Characterization of HIF-1alpha Knockout Primary Human Natural Killer Cells in Allogeneic Glioblastoma 9059

Description Natural killer (NK) cells, key innate effector cells with broad cytotoxicity against tumors, are promising candidates for immunotherapies against cancers, including glioblastoma (GBM). Hypoxia, a common feature of solid tumors, induces the upregulation of hypoxia-inducible factor-1alpha (HIF-1A) in both tumor and normal cells, impairing the function of anti-tumor effector cells such as NK cells. We knocked out HIF-1A in human primary NK cells using clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9). Then, cellular characterizations were conducted in normoxic and hypoxic conditions. Electroporation of two HIF-1A-targeting guide RNA–Cas9 complexes effectively suppressed HIF-1A expression in expanded NK cells. Under hypoxic conditions, HIF-1A knockout NK cells (HIF-KO-NKP) inhibited the growth of allogeneic GBM cells and induced apoptosis in GBM-derived spheroids compared to mock NK cells. RNA sequencing revealed that enhanced cytotoxicity of HIF-KO-NKP in hypoxic conditions was associated with increased perforin expression and improved mitochondrial function. Furthermore, intracranial administration of HIF-KO-NKP in a xenograft brain tumor mouse model prolonged survival compared to mock NK cells. These findings demonstrate that HIF-KO-NKP improves anti-tumor effects in conditions mimicking the hypoxic GBM environment. CRISPR-Cas9-mediated HIF-KO-NKP represents a promising immunotherapeutic strategy for GBM patients. Funding Sources This study was supported by JSPS KAKENHI (grant no. JP 22K09213) and Kurata Grant of the Kurata Memorial Hitachi Science and Technology Foundation.