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

Abstract

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 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 28 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.

SHP2 inhibition enhances Yes-associated protein–mediated liver regeneration in murine partial hepatectomy models

“…To explore this, we performed hepatocyte-specific deletion of YAP and its paralog TAZ concurrently, utilizing Yap fl/fl / Taz fl/fl mice injected i.v. with hepatocyte tropic AAV8 viral particles expressing Cre recombinase ( 38 ). These Yap Δhep / Taz Δhep mice then underwent treatment with NSC or vehicle perioperatively ( Supplemental Figure 4E ).…”

“…We further evaluated if YAP activation was a major contributing mechanism by which accelerated liver regeneration occurred following NSC administration. To explore this, we performed hepatocyte-specific deletion of YAP and its paralog TAZ concurrently utilizing Yap fl/fl /Taz fl/fl mice injected intravenously with hepatocyte tropic AAV-8 viral particles expressing Cre recombinase (38). These Yap ∆hep /Taz ∆hep mice then underwent treatment with NSC or vehicle perioperatively (Supplemental Figure 4E).…”

“…Yap/Taz double floxed mice (Yap fl/fl /Taz fl/fl ) were obtained from Jackson Laboratory (Strain #030532). Eight-week-old Yap fl/fl /Taz fl/fl mice were administered 1x10 11 AAV8 particles expressing AAV.TBG.PI.Cre.rBG, a gift from James M. Wilson (University of Pennsylvania, Philadelphia, PA) (Addgene viral prep # 100787-AAV8) as previously described, intravenously by tail vein injection (38). Four weeks following administration mice underwent 70% partial hepatectomy as described above.…”

Constitutively active STAT5b feminizes mouse liver gene expression

“…To determine whether the control of sex-biased liver gene expression by plasma GH pulses (male GH pattern) vs persistent GH stimulation (female GH pattern) is due to the downstream effects of pulsatile vs persistent STAT5 activation per se , we expressed a constitutively active form of STAT5 (STAT5 CA ) in male mouse liver to mimic the pattern of persistent STAT5 activation that occurs in female mouse liver (9). STAT5 CA cDNA expressed from the hepatocyte-specific thyroxin binding globulin promoter (45,46) was delivered using an AAV serotype 8 vector, which has high intrinsic tropism for the liver (30,31). We validated the AAV8-STAT5 CA viral vector by its ability to target STAT5 protein to the nucleus ( Fig.…”

Hepatocyte PPARγ contributes to the progression of non-alcoholic steatohepatitis in male and female obese mice

“…This approach has the potential to unveil new roles of the studied gene which could be masked by developmental compensation induced by the knockout. Here, we used adeno-associated virus (AAV) to selectively, and efficiently [48], knockout PPAR in hepatocytes in mice with pre-established diet-induced obesity. Also, we fed adult (>10 weeks of age) mice, instead of peripuberal mice (~ 6 weeks), with a HFD to promote metabolic dysfunction and to prevent an increase of lean mass by HFD [49].…”