Spatiotemporal liver dynamics shape hepatocellular heterogeneity and impact in vivo gene engineering

Background & Aims: Hepatocytes are the liver's main functional cells and are key targets for in vivo gene therapy to treat monogenic diseases. Integrating the transgene into the genome is critical for long-term expression from a single early-life dose, which is achievable via integrating vectors or genome editing. To ensure persistence through liver growth and cell turnover, it is also necessary to target the hepatocytes driving these processes. While liver regeneration and homeostasis have been studied extensively, hepatocyte growth and maturation remain less well understood. Here, we investigate how hepatocyte heterogeneity evolves during liver growth and its implications for in vivo gene engineering. Methods: We performed clonal tracing, as well as single-cell and spatial transcriptomics, on mouse livers of various ages. We evaluated the efficiency, stability, and lobule distribution of lentiviral gene transfer and targeted transgene integration. Results: We found that a subset of clonogenic hepatocytes (15-20%) in the newborn liver generates >90% of the adult tissue and co-localizes with hematopoietic islands within a spatial niche. Preferential gene editing of these clonogenic hepatocytes resulted in an increased proportion of the gene-engineered liver area, supporting their role in liver growth. Age-dependent hepatocellular heterogeneity affected the efficiency of lentiviral gene delivery in vivo and its distribution throughout the hepatic lobule. The gradual establishment of metabolic zonation after weaning and elevated proteasome activity in the peri-central area in adults influenced the observed age-related outcomes. Conclusion: These insights into spatiotemporal hepatocyte dynamics enhance our understanding of liver biology and have important implications for therapeutic strategies. Impact and implications: We provide new insights into the spatiotemporal dynamics of the mouse liver during postnatal growth, highlighting both proliferative and transcriptomic heterogeneity among hepatocytes and their impact on the efficiency and distribution of in vivo lentiviral gene delivery and targeted gene editing. Understanding and manipulating the biological processes behind this heterogeneity can enhance gene transfer outcomes. We report that not all hepatocytes contribute equally to liver growth, indicating that effectively targeting clonogenic hepatocytes in the newborn liver is crucial for the long-term maintenance of therapeutic genetic modifications. Furthermore, this phenomenon can be leveraged to expand the pool of genetically corrected cells, as illustrated here by a targeted gene editing strategy. Finally, we reveal the existence of a tissue niche that supports the proliferation of both clonogenic hepatocytes and hematopoietic progenitors in neonatal livers. Gaining a deeper understanding of this niche and its signals may be beneficial for regenerative purposes.