Enhancer AAVs for targeting spinal motor neurons and descending motor pathways in rodents and macaque.

Experimental access to cell types within the mammalian spinal cord is severely limited by the availability of genetic tools. To enable access to spinal motor neurons (SMNs) and SMN subtypes, we generated single-cell multiome datasets from mouse and macaque spinal cords and discovered putative enhancers for each neuronal population. We cloned these enhancers into adeno-associated viral vectors driving a reporter fluorophore and functionally screened them in the mouse. We extensively characterized the most promising candidate enhancers in rat and macaque and developed an optimized pan-SMN enhancer virus. Additionally, we generated derivative viruses expressing iCre297T recombinase or ChR2-EYFP for labeling and functional studies, and we created a single vector with combined enhancer elements to achieve simultaneous labeling of layer 5 extratelencephalic projecting neurons and SMNs. This unprecedented SMN toolkit will enable future investigations of cell type function across species and potential therapeutic interventions for human neurodegenerative diseases.