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<p>Recombinant adeno-associated viruses (rAAVs) are a platform of choice for gene therapy. However, liver-directed transduction has been hindered by immune responses unpredicted in the pre-clinical models, resulting in therapy failure. Liver immune responses are strictly regulated by the interactions between hepatocytes and non-parenchymal liver cells, such as Kupffer cells (the liver-resident macrophages) but how rAAVs induce such responses remains largely unknown. Therefore, human models recapitulating such interactions are required to address innate immune responses. Here, we developed a human 3D model to characterize the contribution of hepatocytes and Kupffer cells to the innate immune response. We developed a strategy for the differentiation of Kupffer-like cells from circulating monocytes based on cell–cell contact with primary human hepatocytes. We fine-tuned critical co-culture parameters to obtain a Kupffer-like phenotype while retaining hepatocyte viability and identity. Functional assessment of the differentiated 3D co-cultures showed that the model is responsive to classical pathogen-associated molecular pattern molecules, at a gene expression and secretory level. Moreover, we observed increased proinflammatory cytokine expression and secretion when challenged with a rAAV vector. Our data indicate the suitability of the novel model to investigate hepatocyte-Kupffer cell interactions and address innate immune responses within a human liver microenvironment.</p>

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3D co-cultures of primary human hepatocytes and Kupffer-like cells to address innate immune responses to rAAV

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月6日

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  • Isabella Ramella Gal,
  • Francisca Arez,
  • Inês P. Correia,
  • Giacomo Domenici,
  • Sofia Fernandes,
  • Gabriela Silva,
  • Inês Saldanha,
  • Nadia Duarte,
  • Catarina Freitas,
  • Paula M. Alves,
  • Udo Maier,
  • Ana Sofia Coroadinha,
  • François du Plessis,
  • Catarina Brito

摘要

Recombinant adeno-associated viruses (rAAVs) are a platform of choice for gene therapy. However, liver-directed transduction has been hindered by immune responses unpredicted in the pre-clinical models, resulting in therapy failure. Liver immune responses are strictly regulated by the interactions between hepatocytes and non-parenchymal liver cells, such as Kupffer cells (the liver-resident macrophages) but how rAAVs induce such responses remains largely unknown. Therefore, human models recapitulating such interactions are required to address innate immune responses. Here, we developed a human 3D model to characterize the contribution of hepatocytes and Kupffer cells to the innate immune response. We developed a strategy for the differentiation of Kupffer-like cells from circulating monocytes based on cell–cell contact with primary human hepatocytes. We fine-tuned critical co-culture parameters to obtain a Kupffer-like phenotype while retaining hepatocyte viability and identity. Functional assessment of the differentiated 3D co-cultures showed that the model is responsive to classical pathogen-associated molecular pattern molecules, at a gene expression and secretory level. Moreover, we observed increased proinflammatory cytokine expression and secretion when challenged with a rAAV vector. Our data indicate the suitability of the novel model to investigate hepatocyte-Kupffer cell interactions and address innate immune responses within a human liver microenvironment.