RNA is a critical component for both molecular and cellular biology research. Because of its inherent instability, RNA is generally stored frozen to preserve activity and to avoid degradation. However, even when stored frozen at ultracold temperatures (− 80 °C and below), the RNA may not maintain full activity when stored for extended periods. The limited shelf-life and complex logistics associated with cold-storage have a significant negative impact on both the financial costs and manpower required to execute laboratory studies with RNA. Likewise, RNA-based vaccines and therapeutics also suffer from these issues. One critical example is the impact of cold storage requirements on the availability of COVID-19 vaccines in low-income countries. Some estimates suggest that as a result of the shorter shelf-life and limited availability of cold storage in the developing world, as much as half of the mRNA vaccines produced to date have been wasted (4). The RNA markers degradation during long-term storage of forensic and clinical samples are also unsolved issues (9, 10).
Additionally, ultra-low-temperature freezers used to store RNAs cost up to $20,000/year to run continuously and each freezer releases up to 35,000 pounds of carbon dioxide (11). Freeze-drying or lyophilization can mitigate some of the issues with frozen storage, but often requires product-specific optimization, is both capital- and energy-intensive, requires long drying cycle time, and may not be suitable for complex formulations including those containing complex lipid assemblies (12,13,14). The storage of RNA in RNAshell minicapsules has been demonstrated as an alternative to lyophilization (5, 15). However, these processes cannot be easily adapted for therapeutic applications, and it is unclear whether the stabilization solution may interfere with some analytical applications including both cell-based assays and in vivo potency testing of drugs and vaccines. Development of RNA formulations that are stable under ambient conditions, suitable for use in in vivo and therapeutic applications, and can be utilized in regions where the temperatures routinely exceed 25 °C and are tolerant of temperature excursions that can occur during shipping and storage are needed. Compared to existing RNA stabilization methods, the CMV process is highly flexible and is compatible with in vivo applications. The scaffold can be designed to fit into a variety of containers ranging from standard lyophilization vials, microcentrifuge tubes, and disposable plastic ware or can be incorporated into novel containers that are process and work-flow specific. Additionally, the CMV process leverages only common excipients that are considered safe for both human health products and animal studies, while simultaneously minimizing the risk of analytical matrix interference.
In this study, we used the novel CMV process to preserve GFP-mRNA. CMV leverages a porous scaffold to increase surface area and enables capillary evaporation, enabling a rapid drying cycle without the risk of boiling or the need for a freezing step (7). CMV-preserved GFP-mRNA maintained both its integrity and transfection activity when stored long-term at 25 °C and higher temperature. The CMV processed samples had comparable transfection efficiency when stored at either room temperature or 55 °C, respectively, compared to the protein expression (fluorescence signal) observed for a frozen mRNA control (16, 17).
Although further stability studies are required, this data suggests CMV process may enable long-term storage of mRNA at the room or above temperatures and could provide an opportunity to reduce the cold chain requirements for mRNA vaccines. However, the impact of the CMV process on the structure of the lipid nanoparticle (LNP) formulation must be fully characterized to ensure that there is no change to the LNP size, shape, and product potency.
This proof-of-concept study also demonstrates the potential for using CMV to simplify the storage and transportation of analytical and clinical samples. As an example, the CMV process could be used to enhance the stability of neonatal blood spots which although they are stored dried, currently require long-term, frozen storage. The CMV process may also allow samples collected at international clinical study sites to be shipped under ambient conditions to a central testing laboratory for processing.
The CMV technology is expected to be applicable to a wide range of biological molecules. Previous studies have shown that a similar process, including the same excipient mixture and vacuum cycle, can also be used to stabilize proteins and enzymes (18,19,20). Thus, the process is believed to be easily adaptable to different types of biomolecules (7) and may enable storage, distribution, and deployment of RNA-based reagents, clinical samples, and therapeutics in regions where the cold chain is not readily available.