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Spatial Transcriptomics and Single-Nucleus RNA Sequencing Reveal rAAV2- and rAAV9-Specific Transduction Signatures in the Mouse Liver

Amberg, B.; Koechl, F.; Kumpesa, N.; Prasad, M.; Bochner, F.; Hernandez-Obiols, M.; Otteneder, M. B.; Stalder, L.; Shaffo, F.; Nowrouzi, A.; Markusic, D.; Haegel, H.; Xicluna, R.; Sultan, M.; Jacobsen, B.; Rottenberg, S.; Valdeolivas Urbelz, A.; Schwalie, P. C.; Hahn, K.

2025-03-15 molecular biology
10.1101/2025.03.13.643011 bioRxiv
Show abstract

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 remain 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 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 deregulated independent of zonation, serotype, and sex, while Srebf1, Tlcd4, Cpt2, and Acot1 exhibited sex-specific patterns. Circadian clock modulators (Dbp, Tef, Arntl, Nfil3, Nr1d1/Nr1d2) were altered independent of zonation. The study found sex-specific downregulation of immune and stress-response genes and pathways, including Gadd45g and hypoxia pathways. TGF-{beta} 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/643011v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@af87b9org.highwire.dtl.DTLVardef@1f79165org.highwire.dtl.DTLVardef@6aa2ccorg.highwire.dtl.DTLVardef@e6274c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Published in Gene Therapy · not in our set (fewer than 10 published preprints to learn from) · training set

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