biostudies-literatureCoughlin GMGouvernement du Canada | Natural Sciences and Engineering Research Council of CanadaU.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)Michael J. Fox Foundation for Parkinson's Research (Michael J. Fox Foundation)Chan Zuckerberg Initiative Neurodegeneration Challenge NetworkNIMH NIH HHSNINDS NIH HHSU.S. Department of Health & Human Services | NIH | National Institute of Mental HealthMichael J. Fox Foundation for Parkinson's Research133-145https://www.ebi.ac.uk/biostudies/studies/S-EPMC12807873biostudies-literatureUnknown44(1)Cell-type-specific regulatory elements such as enhancers can direct expression of recombinant adeno-associated viruses (AAVs) to specific cell types, but this approach is limited by the relatively small packaging capacity of AAVs. In this study, we used spatial genomics to show that transcriptional crosstalk between individual AAV genomes provides a general method for cell-type-specific expression of large cargo by separating distally acting regulatory elements into a second AAV genome. We identified and profiled transcriptional crosstalk in AAV genomes carrying 11 different enhancers active in mouse brain. We developed spatial genomics methods to identify and localize AAV genomes and their concatemeric forms in cultured cells and in tissue, and we demonstrate here that transcriptional croNature biotechnologySpatial genomics of AAV vectors reveals mechanism of transcriptional crosstalk that enables targeted delivery of large genetic cargo.PMC12807873DP1 NS111369ASAP-020495UF1MH128336UF1 MH128336Barcelona BHAppling NMackey EDGradinaru VKumar SREser RAChen XBorsos MCoughlin GMMayfield AMHJackson CRfalseSpatial genomics of AAV vectors reveals mechanism of transcriptional crosstalk that enables targeted delivery of large genetic cargo.Cell-type-specific regulatory elements such as enhancers can direct expression of recombinant adeno-associated viruses (AAVs) to specific cell types, but this approach is limited by the relatively small packaging capacity of AAVs. In this study, we used spatial genomics to show that transcriptional crosstalk between individual AAV genomes provides a general method for cell-type-specific expression of large cargo by separating distally acting regulatory elements into a second AAV genome. We identified and profiled transcriptional crosstalk in AAV genomes carrying 11 different enhancers active in mouse brain. We developed spatial genomics methods to identify and localize AAV genomes and their concatemeric forms in cultured cells and in tissue, and we demonstrate here that transcriptional cro2026-01-01T00:00:00Z2026 Jan2026-06-06T13:59:05.282Z2026-05-31T03:09:56.426ZS-EPMC128078734011395310.1038/s41587-025-02565-4