Background: Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematologic malignancies but remains limited by complex ex vivo manufacturing and high costs. The development of in vivo CAR-T cell engineering platforms, which generate therapeutic T cells directly within the patient’s body, offers a scalable, cost-effective, and widely accessible alternative.

Methods: We developed a novel in vivo T cell engineering platform based on cell-derived vesicles (CDVs) produced via a specialized extrusion process. Unlike conventional systems that utilize synthetic in vitro transcribed (IVT) mRNA, our platform employs CAR mRNA that is endogenously transcribed within the source cells. By co-expressing CAR mRNA containing MS2-binding loops and the MS2-LAMP1 fusion protein, we achieved active intracellular loading of native mRNA into the CDVs. To ensure precise T-cell targeting, the exterior of the CDV membrane was decorated with anti-CD3 single-chain variable fragments (scFvs)

Results: This delivery platform demonstrated efficient encapsulation of cell-expressed mRNA and specific binding to target T cells via the anti-CD3 scFv. Our data indicate that CDVs can effectively deliver these natural genetic payloads into endogenous T cells, enabling robust expression of CAR molecules on the cell surface. Notably, the delivery of endogenously processed mRNA facilitated the functional conversion of resting T cells into active effector cells in situ. These generated CAR-T cells exhibited potent antigen-specific cytotoxicity, effectively inducing cell death in target-positive tumor cells. This functional activity confirms that the CDV platform not only delivers genetic payloads but also supports the full therapeutic reprogramming of T cells.

Conclusion: The CDV-mediated delivery of endogenously expressed CAR mRNA and anti-CD3 scFvs provides a novel and powerful strategy for direct in vivo T cell conversion. This “off-the-shelf” platform technology has the potential to simplify the CAR-T therapeutic workflow and significantly broaden patient access to immunotherapy.