We have used high-titer (10(8) ffu/ml) recombinant retroviral vectors to transfer the beta-galactosidase (beta-Gal) gene to rat hepatocytes in vivo. In animals injected twice in the portal blood stream the next day after partial hepatectomy, half of the hepatocytes (46 +/- 17%) expressed the marker at the end of liver regeneration. The number of positive cells closely correlated with the viral titer as well as with beta-Gal enzymatic activity present in the whole liver. Because genes transferred via retroviral vectors in the liver are known to be expressed permanently, our present results open new possibilities for the development of gene therapy protocols for hereditary liver diseases using recombinant retroviral vectors.

The presence of an inactivating heat shock protein 110 (HSP110) mutation in colorectal cancers has been correlated with an excellent prognosis and with the ability of HSP110 to favor the formation of tolerogenic (M2-like) macrophages. These clinical and experimental results suggest a potentially powerful new strategy against colorectal cancer: the inhibition of HSP110. In this work, as an alternative to neutralizing antibodies, Nanofitins (scaffold~7 kDa proteins) targeting HSP110 were isolated from the screening of a synthetic Nanofitin library, and their capacity to bind (immunoprecipitation, biolayer interferometry) and to inhibit HSP110 was analyzed in vitro and in vivo. Three Nanofitins were found to inhibit HSP110 chaperone activity. Interestingly, they share a high degree of homology in their variable domain and target the peptide-binding domain of HSP110. In vitro, they inhibited the ability of HSP110 to favor M2-like macrophages. The Nanofitin with the highest affinity, A-C2, was studied in the CT26 colorectal cancer mice model. Our PET/scan experiments demonstrate that A-C2 may be localized within the tumor area, in accordance with the reported HSP110 abundance in the tumor microenvironment. A-C2 treatment reduced tumor growth and was associated with an increase in immune cells infiltrating the tumor and particularly cytotoxic macrophages. These results were confirmed in a chicken chorioallantoic membrane tumor model. Finally, we showed the complementarity between A-C2 and an anti-PD-L1 strategy in the in vivo and in ovo tumor models. Overall, Nanofitins appear to be promising new immunotherapeutic lead compounds.

Mature hepatocytes actively divide and express ga&a-&tamyl transpeptidase after D-galactosamine liver injury Kitten 0, Ferry N. Mature hepatocytes actively divide and express gamma-glutamyl transpeptidase after D-galactosamine liver injury.Abstract: AindBackground: We studied the fate of hepatocytes in the rat liver after D-galactosamine injury by genetic labeling using recombinant retroviruses carrying the Escherichia coli lacZ gene coupled to a nuclear localization signal. Methods: Hepatocytes were either labeled by direct injection of 2.5 ml high-titer retrovirus-containing medium in the regenerating liver parenchyma after administration of a single dose of D-galactosamine. Alternatively hepatocytes were pre-labeled, 24 h after a two-thirds hepatectomy, by injecting the same volume of retroviral solution in the portal vein and D-galactosamine was administered 15 days later. Gammaglutamyl transpeptidase and p-galactosidase activities were assessed on cryostat sections, along with localization of the hepatocyte-specific HES6 antigen. Results: Morphological observations, as well as p-galactosidase activity detection, showed that hepatocytes actively divide as early as 1 day after D-galactosamine injection. Gamma-glutamyl transpeptidase activity was detected in biliary cells, but also in mature hepatocytes, prelabeled with p-galactosidase before D-galactosamine administration. Conclusions: These experiments demonstrate that hepatocytes can divide to restore the liver mass after D-galactosamine liver injury. Furthermore, we also show that gamma-glutamyl transpeptidase, which has been reported to be expressed only by fetal or preneoplastic hepatocytes, can be re-expressed by mature hepatocytes during the recovery process.