Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells

Abstract

Multiple genetic modifications may be required to develop potent off-the-shelf chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR-Cas nucleases install sequence-specific DNA double-strand breaks (DSBs), enabling gene knock-out (KO) or targeted transgene knock-in (KI). However, simultaneous DSBs provoke a high rate of genomic rearrangements which may impede the safety of the edited cells. Here, we combine a non-viral CRISPR-Cas9 nuclease-assisted KI and Cas9-derived base editing technology for DSB free KOs within a single intervention. We demonstrate efficient insertion of a CAR into the T cell receptor alpha constant (TRAC) gene, along with two KOs that silence major histocompatibility complexes (MHC) class I and II expression. This approach reduced translocations to 1.5% of edited cells. Small insertions and deletion at the base editing target sites indicated guide RNA exchange between the editors. This was overcome by using CRISPR enzymes of distinct evolutionary origins. Combining Cas12a Ultra for CAR KI and a Cas9-derived base editor enabled the efficient generation of triple-edited CAR T cells with a translocation frequency comparable to unedited T cells. Resulting T cell receptor- (TCR-) and MHC-negative CAR T cells resisted allogeneic T cell targeting in vitro. Thus, we demonstrate a solution for safer multiplex-edited cell products and a path towards off-the-shelf CAR therapeutics.

Integration of ζ-deficient CARs into theCD3-zetagene conveys potent cytotoxicity in T and NK cells

“…Although CD3 ζ-editing can be used for both T and NK cells, the respective edits required to improve the functionality of NK cells 59,60 may differ to the ones proposed for T cells 55,61 . Finally, complex editing may require the combination of nuclease-assisted gene transfer with other gene silencing modalities such as base editing 62,63 to reduce the risk for genomic rearrangements with unknown biological impact 52,61,64 .…”

Gene editing ofCD3 epsilongene to redirect regulatory T cells for adoptive T cell transfer

“…Here, the minimized transgene size enables easier co-delivery of therapeutic transgenes. Further, our Cas12a editing strategy would allow combination with SpCas9-derived enzymes in a single transfection for multiplex gene editing with minimal translocations 56 . Finally, multiplex gene editing may allow to combine edits for enhanced functionality, such as drug resistance 52,53 or FOXP3 stabilization 57 , with modifications of HLA 58 and enable successful off-the-shelf Treg applications.…”

Matching or genetic engineering of HLA Class I and II facilitates successful allogeneic ‘off-the-shelf’ regulatory T cell therapy

“…Base editors, such as the employed ABE (29), introduce targeted base changes without inducing DNA breaks and thereby reduce the risk of gross genetic rearrangements in the gene edited cell product. In future, our editing strategy may be adapted to enable multiplexediting in a single manipulation to reduce manufacturing using different nucleases (52) or novel gene editing tools for DSB-free KI (53,54).…”