Oncolytic Vaccinia Virus‐ HSP70 ‐ shRNA Amplifies Viral Replication, ROS /Autophagy, and Immunity to Fight Colorectal Cancer

Colorectal cancer persists as a major contributor to cancer mortality, with immunotherapy offering limited efficacy in immunologically "cold" tumors. To address this issue, we engineered an oncolytic vaccinia virus to express HSP70 shRNA within its TK gene locus (oncoVV-shHSP70). In vitro, oncoVV-shHSP70 significantly suppressed HSP70 expression, enhanced viral replication, and induced apoptosis in CRC cells via Caspase-7/9 activation and Bcl-2 downregulation. OncoVV-shHSP70 also triggered reactive oxygen species (ROS) accumulation by suppressing the antioxidant regulator Nrf2 and stimulated autophagy via the Beclin-1/Vps34 pathway. Notably, a mutually reinforcing cycle between ROS overproduction and autophagy amplified the oncolytic effects of oncoVV-shHSP70, enhancing viral production, promoting apoptosis, and increasing cytokine release. Specifically, oncoVV-shHSP70 boosted cytokine production (TNF-α, IFN-α/β) through ROS/autophagy-dependent activation of AP-1 and IRF-3/7 pathways. In xenograft mouse models, oncoVV-shHSP70 induced cellular necrosis and significantly inhibited tumor growth. These findings demonstrate that oncoVV-shHSP70 enhances antitumor immunity in both syngeneic and humanized mouse models by promoting T-cell and dendritic-cell infiltration and inducing immunogenic cell death. In summary, oncoVV-shHSP70 emerges as a promising multimodal therapy for CRC, leveraging viral oncolysis, apoptosis induction, and immunomodulation to overcome therapeutic resistance.