Airway-applied mRNA vaccine needs tailored sequence design and high standard purification that removes devastating dsRNA contaminant.

The development of mucosal mRNA vaccines is promising but extremely challenging. Major efforts have been focused on optimizing delivery systems, but it is still unknown whether the intrinsic quality of mRNA components significantly impacts the potency of airway-inoculated mRNA vaccines. Here, we systematically demonstrate that mucosal mRNA vaccine requires higher standards of purification and a tailor-designed sequence to fulfill its potency compared to its parenteral-route-inoculated counterpart. Double-stranded RNA (dsRNA) contaminants are prone to trigger the innate immune response in the airway that activates the mRNA degradation mechanism, thereby diminishing mRNA expression and subsequent antigen-specific immune responses. To address these challenges, we developed a strategy that combines optimized untranslated regions (UTRs) screened from endogenous genes of pulmonary cells with affinity chromatography-based purification, which effectively removed dsRNA contaminants. The optimized mRNA administered via the airway route not only demonstrated superior protein expression (30-fold increase) and reduced inflammation in the lung but also promoted robust adaptive immunity comprising significantly elevated systemic, cellular, and mucosal immune responses. This was in stark contrast to the intramuscular-injected counterpart that displayed less-pronounced benefits. Our findings offer new insights into the development of mucosal mRNA therapeutics from an overlooked but crucial perspective of optimizing mRNA components.