Identifying the risks of a potentially negative immune response to an mRNA therapeutic before it is delivered to patients may now be possible thanks to new research from scientists in the Medical Research Council (MRC) toxicology unit at the University of Cambridge and elsewhere. The team identified a sequence in therapeutic mRNAs that if misread by the cell’s machinery can cause an unintended immune response and found a way to prevent this from happening.
Details of the work are published in a Nature paper titled, “N1-methylpseudouridylation of mRNA causes +1ribosomal frameshifting.” To summarize, what they discovered is that the cellular machinery that reads mRNAs “slips” when it meets strings of N1-methylpseudouridinated bases. Modified ribonucleotides like this are an important feature of therapeutic mRNAs as they have been shown to “decrease innate immunogenicity” and “increase mRNA stability.” However, when ribosomes encounter these bases in the mRNA, they slip around 10% of the time causing the mRNA to be misread. This can result in the production of unintended proteins, which can trigger an immune response.
Working with scientists at the Universities of Kent, Oxford, and Liverpool, the MRC team looked for evidence of off-target proteins in people who received Pfizer’s mRNA vaccine against COVID-19. Of the 21 vaccinated patients in the study, about a third had unintended immune activity although it’s important to note that the patients did not experience any ill-effects as a result. The researchers also confirmed that the observed off-target proteins were likely not the result of infection by SARS-CoV-2 but instead a response to the vaccine.
Other ribonucleotide modifications could be used in therapeutics, such as 5-methoxyU. But that particular option “significantly decreased translation efficiency of IVT mRNAs, which may limit clinical translation,” the team wrote. To prevent slips from occurring with use of N1-methylpseudouridine, the team designed so-called “slip-resistant” mRNA sequences that remove the runs of these modified bases. This correction resulted in the production of the intended protein without making off-target ones.
Design modifications like this could one day be used to improve future therapeutics and vaccines and the team believes they should. “[F]or future use of mRNA technology it is important that mRNA sequence design is modified to reduce ribosome frameshifting events, as this may limit its future use for applications that require higher doses or more frequent dosing, such as the in vivo production of hormones,” they wrote.
It is also possible that other causes of protein mistranslation have not yet been identified or investigated. Studies like this are crucial for ensuring that future therapies and vaccines are as safe as they are effective. “These new therapeutics hold much promise for the treatment of a wide range of diseases,” said Anne Willis, PhD, director of the MRC toxicology unit and joint senior author of the paper. “As billions of pounds flow into the next set of mRNA treatments, it is essential that these therapeutics are designed to be free from unintended side-effects.”
James Thaventhiran, MD, an investigator in the MRC toxicology unit and joint senior author on the paper, expressed similar sentiments in his comments. “Research has shown beyond doubt that mRNA vaccination against COVID-19 is safe. Billions of doses of the Moderna and Pfizer mRNA vaccines have been safely delivered, saving lives worldwide,” he noted. “We need to ensure that mRNA vaccines of the future are as reliable. Our demonstration of ‘slip-resistant’ mRNAs is a vital contribution to the future safety of this medicine platform.”
