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<p>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 (<i>Elovl3</i>, <i>Chka</i>, <i>Irs2</i>, <i>Ppard</i>), were consistently deregulated across all zones of the liver lobule in male and female rAAV2- and rAAV9-CMV-EGFP-treated mice, while <i>Srebf1</i>, <i>Tlcd4</i>, <i>Cpt2</i>, and <i>Acot1</i> exhibited sex-specific patterns. Circadian clock modulators (<i>Dbp</i>, <i>Tef</i>, <i>Arntl</i>, <i>Nfil3</i>, <i>Nr1d1/Nr1d2</i>) were altered independently of zonation. The study found sex-specific downregulation of immune and stress-response genes and pathways, including <i>Gadd45g</i> 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 <i>Rpsa</i>, <i>Dpp4</i>, <i>Sdc1</i>, 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月11日

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  • Bettina Amberg,
  • Fabian Köchl,
  • Nadine Kumpesa,
  • Megana Prasad,
  • Filip Bochner,
  • Mar Hernández-Obiols,
  • Michael B. Otteneder,
  • Lucas Stalder,
  • Frances Shaffo,
  • Ali Nowrouzi,
  • David Markusic,
  • Hélène Haegel,
  • Rebecca Xicluna,
  • Marc Sultan,
  • Björn Jacobsen,
  • Sven Rottenberg,
  • Alberto Valdeolivas,
  • Petra C. Schwalie,
  • Kerstin Hahn

摘要

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.