A low-toxicity circular single-stranded DNA platform for safe and potent IL-12 immunotherapy against hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) remains a major global health challenge, largely because of its immunosuppressive "cold" tumor microenvironment (TME) that resists therapy. Interleukin-12 (IL-12) is a potent immune-activating cytokine, but its clinical application has constrained by severe systemic toxicity. Therefore, a delivery strategy that enables tumor-specific IL-12 expression is urgently needed. Here, a novel platform IL-12-cssDNA-LNP is developed using circular single-stranded DNA (cssDNA) encapsulated in lipid nanoparticles for controlled IL-12 delivery in HCC. The cssDNA demonstrates a clear expression advantage over plasmid DNA (pDNA) in HCC cells both in vitro and in vivo. Critically, our cssDNA-LNP platform is far less immunogenic and toxic than pDNA. In both subcutaneous and highly clinically relevant c-MYC/p53-driven primary HCC models, IL-12-cssDNA-LNP treatment significantly reprograms the "cold" TME and successfully suppresses tumor growth, and this is achieved without the severe off-target hepatic inflammation observed in the pDNA-treated group. The treatment also establishes durable immunological memory, conferring 100% protection against tumor rechallenge in cured mice. Our work establishes cssDNA-LNP as a versatile platform uncoupling high-efficiency hepatic gene expression from severe vector-driven toxicity. It is feasible to extend this novel gene delivery platform using cssDNA for HCC immunotherapy and other liver-associated diseases requiring precise and sustained gene delivery.