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, whereas it is still unknown whether the intrinsic quality of mRNA components significantly impacts the potency of airway inoculated mRNA vaccines. Here, we systematically demonstrate mucosal mRNA vaccine requires higher standards of purification and tailor-designed sequence to fulfil its potency compared to parenteral-route inoculated counterpart. Double-stranded RNA (dsRNA) contaminants are prone to trigger innate immune response in the airway that activates the mRNA degradation mechanism, thereby diminishing the 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 airway route not only demonstrated superior protein expression (30-fold increase) and reduced inflammation in the lung, but also promoted robust immunity comprising significantly elevated systemic, cellular, and mucosal immune responses, which was in stark contrast to intramuscular injected counterpart that displays 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.