Dual-Targeting OH2 Oncolytic Virus Therapy Overcomes Immune Resistance in TNBC via PD-L1/DDR1 Silencing and GM-CSF Expression

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer, accounting for between 15% and 20% of cases and remains resistant to conventional treatments due to the absence of active receptors, high metastatic potential and a strongly immunosuppressive tumour microenvironment. Here, we propose the oncolytic herpes simplex virus type 2 (OH2), which was rationally designed as a dual-purpose therapeutic platform, Recombinant OH2 was designed to express a small forked RNA (shRNA) that silences both the discoid domain receptor (DDR1) and programmed death ligand 1 (PD-L1) , while incorporating the surviving promoter to limit viral replication in cancer cells and thus reduce off-target cytotoxicity. In addition, OH2 encodes granulocyte-macrophage colony-stimulating factor (GM-CSF) to enhance dendritic cell activation and immune cell capture . Simultaneous silencing of DDR1 and PD-L1 alters extracellular matrix remodelling and weakens tumour immune evasion. In addition, OH2-mediated suppression of β- catenin signalling indirectly relieves T cell blockade, collectively stimulating the infiltration of CD8⁺ T lymphocytes and natural killer cells into the tumour microenvironment. In vitro, therapeutic efficacy is evaluated using human and murine TNBC cell lines (e.g., MDA-MB-231, 4T1) with Western blotting, qPCR, ELISA and luciferase assays. In vivo, we use xenograft models in humanised NSG mice, which have been reconstituted with CD34+ haematopoietic stem cells , as well as genetically modified Myc;Ptenfl/fl mice, which develop spontaneous breast tumours. Translational challenges — including potential immune clearance of the viral vector, tumour heterogeneity, and intratumoral delivery constraints — are considered in the experimental design and discussed as directions for future refinement. This strategy combines tumour-specific oncolytic action, modulation of immune checkpoints, and matrix reprogramming to increase tumour immunogenicity and overcome the resistance mechanisms characteristic of TNBC. Our results indicate that this dual-purpose OH2 construct represents a promising new-generation oncolytic viral therapy. Together, these findings establish OH2 as a multifunctional immuno-oncolytic platform with strong potential for clinical translation in refractory TNBC.