Michael Ott

Rapid clearance of syngeneic transplanted hepatocytes following transduction with E-1-deleted adenovirus indicates early host immune responses and offers novel ways for studying viral vector, target cell and host interactions.

To distinguish between transduced cell clearance and transgene regulation following adenoviral gene transfer, we infected F344 rat hepatocytes with an E-1-deleted adenovirus (Adβgal) and studied cell survival in the liver of dipeptidyl peptidase IV-deficient (DPPIV−) F344 rats. Transplanted cells were localized with histochemical staining for DPPIV and transgene expression localized with staining for β-galactosidase (lacZ). The transgene was expressed in 90–100% hepatocytes without impairment in cell viability in vitro, although transplanted cells were cleared mostly within 1 day by infiltrates containing activated macrophages, CD4+ or CD8+ lymphocytes, and phagocytes. When Adβgal-transduced hepatocytes were transplanted repeatedly at 14-day intervals, transplanted cells were cleared rapidly each time. LacZ expression following Adβgal administration to intact animals was associated with apoptosis and unscheduled DNA synthesis in the liver. To determine whether adenoviral antigen expression activated consequential MHC-restricted liver injury, we transplanted Adβgal-hepatocytes followed subsequently by transplantation of nontransduced hepatocytes. Transplanted cells expressing Adβgal were rapidly cleared as before, whereas nontransduced hepatocytes engrafted with progressive liver repopulation. The findings indicated that adenovirally transduced cells are cleared early in the host liver. Use of ex vivo strategies will facilitate analysis of modified adenoviral vectors in the context of immunoregulatory, cellular and viral mechanisms.

Control of Hepatitis C Virus Replication in Mouse Liver-Derived Cells by MAVS-Dependent Production of Type I and Type III Interferons

ABSTRACT Hepatitis C virus (HCV) efficiently infects only humans and chimpanzees. Although the detailed mechanisms responsible for this narrow species tropism remain elusive, recent evidence has shown that murine innate immune responses efficiently suppress HCV replication. Therefore, poor adaptation of HCV to evade and/or counteract innate immune responses may prevent HCV replication in mice. The HCV NS3-4A protease cleaves human MAVS, a key cellular adaptor protein required for RIG-I-like receptor (RLR)-dependent innate immune signaling. However, it is unclear if HCV interferes with mouse MAVS function equally well. Moreover, MAVS-dependent signaling events that restrict HCV replication in mouse cells were incompletely defined. Thus, we quantified the ability of HCV NS3-4A to counteract mouse and human MAVS. HCV NS3-4A similarly diminished both human and mouse MAVS-dependent signaling in human and mouse cells. Moreover, replicon-encoded protease cleaved a similar fraction of both MAVS variants. Finally, FLAG-tagged MAVS proteins repressed HCV replication to similar degrees. Depending on MAVS expression, HCV replication in mouse liver cells triggered not only type I but also type III IFNs, which cooperatively repressed HCV replication. Mouse liver cells lacking both type I and III IFN receptors were refractory to MAVS-dependent antiviral effects, indicating that the HCV-induced MAVS-dependent antiviral state depends on both type I and III IFN receptor signaling. IMPORTANCE In this study, we found that HCV NS3-4A similarly diminished both human and mouse MAVS-dependent signaling in human and mouse cells. Therefore, it is unlikely that ineffective cleavage of mouse MAVS per se precludes HCV propagation in immunocompetent mouse liver cells. Hence, approaches to reinforce HCV replication in mouse liver cells (e.g., by expression of essential human replication cofactors) should not be thwarted by the poor ability of HCV to counteract MAVS-dependent antiviral signaling. In addition, we show that mouse MAVS induces both type I and type III IFNs, which together control HCV replication. Characterization of type I or type III-dependent interferon-stimulated genes in these cells should help to identify key murine restriction factors that preclude HCV propagation in immunocompetent mouse liver cells.

Overexpression of Mad transcription factor inhibits proliferation of cultured human hepatocellular carcinoma cells along with tumor formation in immunodeficient animals

Dominant negative regulation of critical cell cycle molecules could perturb survival of cancer cells and help develop novel therapies.

Homologous recombination mediates stable Fah gene integration and phenotypic correction in tyrosinaemia mouse-model

AIM To stably correct tyrosinaemia in proliferating livers of fumarylacetoacetate-hydrolase knockout (Fah-/-) mice by homologous-recombination-mediated targeted addition of the Fah gene. METHODS C57BL/6 Fah∆exon5 mice served as an animal model for human tyrosinaemia type 1 in our study. The vector was created by amplifying human Fah cDNA including the TTR promoter from a lentivirus plasmid as described. The Fah expression cassette was flanked by homologous arms (620 bp and 749 bp long) of the Rosa26 gene locus. Mice were injected with 2.1 × 108 VP of this vector (rAAV8-ROSA26.HAL-TTR.Fah-ROSA26.HAR) via the tail vein. Mice in the control group were injected with 2.1 × 108 VP of a similar vector but missing the homologous arms (rAAV8-TTR.Fah). Primary hepatocytes from Fah-/- recipient mice, treated with our vectors, were isolated and 1 × 106 hepatocytes were transplanted into secondary Fah-/- recipient mice by injection into the spleen. Upon either vector application or hepatocyte transplantation NTBC treatment was stopped in recipient mice. RESULTS Here, we report successful HR-mediated genome editing by integration of a Fah gene expression cassette into the “safe harbour locus” Rosa26 by recombinant AAV8. Both groups of mice showed long-term survival, weight gain and FAH positive clusters as determined by immunohistochemistry analysis of liver sections in the absence of NTBC treatment. In the group of C57BL/6 Fah∆exon5 mice, which have been transplanted with hepatocytes from a mouse injected with rAAV8-ROSA26.HAL-TTR.Fah-ROSA26.HAR 156 d before, 6 out of 6 mice showed long-term survival, weight gain and FAH positive clusters without need for NTBC treatment. In contrast only 1 out 5 mice, who received hepatocytes from rAAV8-TTR.Fah treated mice, survived and showed few and smaller FAH positive clusters. These results demonstrate that homologous recombination-mediated Fah gene transfer corrects the phenotype in a mouse model of human tyrosinaemia type 1 (Fah-/- mice) and is long lasting in a proliferating state of the liver as shown by withdrawal of NTBC treatment and serial transplantation of isolated hepatocytes from primary Fah-/- recipient mice into secondary Fah-/- recipient mice. This long term therapeutic efficacy is clearly superior to our control mice treated with episomal rAAV8 gene therapy approach. CONCLUSION HR-mediated rAAV8 gene therapy provides targeted transgene integration and phenotypic correction in Fah-/- mice with superior long-term efficacy compared to episomal rAAV8 therapy in proliferating livers.