(M1130-01-06) Structural Profiling of mRNA Vaccine Constructs: Applications for mRNA Vaccine Design and Quality Assessment

Purpose: The emergence of mRNA-based vaccines in recent years can be attributed to their unique advantages over inactivated and live-attenuated platforms, including an improved safety profile, high translation efficacy, and rapid, inexpensive, and scalable production. Additional improvements in platform technology to address mRNA translation and stability, immunogenic response, and developments in delivery systems, such as lipid nanoparticles (LNPs), have also strengthened the field. The successes of both the BNT162b2 and mRNA-1273 LNP-mRNA vaccines aimed at alleviating the COVID-19 pandemic have further underscored the potential of this technology. Despite the promise of this therapeutic platform, an open question remains about how the structural morphology of an mRNA construct relates quality attributes such as translation efficiency. New analytical methods are needed to generate a more complete and robust understanding of the structural profiles of these mRNA constructs.

Methods: To begin addressing these issues, here we demonstrate a structural ‘fingerprint’ of model mRNA constructs using the chemical probing technique, selective 2’-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) metrology.

Results: The degree of reactivity of each 2’-hydroxyl group is directly related to its solvent exposure and has been used to generate force fields for structural modeling. However, when used in a comparative mode, this method generates nucleotide-level ‘fingerprints’ that can be used to directly examine the effect of nucleoside modifications and open reading frame substitutions. Using model mRNA constructs with the same untranslated regions (UTRs), we demonstrate how the SHAPE-MaP technology reveals structural differences in the UTRs. Such information will allow for more rational mRNA drug design. Further, using SHAPE-MaP in comparative mode may also allow for developing metrics for quality assessment, to ensure the safety and efficacy of mRNA vaccine products.

Conclusion: Taken together, the SHAPE-MaP metrology seeks to fulfill a gap in mRNA analytical characterization by taking the first steps in establishing methods to assess mRNA structure that can be harmonized to meet the rigors of regulatory guidelines.



Acknowledgements: Certain commercial equipment, instruments, and materials are identified in this paper in order to specify the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the material or equipment identified is necessarily the best available for the purpose.