A cytotoxic gene therapy targeting SOX9-positive therapy-resistant medulloblastoma

Background The transcription factor SOX9 is associated with poor prognosis and therapy resistance in medulloblastoma, one of the most common malignant pediatric brain tumors. While SOX9 knockdown inhibits tumor recurrence in animal models, no direct SOX9 inhibitors are currently available for clinical use. SOX9 functions as a potent activator of a minimal COL2A1 enhancer that can work as a SOX9 specific response element (S9RE). In this study, we aimed to engineer S9RE to identify therapy-resistant tumor cells and further use it as a gene therapy to selectively target SOX9-expressing medulloblastoma. Methods We employed lentiviral and adeno-associated virus (AAV) vectors, flow cytometry, immunohistochemistry, immunoblotting, in vitro cell models, and in vivo orthotopic mouse models to track and eliminate therapy-resistant medulloblastoma cells. Results We demonstrated that SOX9-active cells can be efficiently labeled using a reporter construct containing four tandem S9REs coupled to a minimal promoter (S9RE-minP) driving expression of enhanced green fluorescent protein (EGFP) and luciferase. Furthermore, S9RE-minP was used in an AAV vector to drive expression of herpes simplex virus thymidine kinase (HSV-TK), enabling a suicide gene therapy approach wherein ganciclovir selectively kills dividing SOX9-positive tumor cells. Conclusion This AAV-based gene therapy approach, when combined with fractionated radiation, synergized with standard therapy to effectively eliminate SOX9-positive medulloblastoma cells in vitro and in vivo. These findings support the potential of SOX9-driven gene therapy as a targeted strategy to overcome therapy resistance in malignant pediatric brain tumors.