In Vitro-Transcribed (IVT)-mRNA CAR Therapy Development

Chimeric antigen receptor (CAR) cancer immunotherapy uses autologous immune system's cells, genetically modified, to reinforce the immune system against cancer cells. Genetic modification is usually mediated via viral transfection, despite the risk of insertional oncogenesis and off target side effects. In vitro-transcribed (IVT)-mRNA-mediated transfection could contribute to a much safer CAR therapy, since IVT-mRNA leaves no ultimate genetic residue in recipient cells. In this chapter, the IVT-mRNA generation procedure is described, from the selection of the target of the CAR T-cells, the cloning of the template for the in vitro transcription and the development of several chemical modifications for optimizing the structure and thus the stability of the produced CAR IVT-mRNA molecules. Among various transfection methods to efficiently express the CAR molecule on T-cells' surface, the electroporation and the cationic-lipid mediated transfection of the CAR IVT-mRNAs are described.