Opportunity at National Institute of Standards and Technology NIST
Structural characterization of LNP/mRNA vaccines by combining CryoEM, SAXS, and SANS
Material Measurement Laboratory, Biomolecular Measurement Division
Please note: This Agency only participates in the February and August reviews.
|Thomas Edgar Cleveland
Lipid nanoparticles (LNPs) can be formulated to deliver nucleic acids to cells; for instance, mRNA for vaccines, siRNA to silence the expression of genes involved in disease processes, or potentially DNA for gene therapy applications. Retention of the nucleic acid cargo in LNPs under conditions of neutral pH, and targeted release upon acidification once internalized into cells, is facilitated through the use of ionizable lipids with carefully chosen pKa values. Typically, LNP formulations also include additional lipids such as DSPC, cholesterol, and a small proportion of lipid with PEGylated head groups. The identity and proportions of these additional lipids can be adjusted in order to optimize LNP properties such as their diameter, net charge, and stability. The utility of LNPs for mRNA delivery has now been strongly demonstrated by their use in SARS-CoV-2 mRNA vaccines. However, many fundamental questions remain about LNP morphology and internal lipid/RNA distribution, and particularly how these relate to the stability and efficacy of drug formulations. For example, LNPs are often not simply uniform and spherical, but can have separate lipid and aqueous sub-compartments containing nucleic acid cargo. The significance of these compartments, and other morphological features, is not yet well-understood. We will use complementary cryogenic electron microscopy (CryoEM) and Small-Angle Neutron and X-ray Scattering (SANS/SAXS) studies in order to obtain complementary structural information on LNP/mRNA assemblies across multiple length scales. This information will then be combined into a unified molecular model of the distribution of LNP/mRNA structures within the formulation. Using SANS/SAXS measurements on formulations prepared (1) in the presence and absence of mRNA; (2) with and without deuteration of the lipid DSPC; and (3) at several solvent D2O concentrations, one can also experimentally measure the distribution of lipids and mRNA within the LNPs. CryoEM will be used as a complementary technique to observe the morphology of LNPs, and to inform and constrain the models used in analyzing the scattering data. Finally, molecular modeling and simulated image calculation will be used to support the assignment of constituent lipid distributions within the larger LNP structure. Correlation of this quantitative understanding of LNP structure and assembly with functional activity and stability will aid in the design of new formulations with improved properties.
LNP; Lipid Nanoparticle; RNA; mRNA; CryoEM; EM; electron microscopy; SAXS; SANS; scattering; lipid
Open to U.S. citizens
Open to Postdoctoral applicants