Specificity and off-target effects of AAV8-TBG viral vectors for the manipulation of hepatocellular gene expression in mice

Mice are a widely used pre-clinical model system in large part due to their potential for genetic manipulation. The ability to manipulate gene expression in specific cells under temporal control is a powerful experimental tool. The liver is central to metabolic homeostasis and a site of many diseases, making the targeting of hepatocytes attractive. Adeno-associated virus 8 (AAV8) vectors are valuable instruments for the manipulation of hepatocellular gene expression. However, their off-target effects in mice have not been thoroughly explored. Here, we sought to identify the short-term off-target effects of AAV8 administration in mice. To do this, we injected C57BL/6J wildtype mice with either recombinant AAV8 vectors expressing Cre recombinase or control AAV8 vectors and characterised the changes in general health and in liver physiology, histology and transcriptomics compared to uninjected controls. We observed an acute and transient trend for reduction in homeostatic liver proliferation together with induction of the DNA damage marker gamma H2AX following AAV8 administration. The latter was enhanced upon Cre recombinase expression by the vector. Furthermore, we observed transcriptional changes in genes involved in circadian rhythm and response to infection. Notably, there were no additional transcriptomic changes upon expression of Cre recombinase by the AAV8 vector. Overall, there was no evidence of liver injury, and only mild T-cell infiltration was observed 14 days following AAV8 infection. These data advance the technique of hepatocellular genome editing through Cre-Lox recombination using Cre expressing AAV vectors, demonstrating their minimal effects on murine physiology and highlight the more subtle off target effects of these systems.

Automated summary

Mice are commonly used in pre-clinical studies due to their genetic manipulability, with AAV8 vectors being valuable tools for manipulating gene expression in hepatocytes. This study found that AAV8 administration in mice led to a transient reduction in liver proliferation and induction of DNA damage markers. Additionally, transcriptional changes were observed in genes related to circadian rhythm and response to infection.