Antigen-encoding circular single-stranded DNA for cancer vaccine

Nucleic acid drugs have increasingly demonstrated their potential in tumor therapy and vaccine development, particularly exemplified by the recent advancements in mRNA-based cancer vaccines. Although mRNA cancer vaccines have shown promising clinical effects in cancer treatment, the inherent susceptibility of mRNA to degradation and its transient expression profile pose significant challenges in achieving long-term prevention of postoperative recurrence and metastasis. Herein, we propose the utilization of circular single-stranded DNA (Css DNA) as a novel gene expression vector to achieve robust expression for at least 315 days, which offers distinct advantages over conventional plasmids. It lacks bacterial-derived redundant sequences that are prone to clearance while exhibiting remarkably low immunogenicity due to its single-stranded conformation, which minimally activates the cGAS-STING pathway. With ovalbumin (OVA) as a model antigen, OVA-encoding Css DNA as a long-acting prophylactic vaccine has been demonstrated to significantly inhibit tumor growth and recurrence, thereby prolonging the survival time. Further combination with IL-12-encoding Css DNA, it elicited up to 7-fold cytotoxic CD8+ T cells and recruited innate immune cells in tumors. Our findings establish Css DNA as a versatile platform for cancer immunotherapy, combining durable antigen expression with potent immune activation to overcome current limitations of nucleic acid vaccines.