Jason Downing- Senior Product Manager, TriLink BioTechnologies®
1. What is mRNA capping and how does it contribute to the production of vaccines and therapeutics?
Although it is a ubiquitous biological molecule, mRNA is notoriously unstable. As we continue to innovate and explore the possibilities of using mRNA in next-generation vaccines and therapeutics, a key consideration requires addressing the stability issue. Adding a cap to mRNA is analogous to having a bumper on a car. While mRNA caps have many other uses, one of their primary functions is acting as a protective barrier for the mRNA payload in the center, preventing degradation within the cell. In addition to mRNA stability, the 5’ cap plays an important role in gene expression and serves to “mark” the mRNA to the innate immune system.
Because of this, capping is a vital component in producing mRNA-based vaccines and therapeutics. Any RNA molecule that does not have a cap would be degraded so rapidly it would be unable to perform its function. As such, successfully bringing an mRNA vaccine or therapeutic to market depends on having an effective capping mechanism that maintains mRNA stability and potency and enables quick and cost-effective scale-up.
2. What are the different mRNA capping technologies currently used in the production of vaccines and therapeutics?
Three major capping technologies used in the production of mRNA vaccines and therapeutics include anti-reverse cap analog (ARCA) technology, enzymatic capping, and CleanCap® technology. Different capping approaches have varying efficiencies and yield different types of caps. The different types of caps that can be produced include the intermediate cap 0 structure, as well as the cap 1, cap 2, and N6 ,2’-O-dimethyladenosine (m6 Am) cap structures found in mature eukaryotic mRNAs. While the immunogenic role of mRNA caps still requires elucidation, cap 1 structures are thought to authenticate mRNAs as “self RNA”. The function of cap 2 remains unclear. Cap 0 RNAs show much lower activity in vivo compared to cap 1 RNAs.
The ARCA method is one of the original capping approaches. This co-transcriptional method creates a cap 0 (non-self ) structure with approximately 80 percent capping efficiency. On the other hand, enzymatic capping involves post-transcriptional capping using capping enzymes to produce a cap 0 or cap 1 structure, depending on the enzymes used. While this method requires multiple reactions and purification steps, these can be optimized for more straightforward processing. Finally, CleanCap® technology is a proprietary cap analog producing a cap 1 (self ) structure incorporated by RNA polymerase in a single in vitro transcription reaction. Additionally, it is available in several various cap structures, including CleanCap® AG 3’ OMe and CleanCap® M6 for improving protein translation, along with CleanCap® AU specifically for saRNA. The design of this method offers several advantages by increasing capping efficiency while reducing process time.
3. How does mRNA capping ensure the stability and efficiency of mRNA molecules in vaccine and therapeutic development?
Natural mRNA caps made endogenously within the cell have the primary function of shutting immature RNA out of the nucleus and facilitating mRNA processing and maturation. However, when mRNA has been produced exogenously, such as in the context of mRNA-based vaccine and therapeutics development, the cap has another critical role. In this case, capping affords therapeutic mRNA molecules a high degree of stability by preventing its rapid degradation once inside the cell, enabling the mRNA to maintain its therapeutic function and potency. Furthermore, caps with a cap 1 structure do not trigger an immune reaction and therefore do not detract from the efficacy of the vaccine or therapeutic product.
Additionally, regarding manufacturing, different capping methods can also improve how efficiently manufacturers can produce a vaccine or therapeutic product. For example, CleanCap® technology’s one-pot solution reduces manufacturing steps allowing for quicker turnaround times, easier scale-up, and higher transcriptional yields than other methods - all of which have an overall positive impact on manufacturing costs.
4. What are the advantages and limitations of different mRNA capping technologies for producing vaccines and therapeutics?
Each method of mRNA capping offers different advantages and disadvantages. For example, the ARCA approach tends to provide lower transcriptional efficiency and produces a cap 0 structure that is not recognized as self in eukaryotes and thus can cause higher immunogenicity. On the other hand, enzymatic capping can achieve higher capping efficiency rates than ARCA, but it usually requires additional purification steps. In contrast, CleanCap® technology produces a natural cap 1 structure, reducing immunogenicity. Moreover, it is a one-pot solution with fewer manufacturing steps that caps RNA molecules at about 95 percent efficiency.
The importance of choosing the correct mRNA capping technology extends beyond just having technical implications. Manufacturers should remember that a capping reagent impacts both downstream and upstream processes, impacting cost, time, yield, and purity- all of which affect a vaccine or therapeutic’s journey to market.
5. How do mRNA capping technologies impact the immunogenicity and efficacy of mRNA-based vaccines and therapeutics?
A vaccine is a molecule that stimulates the body to mount an immune response against a pathogen of entry, whether a bacterium or a virus. Critical to effective vaccine design is ensuring that the body’s immune system only responds to the specific gene sequence responsible for a particular disease but not other vaccine components. As such, all capping components must be immune-silent to ensure the body generates the desired immune response, limiting the potential for off-target effects and immunogenicity. Since an uncapped 5’-phosphate mRNA is immunogenic, it is critical that the mRNA is capped so it does not cause an immune response. The cap binds to the mRNA translation initiation factor and initiates the mRNA translation.
In practice, vaccine development requires delicately balancing potency with specificity. The goal is to generate an immune response specific to the pathogen of entry and not the vaccine itself.
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