Spatial mapping of barcoded, brain-tropic AAVs using multiplexed RNA <i>in situ</i> hybridization

Gene therapy cargo delivery to specific cell types in the central nervous system (CNS) remains a major challenge for the development of adeno-associated virus (AAV) vectors as a therapeutic modality. Here, we leverage high-plex <i>in situ</i> transcriptomics (10× Genomics Xenium) to spatially map barcoded AAV payloads at subcellular resolution in the intact mouse brain while preserving anatomical context. Tropism profiling of 22 barcoded AAV variants including novel AAV9 derivatives and CNS-targeted capsids revealed that established vectors, AAV9-PHP.eB and AAV9-CAP-B10, demonstrated distinct neuronal and non-neuronal subtype preferences, recapitulating previous findings. Several novel AAV variants candidates predominantly transduced endothelial and vascular cells, suggesting limited blood-brain barrier (BBB) penetration. Notably, three novel variants (AAV9-BTX166, -BTX168, and -BTX175) exhibited enhanced endothelial and mural cell tropism, despite robust CNS activity in bulk assays. Intriguingly, the novel variants AAV9-BTX149 and -BTX001 displayed selective targeting of specific inhibitory neuron subtypes, with a single Trp503Arg substitution in the capsid being sufficient to redirect tropism. Our results establish <i>in situ</i> spatial transcriptomics as a powerful tool for resolving AAV biodistribution and BBB traversal capacities at high resolution, providing a blueprint for capsid engineering to achieve precise cell subtype targeting in the CNS.