Translational implications of off-frame stop codon optimisation in RNA therapeutics

N1-methyl-pseudouridine (m1Ψ) is a modified nucleoside used in therapeutic mRNAs to reduce immune responses; however, its use leads to a higher rate of random frameshift events during translation. Inclusion of stop codons in the +1 reading frame (+1 FSS) is proposed as a mitigation strategy against increased aberrant peptide products arising from frameshift events. The efficacy of mRNA vaccines can be increased through a reduction in off-target antigens. Emerging applications of mRNA therapeutics to genetic disease and cancer require higher dosage levels and treatment frequency; potential side effects could arise from increased burden of aberrant proteins, otherwise manageable by cellular processes at lower concentrations. This study investigates expression levels and kinetics of a Nanoluciferase reporter protein in response to differing levels of +1 FSS. Experimental sequences range in +1 FSS content from zero to the maximum possible given for the same amino acid sequence. Expression results indicated a ~20% reduction in NanoLuc expression at 100% +1 FSS content relative to the commercial sequence, aligning with a higher content of sub-optimal codons resulting from inclusion of +1 FSS. Increased expression was observed at 25% +1 FSS, suggesting a role of improved secondary structure in the findings, whether due to robustness of the structure or accessibility to ribosome complexes. Prolonged protein expression was observed in the 100% +1 FSS sequence at consistently lower expression rates, contradicting estimates from predictive tools. To explain these results, we also studied alterations in secondary structure arising from inclusions of +1 FSS through differential scanning fluorimetry. The findings suggest that in applications requiring sustained production of therapeutic proteins, such as the aforementioned proposed cancer treatments, +1 FSS can be a viable feature of sequence design, particularly when de-optimisation is considered for increasing mRNA half-life. Improved translation efficiency resulting from +1 FSS inclusion at low levels may aid broader applications, but further research across diverse amino acid sequences would strengthen evidence.