AUTHOR=Cappabianca Dan , Li Jingling , Zheng Yueting , Tran Cac , Kasparek Kassandra , Mendez Pedro , Thu Ricky , Maures Travis , Capitini Christian M. , Deans Robert , Saha Krishanu 

TITLE=Non-viral expression of chimeric antigen receptors with multiplex gene editing in primary T cells

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=Volume 12 - 2024

YEAR=2024

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2024.1379900

DOI=10.3389/fbioe.2024.1379900

ISSN=2296-4185

ABSTRACT=Efficient engineering of T cells to express exogenous tumor-targeting receptors such as chimeric antigen receptors (CARs) or T cell receptors (TCRs) is a key requirement of effective adoptive cell therapy for cancer. Genome editing technologies, such as CRISPR/Cas9, can further alter the functional characteristics of therapeutic T cells through knockout of genes of interest while knocking-in synthetic receptors that can recognize cancer cells. Performing multiple rounds of gene transfer with precise genome editing, termed multiplexing, remains a key challenge, especially for non-viral delivery platforms. Here, we demonstrate the efficient production of primary human T cells incorporating the knockout of three clinically relevant genes (B2M, TRAC, PD1) along with non-viral transfection of a CAR targeting the disialoganglioside GD2.Multiplexed knockout results in high on-target deletion for all three genes, with low off-target editing and chromosome alterations. Incorporating non-viral delivery to knock-in a GD2-CAR resulted in a TRAC-B2M-PD1 deficient GD2 CAR T cell product with a central memory cell phenotype and high cytotoxicity against GD2-expressing neuroblastoma target cells. Multiplexed gene-editing with non-viral delivery by CRISPR/Cas9 is feasible and safe with high potential for the rapid and efficient manufacturing of highly potent allogeneic CAR T cell products.