m6A-modified mRNA–LNP system enables in vivo CAR expression and is associated with antitumor immune responses

Messenger RNA (mRNA) therapeutics have emerged as a versatile modality for transient gene delivery, but strategies to incorporate epitranscriptomic regulation into synthetic mRNA design remain limited. Among RNA modifications, N6-methyladenosine (m 6 A) is a major regulator of mRNA stability, translation, and cellular signaling through coordinated actions of methyltransferases, binding proteins, and demethylases. However, the use of engineered m 6 A-modified mRNAs to modulate immune responses in vivo has been relatively unexplored. Here, we developed a tunable lipid nanoparticle (LNP) delivery system using m 6 A-modified mRNAs encoding chimeric antigen receptors (CARs) targeting fibroblast activation protein (FAP). Administration of these mRNA–LNPs was associated with altered RNA methylation states in immune cells and elicited antitumor immune responses. Transcriptomic analyses indicated activation of immune-related pathways and signatures consistent with immune memory. The formulation design was modular, allowing modification of LNP composition. Substitution with pH-responsive polybetaines maintained therapeutic activity without substantial loss of efficacy. In addition, replacing the FAP-targeting sequence with cancer stem cell (CSC) markers, including EpCAM, LGR5, and CD133, preserved antitumor responses. Targeting CSC-associated antigens was also accompanied by suppression of tumor-intrinsic SPP1 signaling pathways. Together, these results suggest that mRNA–LNPs provide a flexible and programmable system for CAR targeting and immunotherapy, influencing epitranscriptomic states while enabling modulation of nanoparticle composition and antigen specificity. This work establishes a framework for the development of programmable mRNA-based cancer immunotherapies through synthetic RNA engineering and controlled m 6 A modification of exogenous mRNA.