Synthetic super-enhancers enable precision viral immunotherapy

Cell type-specific promoters are used in gene therapy to restrict therapeutic payload expression but often have suboptimal strength, selectivity, and size. Here, leveraging recent insights into enhancer function, we developed synthetic super-enhancers (SSEs) by assembling functionally validated enhancer fragments into multi-part arrays. Focusing on the core SOX2- and SOX9-driven transcriptional regulatory network in glioblastoma stem cells (GSCs)?, we engineered SSEs with robust activity and high selectivity??. Single cell profiling, biochemical analysis, and genome-binding data, indicate that SSEs integrate neurodevelopmental and signalling state transcription factors (TFs), triggering formation of large multimeric TF complexes. We show that the GSC-selective expression of a combination of cytotoxic and immune-modulatory payloads (HSV-TK/ganciclovir and IL-12), delivered with adeno-associated virus (AAV) vectors as a single treatment, is curative in an aggressive mouse glioblastoma model. Notably, IL-12 induced an immunological memory that prevented tumour recurrence. AAV-SSE activity and selectivity were validated using primary human glioblastoma (GBM) tissue and normal cortex. SSEs, therefore, harness the unique core transcriptional programs that define the GSC phenotype, and enable precision immune activation. This approach may have broader applications in other contexts when precise control of transgene expression from within specific cell states is necessary.