Capillary-Mediated Vitrification: Preservation of mRNA at Elevated Temperatures.

ABSTRACT

RNA is a fundamental tool for molecular and cellular biology research. The recent COVID-19 pandemic has proved it is also invaluable in vaccine development. However, the need for cold storage to maintain RNA integrity and the practical and economic burden associated with cold chain logistics highlight the need for new and improved preservation methods. We recently showed the use of capillary-mediated vitrification (CMV), as a tool for stabilizing temperature-sensitive enzymes. Here, we demonstrate the use of CMV as a method to preserve mRNA. The CMV process was performed by formulating a green fluorescent protein (GFP)-encoding mRNA with common excipients, applying the solution to a porous support, referred to as the scaffold, and drying the samples under vacuum for 30 min. The CMV preserved samples were stored at 55 °C for up to 100 days or 25 °C for 60 days and analyzed by electrophoresis and for transfection efficiency in a cell-based assay. The 55 °C-stressed mRNA exhibited comparable electrophoresis banding patterns and band intensity when compared to a frozen, liquid control. Additionally, the CMV stabilized mRNA maintained 97.5 ± 8.7% transfection efficiency after 77 days and 78.4 ± 3.9% after 100 days when stored 55 °C and analyzed using a cell-based assay in the CHO-K1 cell line. In contrast, a liquid control exhibited no bands on the electrophoresis gel and lost all transfection activity after being stored overnight at 55 °C. Likewise, after 60 days at 25 °C, the CMV-processed samples had full transfection activity while the activity of the liquid control was reduced to 40.1 ± 4.6%. In conclusion, CMV is a simple formulation method that significantly enhances the thermal stability of mRNA, requires minimal processing time, and could enable formulation of mRNA that can tolerate exposure to temperatures well above 25 °C during shipment and deployment in extreme environments.