In the manufacturing process of mRNA vaccines and drugs, in vitro transcription (IVT) technology is utilized to produce mRNA on a large scale. To reduce mRNA's immunogenicity and enhance mRNA drug stability, researchers need to introduce modified nucleotides, including N1-methyl-pseudouridine (1-methylΨ), during the IVT process. This successful application has allowed mRNA drugs, once considered challenging to develop, to reach households worldwide, providing an effective means for preventing and treating diseases.
However, scholars from the University of Cambridge recently found that 1-methylΨ modification can lead to +1 frameshifting and off-target T-cell immune responses in both animals and humans, suggesting a potential "defect" in mRNA vaccine technology.
To investigate the impact of nucleotide modifications on translation, researchers constructed a luciferase system capable of reporting +1 frameshifting. This system can express detectable luciferase upon the occurrence of +1 frameshifting. After introducing nucleotide modifications such as 5-methylcytidine (5-methylC) and 1-methylΨ during mRNA processing, the researchers observed a significant reduction in the synthesis of wild-type luciferase. However, the abnormal synthesis levels induced by 5-methylC and 1-methylΨ were less than other modifications, indicating that these two modifications only partially affect mRNA translation.
In the +1 frameshifting luciferase reporting system, high-intensity luminescence was detected only in the 1-methylΨ modification group. This confirms that 1-methylΨ modification leads to a significant amount of +1 frameshifting.
Due to the use of 1-methylΨ in commercially available mRNA vaccines, researchers investigated whether the frameshifting and resulting mistranslation induced off-target T-cell immune responses. Researchers detected T-cell responses targeting frameshifted products in mice vaccinated with the BNT162b2 mRNA COVID-19 vaccine. The results showed that mice immunized with mRNA vaccines exhibited a higher immune response against +1 frameshifted spike proteins than those immunized with the Oxford-AstraZeneca COVID-19 vaccine (viral vector). Although both vaccines could elicit correct immune responses, the +1 frameshifted products in mRNA vaccines led to unexpected off-target immune responses.
Subsequently, researchers tested the IFNγ response levels in 21 individuals vaccinated with the BNT162b2 mRNA COVID-19 vaccine and 20 individuals immunized with the Oxford-AstraZeneca COVID-19 vaccine. They found that individuals vaccinated with mRNA vaccines showed higher IFNγ response levels against +1 frameshifted products, confirming the results observed in animal experiments. Since natural translation of the SARS-CoV-2 virus only involves -1 frameshifting, researchers believed that the cellular immune response against +1 frameshifted products was likely induced by mRNA COVID-19 vaccines.
To further explore the mechanism of +1 frameshifting, researchers purified +1 frameshifted products and identified six in-frame peptides and nine peptides related to +1 frameshifting. Sequence analysis revealed that a linker peptide could connect in-frame products and +1 frameshifted products, resulting in an extended peptide.
Simultaneously, the study raised the question of whether nucleotide modifications might affect protein synthesis by reducing the accuracy of the transcription process. Through quantitative analysis of the transcription process, researchers found that the mutation frequency of 1-methylΨ during transcription was not significantly different from unmodified mRNA, proving that the elongated products observed in the 1-methylΨ group were not caused by abnormalities in the transcription process but rather by a post-transcriptional mechanism.
Ribosomal frameshifting is a common translation error in the process of translation. Previous studies have reported ribosomal stalling in the context of ribosomal frameshifting. Therefore, researchers suspected that the introduction of 1-methylΨ interfered with the process of ribosomal translation, ultimately leading to frameshifting. To investigate this, researchers used isotopically labeled methionine to study the translation process. The results showed that the translation speed of 1-methylΨ mRNA was significantly slower than that of unmodified mRNA, and the slower translation speed was likely caused by ribosomal stalling. This ribosomal stalling might be due to differences in the decoding function of aminoacyl tRNA. Erythromycin, which can alter the conformation of the decoding center, was used to intervene in the translation of 1-methylΨ mRNA. The results showed that erythromycin significantly increased the translation speed of 1-methylΨ mRNA while inhibiting the translation of unmodified mRNA. These data suggest that the slower translation speed of 1-methylΨ mRNA is caused by ribosomal stalling, and the reason for ribosomal stalling is the altered dynamics of aminoacyl tRNA binding.
TIn the final part of the study, researchers explored the "slippery sequences" that led to ribosomal stalling. These sequences result in faster ribosome movement, leading to misreading and frameshifting. Through sequence analysis, researchers identified three potential "slippery sequences." When 1-methylΨ was replaced with C at positions 187 and 208, the level of +1 frameshifted products generated during translation significantly decreased. Double mutations at positions 187 and 208 reduced +1 frameshifted yields to undetectable levels. Mutations targeting both positions did not significantly affect the efficiency of translation. These pieces of evidence indicate that introducing 1-methylΨ at these positions is a crucial factor causing ribosomal +1 frameshifting.
We cannot deny the significant role that mRNA drugs have played in the COVID-19 pandemic, and abundant clinical data also attest to the fact that the ribosomal +1 frameshifting induced by them has not resulted in obvious adverse effects on the human body. However, as mRNA therapy continues to advance, and with the emergence of higher doses of mRNA drugs, these +1 frameshifted products may potentially pose unexpected risks to patients. Developers of mRNA drugs should be vigilant about "slippery sequences" and strive to avoid their impact on drug safety and efficacy during the drug development process.
[1] Mulroney, T.E., Pöyry, T., Yam-Puc, J.C. et al. N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting. Nature (2023). https://doi.org/10.1038/s41586-023-06800-3.
