Building a novel TRUCK by harnessing the endogenous IFN-gamma promoter for cytokine expression.

Despite the remarkable success of CAR T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous IFN-γ promoter for transgenic IL-15 expression. We demonstrate that IFN-γ expression is tightly regulated by TCR signaling. By introducing IRES-IL15 into the 3'-UTR of the IFN-γ gene via HDR-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen-stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Lastly, as part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells.