A New Approach to In Vivo Transformation of Killer T Cells.

Abstract

Chimeric antigen receptor (CAR) T cell therapy is a relatively new and powerful way of transforming T cells with receptors needed to recognize and kill diseased cells. Traditionally, it involves extraction of T cells from a patient, ex vivo transformation of them with CARs, expansion, and subsequent re-infusion into the patient. Recent developments aim to avoid this lengthy, costly patient-specific procedure by using various viral and non-viral vector particles for direct in vivo delivery of CAR-encoding genes. In this paper we highlight several fundamental connections between in vitro and in vivo aspects of this process. We discuss the proposed use of in vitro-reconstituted virus-like particles (VLPs), prepared from purified CAR-encoding mRNA and viral capsid protein, and functionalized with a T cell-targeting antibody. We compare and contrast these particles - and their use as gene vectors - with the several modalities currently employed that involve in cellulo generation of lentiviral or AAV vectors or in vitro complexation of nucleic acids with cationic polymers or lipid vesicles. We report the unique stoichiometric preciseness and thermodynamic stability of VLPs formed from anti-HIV-glycoprotein CAR-encoding mRNA and the capsid protein from a plant virus, and quantify the extent to which these monodisperse spherical VLPs are RNase resistant and lead to strong CAR expression in T cells. Further, in vitro cell-killing experiments are proposed, in which these CAR VLP-transformed T cells are mixed with HIV-infected cells, to be followed by in vivo experiments involving injection of the particles into HIV-infected humanized mice.