The tumor microenvironment enhances the expression of cssDNA by modulating cell cycle signaling pathways via SKP2.

Circular single-stranded DNA (cssDNA), an emerging nucleic acid vector, exhibits significant clinical potential for treating genetic disorders, enabling gene editing, and advancing oncotherapy. Its unique attributes, including high stability, structural simplicity, conformational flexibility, and low molecular mass, establish it as a promising gene therapy tool. Our study reveals that cssDNA demonstrates superior expression efficiency over conventional plasmids across diverse tumor cell lines. Notably, cssDNA expression is enhanced under certain tumor microenvironment (TME) conditions (glucose deficiency, glutamine deficiency and hypoxia) compared to normal condition. Mechanistically, these TME conditions induce significant cell cycle perturbations, particularly pronounced G1 arrest. Intriguingly, transfected cssDNA expression peaks during the late G2/M phase, immediately preceding entry into the G1 phase. We further identify S-phase kinase-associated protein 2 (SKP2) as a critical regulator of cssDNA expression under TME conditions. SKP2 inhibition directly or indirectly notably enhances the expression levels of cssDNA. These findings confirm cssDNA's advantages as a gene expression vector and how specific TME conditions modulate its expression via cell cycle and SKP2-dependent mechanisms in vitro. This work provides a scientific foundation for cssDNA-based cancer therapy and opens new avenues for future clinical translation.