Scarless circular mRNA-based CAR-T cell therapy elicits superior antitumor efficacy.

Messenger RNA (mRNA)-based transient expression of chimeric antigen receptors (CARs) results in optimal safety profiles and provides promising opportunities to address existing challenges associated with viral vector-based CAR-T-cell therapies and to meet emerging medical needs for noncancerous indications. Conventional linear mRNAs, however, are intrinsically unstable and typically support short-lived protein expression, which can constrain therapeutic activity. Here, we engineered a high-efficiency permuted intron exon (PIE) platform to synthesize scarless circular mRNAs (cmRNAs) that drive robust CAR expression with extended durability. The scarless design avoids extraneous junction sequences, streamlining manufacturability and potentially reducing innate immune sensing. Compared with linear mRNAs, cmRNAs significantly increased both the magnitude and duration of anti-CD19 CAR and anti-GPRC5D CAR expression in primary human T cells. Functionally, cmRNA-based CAR-T cells elicited superior antitumor efficacy over their linear mRNA counterparts, as demonstrated by parallel lines of evidence, including in vitro antigen-specific cytotoxicity, cytokine release, and transcriptomics patterns consistent with sustained activation and absence of exhaustion signatures, as well as in vivo models demonstrating tumor elimination and prolonged survival benefits. Collectively, these findings position cmRNA as a next-generation mRNA modality for potent and controllable CAR expression, thereby providing a robust platform to unleash the full potential of mRNA technologies in cellular immunotherapy and precision medicine.