A T7 autogene-mediated DNA vaccine platform for SARS-CoV-2: Overcoming DNA vaccine limitations with enhanced spike...

The COVID-19 pandemic has underscored the urgent need for innovative vaccine platforms that combine potent immunogenicity, thermal stability, and global accessibility. While mRNA vaccines have proven highly efficacious, their fragility and reliance on ultracold storage complicates distribution in resource-constrained settings. DNA vaccines, by contrast, are more stable and easier to distribute but suffer from low immunogenicity due to inefficient nuclear translocation. Here, we introduce the mRNA Factory (mRF) vaccine platform, a DNA-based system that enables direct cytoplasmic transcription of spike mRNA via self-amplifying T7 RNA polymerase. The Rpol/spk mRF vaccine elicited markedly enhanced immunogenicity compared to conventional plasmid DNA vaccines, as evidenced by elevated spike protein expression, robust T cell activation, Th1-biased cytokine profiles, and higher titers of SARS-CoV-2-specific IgG. Compared with an IVT spike mRNA vaccine, the mRF vaccine elicited comparable levels of overall immune responses, including T effector cell activation, IFN_γ⁺CD8⁺ cytotoxic T cell activation, Th1 polarization, and specific IgG production. These results highlight the mRF platform's potential to overcome key limitations of conventional DNA vaccines while addressing distribution challenges associated with mRNA vaccines. Our findings support the mRF platform as a promising strategy for global vaccine deployment and future pandemic preparedness.