High-fidelity genome editing using NextGEN CRISPR (Clo51-dCas9) system for the production of allogeneic CAR-T cells.

3048Background: Autologous chimeric antigen receptor (CAR) T therapies are highly efficient at targeting hematological malignancies, but the clinical applications have been limited by individualized manufacturing. Furthermore, there has been little success in treating solid tumors due to immunosuppressive microenvironments. Currently, genome editing technologies are being used to address both issues. However, the CRISPR/Cas9 system has significant safety concerns due to high incidence of off-target mutations and TALEN only works sufficiently in activated cells. A hybrid gene editing system, NextGEN (NG) Clo51-dCas9, can be targeted using gRNA, like CRISPR/Cas9, but exhibits little-to-no off-target cutting like TALEN, thereby overcoming limitations in the genome editing of resting T cells. Methods: We successfully developed a platform for production of allogeneic CAR-T cells with reduced receptivity to inhibitory signaling. Here, T cells were modified by piggyBac-mediated BCMA CAR gene delivery, along with...