Despite the increasing awareness that cell and gene-therapy approaches have tremendous biomedical potential, their broad clinical application has been challenging due to prolonged and expensive production times and the emergence of severe immune- and gene-delivery dependent side effects. In this proposal, we aim to establish a stream-lined and high-throughput protocol for the generation of off-the-shelf cell therapy by combining a novel technological platform for gene delivery with a breakthrough biological concept that will permit to manufacture functional, gene-corrected blood forming stem cells and chimeric antigen receptor (CAR) T cells that can recognize and kill specific tumor cells through this CAR. To achieve this, we will optimize and apply a non-viral gene delivery method for CRISPR-mediated and site-specific gene-editing to obtain controlled CAR expression and for performing gene-correction in induced pluripotent stem cells (iPSCs). These are stem cells that have the intrinsic potential to generate any cell type of the body but that are derived from fully differentiated cells following genetic reprogramming. From these gene-modified iPSCs, we will generate CAR T cells and blood forming stem cells, respectively, by selectively targeting a signaling pathway that we established to be critical in human blood cell development and T cell development. Following functional validation of the generated cell products, we will optimize the current protocols to increase the potential for clinical implementation and establish a high-throughput platform to generate a large number of CAR expressing iPSC lines from different ages, sex and ethnicities to demonstrate the population-wide implementation potential of our approach. This will allow to generate a bank of well-characterized, CAR expressing iPSC lines that can be used to manufacture off-the-shelf cell therapy products in the absence of any rejection-related issues, thereby significantly advancing the currently implemented adaptive CAR T cell approaches by reducing the production costs and time, by selectively targeting the CAR into a well-controlled location which will prevent variability and by facilitating the production and evaluation of novel CARs for other cancer entities such as solid tumors.
