Abstract
ABSTRACTMice 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 wild-type 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 γH2AX 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.
Highlights
Animal models have improved our understanding and therapies for human disease
These data show, in agreement with other studies (Wang et al, 2010; Bell et al, 2011b), that associated virus 8 (AAV8)-Thyroxin Binding Globulin (TBG)-mediated gene targeting is highly specific for hepatocytes with negligible targeting of extra-hepatic tissues
AAV8-TBG vectors are an established means for hepatocyte-specific manipulation of gene expression in vivo
Summary
Received 26 February 2021; Accepted 17 August 2021 efficiency and the range of available genetic models. Due to the latter mice have become the most widely used in vivo pre-clinical model system (Rosenthal and Brown, 2007). Manipulation of gene expression in this model organism has come a long way from whole body knockout (KO) to the current point that we are able to introduce point mutations in a tissue specific manner through CRISPR-Cas genomic editing (Sauer and Henderson, 1988; Wilson, 1996; Lee et al, 2020a; Lundin et al, 2020). The Cre-Lox system, less flexible compared to CRISPR, remains widely used for the manipulation of gene expression in mice and is a readily applicable means of genomic editing with high reproducibility
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