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Advanced characterization of human hepatocytes xenotransplanted mice as predictive pharmacological tool system for human liver-targeted gene therapy

Primary human hepatocytes xenotransplanted into mice represent an increasingly interesting system to investigate the translational potential of liver-targeted gene therapy solutions and can in many cases provide an alternative to the use of non-human primates during early efficacy evaluation.
Here we present a standardized workflow for the screening of AAV-derived viral vectors in xenotransplanted mice, with a focus on improved human liver tropism and species-specific transduction of the payload. We describe the application of histological and molecular techniques to study biodistribution and pharmacodynamics with resolution not limited to the tissue-level but refined to the individual human hepatocyte.

In particular, we have established the following techniques:

  • in vivo as well as ex vivo expression analysis of a reporter payload in mice, and precisely within the xenografted (human) vs. resident (murine) populations of liver cells
  • combined immunofluorescent labelling of the payload and detection of cell-identifying markers further allows for the analysis of spatial biodistribution within the organ, such as cell-type targeting and zonation effects
  •  single-nuclei RNA sequencing on isolated tissue specimens, allowing molecular identification of transduced cell populations and associated disturbances to the gene expression profile, with the potential to highlight altered molecular pathways that can inform either pharmacodynamic or toxicological outcomes.
    In summary, we disclose the application of xenotransplanted mice and associated analytics vastly improving the translational value of the model system in the development of gene therapies. This scientific poster was recently presented at ESGCT 2022.

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