Intratumoral Administration of Engineered circRNAs Encoding Cytosine Deaminase–Uracil Phosphoribosyltransferase and IL-15 Elicits Superior Antitumor Efficacy

Abstract Gene-directed enzyme prodrug therapy represents a promising antitumor strategy owing to its low systemic toxicity. However, clinical translation has been hindered by challenges in suicide gene delivery. Artificially engineered circular RNA (circRNA) demonstrates exceptional potential for gene delivery when combined with lipid nanoparticle technology, exhibiting high stability, prolonged protein-coding capacity, and cost-effective production. We developed an in vitro synthesized circRNA encoding the cytosine deaminase–uracil phosphoribosyltransferase (circCDUPRT). Upon intratumoral administration, circCDUPRT achieved sustained intratumoral expression with minimal systemic toxicity. The combination of circCDUPRT and prodrug 5-fluorocytosine showed significant antitumor efficacy in both in vitro and in vivo tumor models. In advanced melanoma models, combining circCDUPRT/5-fluorocytosine with IL-15–expressing circRNA potently enhanced the expansion and activation of CD8+ T and NK cells. Collectively, these findings establish the synthetic circRNA platform as a cost-effective, high-efficiency delivery system for gene-directed enzyme prodrug therapy and immunotherapy applications.