Lipid nanoparticle (LNP)-mediated cytoplasmic expression of single-stranded DNA and its application in Mpox vaccine

Lipid Nanoparticle (LNP)-delivered mRNA has limited therapeutic potential due to short half-life and transient expression, while self-amplifying RNA (saRNA) faces challenges with large size, immunogenicity, and formulation. DNA-based systems offer stability but suffer from inefficient delivery, nuclear dependence, and genomic integration risks. Here, we present a proof-of-concept study of a novel gene delivery system, termed LNP-ssDNA-T7RNAP, which combines the complementary technological niches of both systems, leveraging the efficiency of LNP-mediated delivery and cytoplasmic expression, and the prolonged stability of DNA vectors with reduced genomic integration. This approach uses LNPs to co-deliver single-stranded DNA (ssDNA) templates and T7 RNA polymerase (T7RNAP), enabling transcription and translation entirely within the cytoplasm. Robust gene expression in mammalian cells was achieved through systematic optimization, by tuning the primer length (for initiation of T7RNAP-driven transcription), adjusting primer-to-template ratios, 5'-phosphorothioate modification of ssDNA templates, and co-delivery of T7RNAP either by mRNA or DNA. We further demonstrated its utility by constructing a Mpox vaccine with two antigens, L1R and A33R. A single-dose administration in mice elicited strong antigen-specific immune responses without detectable tissue toxicity. These findings highlight the LNP-ssDNA-T7RNAP system as a safe and versatile gene delivery strategy for vaccines and other therapeutic applications requiring sustained expression.