Background Ex-vivo modification of immune cells to express Chimeric Antigen Receptor (CAR) has shown tremendous clinical and commercial success as a cancer treatment. Despite its widespread adoption, ex-vivo CAR-T approaches face challenges such as soaring production costs, extended timelines, inherent toxicity risks and operational complexities. These challenges limit the accessibility of CAR-T treatment and highlight the need for improved methods. Here, we demonstrate a novel non-viral vector capable of permanently modifying T-cells to express CAR by direct intra venous injection or short transfection in whole blood (extra-corporeal transfection). We will discuss the capabilities of this vector in vitro and in vivo. We believe this cost-effective and efficient vector has the potential to solve the challenges associated with CAR-T generation resulting in an increased accessibility of CAR-T treatment.

Methods We developed a non-viral vector consisting of a targeted lipid nanoparticle formulation (tLNP) loaded with minicircle DNA encoding for CAR, mRNA encoding for transposase and coated with both T cell targeting and T cell activating proteins. This novel vector was used to engineer CAR-T cells both in whole blood and in vivo. CAR expression was analyzed using flow cytometry and functionality was assessed using target cell killing assays. The vector was then tested in vivo using a huPBMC NSG mouse model of human leukemia. CAR expression in blood and lymphoid tissues was measured using flow cytometry and tumor outgrowth was assessed using bioluminescence imaging of luciferase signal derived from luciferase positive Nalm-6 leukemia cells.

Results tLNPs activated resting primary T cells in whole blood, enabling their transfection with minicircle DNA encoding for a CAR construct without exogenous activation. Transposase encoded by mRNA co-loaded into the tLNP facilitated stable integration of this construct, resulting in the generation of fully functional CAR-T cells. In vivo, a single administration of tLNP resulted in the generation of a high number of persistent circulating CAR-T cells. These CAR-T cells were functional as evidenced by a significant reduction in tumor growth and significantly extended survival in treated animals compared to vehicle control mice.

Conclusions Here we demonstrate successful non-viral CAR-T generation both in vitro and in vivo using a tLNP vector. This approach shows exciting potential and could significantly increase the accessibility of CAR-T treatments in the future.