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

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

  • Mol Ther. 2025 May 28:S1525-0016(25)00394-6. doi: 10.1016/j.ymthe.2025.05.024.
Jingjing Zhang 1,  Chao Li 1,  Yuheng Liu 2,  Rui Liao 1,  Dian He 3,  Lifeng Xu 1,  Tingting Chen 4,  Qin Xiao 1,  Mingxing Luo 1,  Yang Chen 1,  Yali Li 5,  Huaxing Zhu 5,  Joseph Rosenecker 6,  Xiaoyan Ding 7,  Shuchen Pei 8,  Shan Guan 9
Affiliations

Affiliations

  • 1 National Engineering Research Center of Immunological Products, Third Military Medical University, Chongqing 400038, China.
  • 2 National Engineering Research Center of Immunological Products, Third Military Medical University, Chongqing 400038, China; Department of Pharmacology, College of Pharmacy, Chongqing Medical University, Chongqing 400016, China.
  • 3 Department of Neurology, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China.
  • 4 Pathogen Biology and Immunology Laboratory, Lab Teaching & Management Center, Chongqing Medical University, Chongqing 400016, China.
  • 5 Novoprotein Scientific, Inc., Shanghai 215200, China.
  • 6 Department of Pediatrics, Ludwig-Maximilians University of Munich, 80337 Munich, Germany.
  • 7 Department of Pediatrics, Ludwig-Maximilians University of Munich, 80337 Munich, Germany. Electronic address: xiaoyan.ding@med.uni-muenchen.de.
  • 8 College of Chemistry and Chemical Engineering, Chongqing University of Science and Technology, Chongqing 401331, China. Electronic address: peishuchen928@163.com.
  • 9 National Engineering Research Center of Immunological Products, Third Military Medical University, Chongqing 400038, China; Department of Pediatrics, Ludwig-Maximilians University of Munich, 80337 Munich, Germany. Electronic address: guanshan87@163.com.
PMID: 40443030 DOI: 10.1016/j.ymthe.2025.05.024
Abstract

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.

Keywords

affinity chromatography purification; airway; dsRNA; in vitro transcribed mRNA sequence design; lung disease; mRNA vaccine; mucosal immunity.

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