Spatial transcriptomics and single-nucleus RNA sequencing reveal rAAV2- and rAAV9-specific transduction signatures in the mouse liver.

The liver is a primary target for recombinant adeno-associated viral (rAAV) vectors, yet the influence of serotype, sex, and liver zonation on transduction and transcriptomic changes remains incompletely understood. This proof-of-concept study employs spatial transcriptomics alongside single-nucleus RNA sequencing to map the spatial distribution and impacts of rAAV2- and rAAV9-CMV-EGFP vectors in male and female mouse livers. Spatial transcriptomics provided precise transgene mapping and highlighted that CMV-EGFP rAAV vectors deregulate hepatocellular lipid metabolism, the circadian clock, and the immune/stress response with sex specific differences. Lipid metabolism genes (Elovl3, Chka, Irs2, Ppard), were consistently deregulated across all zones of the liver lobule in male and female rAAV2- and rAAV9-CMV-EGFP-treated mice, while Srebf1, Tlcd4, Cpt2, and Acot1 exhibited sex-specific patterns. Circadian clock modulators (Dbp, Tef, Arntl, Nfil3, Nr1d1/Nr1d2) were altered independently of zonation. The study found sex-specific downregulation of immune and stress-response genes and pathways, including Gadd45g and hypoxia pathways. TGF-β and EGFR pathways were upregulated sex-independently. Spatial transcriptomics further enabled examination of transgene and rAAV entry factor co-expression, identifying known and novel factors like Rpsa, Dpp4, Sdc1, and solute carrier proteins, highlighting its role in supporting targeted screening. Our findings demonstrate spatial transcriptomics as a powerful tool in gene therapy research and reveal novel rAAV vector effects on liver biology.