The regulatory code of injury responsive enhancers enables precision cell state targeting in the CNS [enhancer_aav]

Enhancer elements in the genome direct cell type-specific gene expression programs. In response to injury, cells undergo dynamic changes in gene expression that drive adaptation and initiate tissue repair. Here, we investigate how injury-induced gene expression programs are encoded within enhancer elements in the mammalian CNS. Leveraging single-nucleus transcriptomics and chromatin accessibility profiling, we identify thousands of injury-induced, cell type-specific enhancers in the mouse spinal cord. These enhancers are more abundant in glial cells and retain cell type specificity, even when regulating shared wound response genes. By modeling glial injury-responsive enhancers using deep learning, we reveal that their architecture encodes cell type specificity by integrating generic stimulus response elements with cell identity programs. Finally, through in vivo enhancer screening, we demonstrate that injury-responsive enhancers can selectively target reactive astrocytes bordering lesion sites across the CNS using therapeutically relevant gene delivery vectors. Our decoding of the principles of injury-responsive enhancers enables the design of sequences that can be programmed to therapeutically target disease related cell states. Overall design: Animals from a Cx30-Tom transgenic line were injected with an enhancer AAV library to test the activity of injury-responsive enhancers identified in our analysis. Mice were then injured with a spinal cord contusion model (14dpi) and samples were collected after 3 days (or from uninjured controls). Single cells were obtained through tissue digestion and sorting and were processed using a transcriptomics experiment that included enrichment for barcodes associated with the enhancer viruses. Each condition encompassed 1-2 animals per sex.