<p>Lipid nanoparticles (LNPs) for mRNA delivery have advanced significantly, but LNP-mediated DNA delivery still faces clinical challenges. This study compared various LNP formulations for delivering DNA-encoded biologics, assessing their expression efficacy and the protective immunity generated by LNP-encapsulated DNA in different models. The LNP formulation used in Moderna’s Spikevax mRNA vaccine (LNP-M) demonstrated a stable nanoparticle structure, high expression efficiency, and low toxicity. Notably, a DNA vaccine encoding the spike protein, delivered via LNP-M, induced stronger antigen-specific antibody and T cell immune responses compared to electroporation. Single-cell RNA sequencing (scRNA-seq) analysis revealed that the LNP-M/pSpike vaccine enhanced CD80 activation signaling in CD8⁺ T cells, NK cells, macrophages, and DCs, while reducing the immunosuppressive signals. The enrichment of TCR and BCR by LNP-M/pSpike suggested an increase in immune response specificity and diversity. Additionally, LNP-M effectively delivered DNA-encoded antigens, such as mouse PD-L1 and p53^R172H, or monoclonal antibodies targeting mouse PD1 and human p53^R282W. This approach inhibited tumor growth or metastasis in several mouse models. The long-term anti-tumor effects of LNP-M-delivered anti-p53^R282W antibody relied on memory CD8⁺ T cell responses and enhanced MHC-I signaling from APCs to CD8⁺ T cells. These results highlight LNP-M as a promising and effective platform for delivering DNA-based vaccines and cancer immunotherapies.</p>