Among all the vaccines under development, the faster and more promising mRNA vaccine has received focused attention globally as an emerging technology. However, some intrinsic features of mRNA molecules demand special strategies to guarantee the stability, efficacy, and safety of mRNA vaccines. First, mRNA is intrinsically unstable and prone to degradation due to the omnipresence of RNases in the serum and plasma. Second, the cellular machinery recognizes exogenous RNA molecules as immunological mimic of viral infection, which results in an immediate immune response. Therefore, optimization of mRNA vaccines is a prerequisite to maximize RNA stability and translation efficiency and to avoid innate immune responses in host cells.
Main optimization strategies
mRNA vaccines comprise synthetic mRNA molecules that direct the production of the antigen that will generate an immune response. In vitro-transcribed (IVT) mRNA mimics the structure of endogenous mRNA, with five sections, from 5ʹ to 3ʹ: 5ʹ cap, 5ʹ untranslated region (UTR), an open reading frame (ORF) that encodes the antigen, 3ʹ UTR and a poly(A) tail. Each of these elements can be optimized and modified in order to modulate the stability, translation capacity, and immune-stimulatory profile of mRNA.
Figure 1. Structural features of IVT mRNA 
Mature mRNA requires a 5'-cap for gene expression and mRNA stability. There are two methods to add a cap in vitro: via a two-step multi-enzymatic reaction or co-transcriptionally. Co-transcriptional methods minimize reaction steps and enzymes needed to make mRNA when compared to enzymatic capping. In co-transcriptional capping, a cap analog is introduced into the transcription reaction, along with the four standard nucleotide triphosphates, in an optimized ratio of cap analog to GTP 4:1.
The 5' and 3' UTR elements flanking the coding sequence are associated with the mRNA replication and translation processes, both of which are critical concerns for vaccines. UTRs must be carefully chosen because they may also impair translation or mRNA stability. UTRs with highly expressed genes are generally selected, but optimized UTRs are also selected based on the fact that the performance of UTRs varies with cell type. These treated UTRs will reduce mRNA degradation by excluding mi-RNA binding sites and AU-rich regions on the 3'UTR. In addition, they will also reduce regions that prevent ribosomes from scanning the mRNA transcript.
The ORF of the mRNA vaccine is the most crucial component because it contains the coding sequence that is translated into protein. Although the ORF is not as malleable as the non-coding regions, it can be optimized to increase translation without altering the protein sequence by replacing rarely used codons with more frequently occurring codons that encode the same amino acid residue.
- Optimization of poly(A) tail
The polyadenylation tail stabilizes mRNA and increases protein translation. A sufficiently long tail (100–150 bp) is necessary to interact with poly(A) binding proteins that form complexes necessary for initiating translation and protecting the cap from degradation by decapping enzymes. Therefore, the length of poly (A) needs to be adjusted to optimize the translation efficiency of mRNA.
Solutions for mRNA vaccine optimization
The optimization of mRNA vaccines is necessary for the stable expression of mRNA in vivo. Alfa Chemistry has in-depth knowledge of nucleic acid theory and advanced mRNA vaccine technology to synthesize "cap" like structures, add regulatory sequences to the 5' and 3' UTR regions, and modify the poly (A) tail.
Why choose us
Alfa Chemistry guarantees to provide customers with efficient and high-quality mRNA vaccine optimization solutions. If you have any questions related to mRNA vaccine production, please feel free to contact us.
- Chaudhary. N.; et al. mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nature Reviews Drug Discovery. 2021, 20: 817-838.
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