Pure capped mRNA vaccine opens the door to more effective vaccines with lower chances of inflammation

Newswise — A Japanese research team has devised a strategy to generate extremely potent mRNA vaccines with exceptional purity by employing a distinctive cap for effortless isolation of the desired capped mRNA. This innovative 'Purecap' approach achieved the extraction of completely pure Cap2-type mRNA, demonstrating a 3-4 fold enhancement in the production of the immune-stimulating protein. These outcomes introduce the potential for more refined vaccines, reducing the likelihood of inflammation due to contaminants. The team's discoveries were documented in Nature Communications.

The efficacy of mRNA vaccines in combating various strains of the coronavirus has provided researchers with optimism regarding their prospective application as a cancer vaccine. Nonetheless, the purity of these vaccines poses a hindrance to this objective as impurities have the potential to activate the immune system, potentially leading to inflammation in the vicinity of the injection site—a frequently encountered vaccination side effect.

The introduction of impurities in mRNA vaccines commonly occurs during the capping process. This crucial step involves the addition of a cap structure that enhances mRNA translation while providing protection and stability. To achieve optimal purity, vaccines should ideally consist of 100% pure single-stranded mRNA, as caps can only be attached to single-stranded molecules. However, the presence of undesired double-stranded mRNA can diminish the overall purity of the vaccine.

By exploiting the distinct properties of single- and double-stranded mRNAs, the separation process can be accomplished through a method known as reversed-phase high-performance liquid chromatography (RP-HPLC). This technique effectively segregates mRNAs based on their hydrophobic or hydrophilic characteristics, which determine their affinity or aversion to water. Consequently, the separation mechanism relies on the differential attraction or repulsion of the mRNA molecules towards water.

Professor Hiroshi Abe (he/him), Project Assistant Professor Masahito Inagaki (he/him), and Project Associate Professor Naoko Abe (she/her) from the Graduate School of Science at Nagoya University, in collaboration with Tokyo Medical and Dental University, spearheaded a groundbreaking study. They employed a distinctive method called PureCap, which involved the incorporation of a hydrophobic tag during the capping process. This hydrophobic tag facilitated the straightforward separation of the tagged mRNA during the RP-HPLC stage. Subsequently, through a simple light treatment, the tag was easily eliminated, resulting in the production of a vaccine with an impressive purity level of 98%-100%.

"The moment we observed complete separation of capped and uncapped RNAs on the chart during the RP-HPLC process, we were genuinely thrilled by the outcome," expressed Hiroshi Abe. He continued, "Using the PureCap method, we achieved remarkable success in generating capped mRNA with a purity exceeding 98% for a coronavirus mRNA, which spans 4247 bases in length." The researchers were undoubtedly pleased with the high level of purity achieved through their innovative approach.

The research group devoted special attention to a group of cap structures, namely Cap0, Cap1, and Cap2, which are present in both animal and plant cells. However, the assessment of Cap2's functionality has posed challenges due to the unavailability of pure capped mRNA required for unbiased experimentation.

"Until now, mRNA vaccines have predominantly employed Cap0 and Cap1 structures as their cap types," explained Abe. "However, our technique enabled us to produce mRNA vaccines with Cap0, Cap1, and Cap2 structures," he added. The highly purified mRNA vaccines synthesized using the PureCap method exhibited reduced immunostimulatory activity compared to those produced using conventional techniques. These findings highlight the potential utilization of highly pure Cap0, Cap1, and Cap2-type mRNA in pharmaceutical applications, emphasizing their significance in the field.

Since viruses predominantly generate Cap1 mRNA, the immune system's response to Cap2 mRNA is comparatively milder. This observation implies that a vaccine utilizing Cap2 may have a reduced likelihood of inducing undesirable side effects like inflammation upon injection. Nonetheless, the Cap2 mRNA would still be capable of transcribing viral proteins necessary for the vaccine's efficacy. Thus, employing Cap2 in vaccines could potentially strike a balance between minimizing adverse reactions while maintaining the desired immune response for effective protection.

Using the Purecap technique, the research group successfully generated Cap2 mRNA and conducted an analysis of its protein synthesis capabilities. The results revealed that Cap2 mRNA exhibited a remarkable protein production capacity, generating 3-5 times more protein compared to Cap1 mRNA. This heightened protein synthesis has the potential to significantly enhance the immune response. Additionally, the researchers demonstrated that their Cap2-type mRNAs induced lower levels of inflammatory response compared to mRNAs produced using conventional methods. This suggests that Cap2-type mRNAs synthesized through the Purecap approach could potentially reduce the risk of inflammation as a side effect, further highlighting their potential in the development of safer and more effective vaccines.

Abe emphasized the limitations of conventional mRNA vaccine production methods in generating highly pure capped mRNA, which raised concerns about compromised protein synthesis and the potential for inflammatory reactions due to impurities. He highlighted that the PureCap method effectively resolves these challenges by selectively purifying only the capped mRNA, thereby ensuring high purity. Additionally, the Cap2-type structure created through this technique demonstrates superior efficiency in protein synthesis and lower immune system irritability. This breakthrough has immense potential to enhance the safety and efficacy of mRNA vaccines, marking a revolutionary advancement towards the practical implementation of mRNA medicine. Furthermore, it deepens our understanding of the fundamental principles of mRNA science, expanding the horizon of scientific knowledge in this field.