, remain elusive, preventing a rational design of improved formulations.

Atomic force microscopy (AFM) imaging, cryogenic transmission electron microscopy (cryo-TEM) as well as small-angle X-ray scattering (SAXS) can in principle be applied to image the individual LNPs, their size and shape, and internal fine structure. Yet, none of these techniques provides molecular-scale insight into the LNP structure nor quantitative compound-specific information.

Our project aims at developing innovative analytical approaches to examine the internal atomic, molecular and nanoscale structure of mRNA-loaded LNPs. Leveraging recent instrumental and methodological breakthroughs in DNP (Dynamic Nuclear Polarisation) and ultra-fast Magic Angle Spinning (MAS) NMR (Nuclear MAgnetic Resonance), the first objective of the LNP-HiRes project is to implement high-sensitivity solid-state NMR approaches to probe the internal architecture of LNPs. Notably we aim at identifying the location of the mRNA cargo, probing its hydration and secondary structural elements as well as its interactions with other LNP components. The development of advanced fluorescence spectroscopy and imaging techniques specifically tailored to LNPs is the second objective. This will include the design of fluorescent probes that will locate in specific LNP environments as well as the development of single particle imaging techniques. The methods will be benchmarked on LNPs of pharmaceutically relevant compositions as well as on innovative formulations, not yet in use in industry, prepared by appropriate microfluidic protocols. The preparation of customized mRNA strands that will incorporate isotopically (15N,13C or 2H) labelled or chemically-modified fluorescent nucleotides will be essential to reach these two first objectives. Improving the current understanding of LNP structure and refining existing models by analyzing jointly the NMR and fluorescence data is the final objective of the project. Links between LNP composition and structural features will be established and put in perspective, whenever possible, with differential biological readouts.
Our project is expected to create new basic knowledge and provide advances beyond current state-of-the-art with the implementation of innovative biochemical strategies for the preparation of customized NMR- and fluorescence-active mRNA strands as well as the introduction of pioneering solid-state NMR and fluorescence methodologies, tailored for the in-depth investigation of multicomponent nano-objects. Unique structural insights into LNPs at the atomic, molecular and nanometer scales will be provided. LNPs represent one of the most widely used and promising platforms for the mRNA therapeutics. The project results are expected to lead to an improved understanding of their organization, stability and (physicochemical and biological) properties, fostering further innovation in the field of mRNA vaccines.