CircRNA-based CD19-targeted CAR-NK therapy for B-cell acute lymphoblastic Leukemia using a Coccidioides immitis-derived group II intron platform.

Chimeric antigen receptor (CAR)-T cell therapy targeting CD19 has demonstrated notable clinical efficacy in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), but its wider clinical applicability is constrained by long manufacturing processes, substantial costs, and severe adverse events. A potentially safer and more accessible alternative is provided by CAR-Natural killer (CAR-NK) cell therapy. Currently, most CAR-NK cells are generated using viral transduction, which is labor-intensive and associated with risks of genomic integration. Electroporation of CAR-encoding mRNA provides a non-integrating alternative but results in only transient CAR expression. Circular RNA (circRNA), owing to its enhanced stability and prolonged protein expression capacity, has recently emerged as a promising alternative to linear mRNA. To overcome the limitations of transient mRNA expression, we generated circRNA using a Group II intron-mediated cyclization system incorporating a newly selected Coccidioides immitis-derived Group II intron. The newly established Coccidioides immitis-derived Group II intron circularization system efficiently generated circRNA and supported more durable EGFP expression than linear mRNA in both HEK293T and NK92 cells. Using this system, we successfully developed a circRNA-based CD19-targeted CAR-NK platform. CircRNA-engineered CD19-targeted CAR-NK92 cells maintained more durable CAR expression and showed stronger antitumor activity at later time points. In mouse models of B-ALL, circRNA-engineered CAR-NK92 cells demonstrated better tumor control and extended survival compared with their linear mRNA-engineered counterparts. These results support the potential of circRNA-based CAR-NK therapy as an effective approach for enhancing the safety and efficacy of cancer immunotherapy.