In Vivo mRNA CAR T Cell Engineering via Targeted Ionizable Lipid Nanoparticles with Extrahepatic Tropism

With six therapies approved by the Food and Drug Association, chimeric antigen receptor (CAR) T cells have reshaped cancer immunotherapy. However, these therapies rely on ex vivo viral transduction to induce permanent CAR expression in T cells, which contributes to high production costs and long-term side effects. Thus, this work aims to develop an in vivo CAR T cell engineering platform to streamline production while using mRNA to induce transient, tunable CAR expression. Specifically, an ionizable lipid nanoparticle (LNP) is utilized as these platforms have demonstrated clinical success in nucleic acid delivery. Though LNPs often accumulate in the liver, the LNP platform used here achieves extrahepatic transfection with enhanced delivery to the spleen, and it is further modified via antibody conjugation (Ab-LNPs) to target pan-T cell markers. The in vivo evaluation of these Ab-LNPs confirms that targeting is necessary for potent T cell transfection. When using these Ab-LNPs for the delivery of CAR mRNA, antibody and dose-dependent CAR expression and cytokine release are observed along with B cell depletion of up to 90%. In all, this work conjugates antibodies to LNPs with extrahepatic tropism, evaluates pan-T cell markers, and develops Ab-LNPs capable of generating functional CAR T cells in vivo. Utilizing an ionizable lipid nanoparticle (LNP) with extrahepatic tropism, this work demonstrates in vivo T cell transfection for CAR T cell engineering. Antibody targeting against the CD3, CD5, and CD7 pan-T cell markers is explored, and following a single intravenous injection, the CD3-LNPs generate transient CAR+ T cells capable of up to 90% B cell depletion.image