Antitumor activity of Oncolytic Herpes Symplex Virus Type 1 and photodynamic therapy in in vitro preclinical models of glioblastoma

Background: Glioblastoma (GBM) remains the most aggressive primary brain tumor in adults, with limited therapeutic options and inevitable recurrence despite maximal standard-of-care treatment. Photodynamic therapy (PDT) and oncolytic virotherapy have independently shown promise as alternative approaches, yet their combined potential in GBM remains incompletely explored. Methods: We engineered a highly neuroattenuated herpes simplex virus type 1-based oncolytic virus (oHSV1), genetically related to talimogene laherparepvec (T-VEC), to express the photosensitizer KillerRed (KR) targeted to the cell plasma membrane (memKR). The cytolytic activity of this recombinant virus was evaluated in multiple human and murine GBM cell lines, patient-derived GBM cells, and three-dimensional (3D) spheroid models. We further assessed whether photoactivation of virus-encoded memKR or of the chemical photosensitizer phthalocyanine could enhance oHSV1-mediated cytotoxicity, including in spheroids infected via monocyte-based viral delivery. Results: The recombinant memKR-encoding virus (oHSV1-KR) efficiently infected and killed GBM cells across 2D and 3D culture systems. Photoactivation of memKR significantly enhanced virus-mediated cytotoxicity in patient-derived GBM spheroids, particularly at lower viral doses. Similarly, phthalocyanine photoactivation augmented oHSV1-induced cell death, accelerating loss of viability in both monolayer cultures and spheroids. Monocyte-mediated delivery of oHSV1-KR resulted in effective viral transfer to GBM spheroids and retained responsiveness to photodynamic activation. Conclusions: These findings demonstrate that combining oHSV1-based virotherapy with photodynamic activation enhances cytotoxic efficacy in advanced preclinical models of GBM. While limited to in vitro and ex vivo systems, this work establishes a modular and controllable therapeutic platform and provides a foundation for future in vivo and immunological studies aimed at translational development.