W167 - Mitigating Genomic Rearrangements in Multiplex Gene-edited CAR T Cells

Christian Flugel; Jonas Kath; Weijie Du; Vanessa Drosdek; Clemens Franke; Maik Stein; Michael Schmueck-Henneresse; Hans-Dieter Volk; Petra Reinke; Dimitrios Wagner

Abstract Text: Multiple genetic modifications may be required to develop potent allogeneic chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR-Cas nucleases install sequence-specific DNA double-strand breaks (DSBs), enabling gene knockout or targeted transgene knock-in. However, simultaneous DSBs provoke a high rate of genomic rearrangements which may impede the safety of the edited cells. Here, we generate T cell receptor (TCR) replaced CAR T cells using CRISPR-Cas9 in combination with Cas9-derived base editing technology for additional DSB free knockouts within a single intervention. To enhance the persistence of allogeneic CAR-T cell products, B2M and CIITA can be knocked out to eliminate HLA-class 1 and 2, respectively. However, we observed guide RNA exchange between the editors by the presence of small insertions and deletions at the base editing target sites, as well as translocations between the edited loci. This is overcome by using CRISPR enzymes of distinct evolutionary origins. Combining Cas12a Ultra for CAR knock-in and a Cas9-derived base editor enables the efficient generation of triple-edited CAR T cells with a translocation frequency comparable to unedited T cells. The ability of resulting TCR- and HLA-negative CAR T cells to resist allogeneic T cell rejection in vitro represents a promising step towards the development of off-the-shelf CAR therapeutics. This single-step procedure for simultaneous non-viral CAR gene transfer and efficient gene silencing using different CRISPR enzymes for knock-in and base editing provides a safe and effective strategy for multiplex gene editing in CAR-T cells.