Installation of dominant-negative mutations in FAS and TGFβR2 via base editing in primary T cells

Adoptive cell transfer (ACT) of engineered T cells is effective against B cell malignancies but has faltered against solid tumors due to the immunosuppressive tumor microenvironment (TME). FAS ligand (FASL) and transforming growth factor β (TGF-β) are key mediators of T cell dysfunction in the TME and overexpressing dominant negative (dn) forms of their receptors in T cells increases anti-tumor efficacy in solid tumor models. However, an approach that directly targets the endogenous genes would be more amenable to multiplex editing and reduce competition with WT alleles. Here, we employ base editing (BE) in primary human T cells to install naturally occurring dominant negative <i>FAS</i> and <i>TGFβR2</i> mutations. <i>In vitro</i> survival and proliferation assays demonstrate that BE T cells are resistant to pro-apoptotic and anti-proliferative effects of FAS and TGF-β signaling. Chimeric antigen receptor (CAR)-T cells with BE-installed dn TGFβR2 or dn FAS exhibit improvements in cytotoxicity, while dn TGFβR2 CAR T demonstrate increased persistence and reduced expression of phenotypic markers of exhaustion compared to controls. Moreover, BE-engineered dn CAR T outperform lentiviral-engineered cDNA over expression counterparts in several functional assays. Considering the efficiency of BE and its amenability for multiplex editing, our approach lends itself to engineering strategies necessary to overcome T cell dysfunction in solid tumors.