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. • RNA activation (RNAa) mediated endogenous upregulation of HIF-1α safely arms CAR-T cells against the hypoxic tumor microenvironment. • HIF1A OE -CAR-T cells exhibit reduced exhaustion and enhanced persistence in hypoxic conditions, 3D tumor spheroids, and orthotopic xenografts. • HIF1A OE -CAR-T cells overcome metabolic stress in hypoxia via concerted glycolytic and mitochondrial reprogramming. • Combination of HIF1A OE -CAR-T cells and checkpoint blockade represents a potent strategy for deep penetration and eradication of solid tumors.
Huang et al. (Fri,) studied this question.