Overexpression of HIF-1α via small activating RNA enhances CAR-T cell function in hypoxic microenvironments

CAR-T cell therapy demonstrates high efficacy in hematological malignancies but remains limited in solid tumors, mainly due to the immunosuppressive hypoxic tumor microenvironment that impairs T cell infiltration and promotes exhaustion. Driven by clinical transcriptomic and protein evidence implicating hypoxia-inducible factor-1α (HIF-1α) in metabolic reprogramming, we developed a non-viral CAR-T cell preparation platform using a piggyBac transposon system to co-deliver a CAR construct and a small activating RNA (saRNA) to drive endogenous HIF-1α overexpression (HIF1A^OE). HIF1A^OE-CAR-T cells exhibited superior tumoricidal activity under hypoxic conditions (1% O₂) and enhanced infiltration in a 3D tumor spheroid model. Importantly, these beneficial effects were successfully validated across independent antigen-targeting CAR models. Metabolomic analyses and Seahorse metabolic flux profiling revealed that HIF-1α remodels cellular metabolism through a dual mechanism: augmenting aerobic glycolysis by upregulating GLUT1 and preserving mitochondrial integrity via the Nrf2/PGC-1α signaling axis. Furthermore, combining HIF1A^OE-CAR-T cells with an anti-CTLA4 nanobody resulted in synergistic tumour regression and improved infiltration in SCID-NOD mice, without significant toxicity. Collectively, activating endogenous HIF-1α in CAR-T cells is proposed as a promising strategy for the safe and effective treatment of solid tumors within hypoxic microenvironments.