Viral Immunoregulatory Genes and Hepatocyte Transplants

DESCRIPTION (provided by applicant): Our objective is to develop safe and efficient methods to genetically modify donor liver cells to abrogate allograft rejection after hepatocyte transplantation without the use of long-term immunosuppression. Adenovirus (Ad) early transcription region three (E3) encodes immunomodulatory proteins, which we have used previously to generate transgenic mice expressing the E3 genes in pancreatic islets. These islets did not undergo autoimmune destruction of beta-cells in model murine systems of type 1 diabetes and were not rejected when transplanted into allogeneic recipients. Our hypothesis is that insertion of some or all of the AdE3 genes into primary or immortalized hepatocytes will prevent their rejection upon transplantation into allogeneic recipient jaundiced Gunn rats, which are an animal model for Crigler-Najjar syndrome type 1 (CN1). CN1 is a potentially lethal inherited deficiency of bilirubin conjugation. Our goals are to determine which of the various potential mechanisms of AdE3 suppression of the immune system are most important in facilitating the survival and function of allogeneic hepatocyte transplants. Among the possible immunologic targets acting alone or together are the downregulation of class I MHC by Ad-gp19; inhibition of TNF cytolysis by Ad E3-14.7K; reduction of cell surface expression of pro-apoptotic receptors such as FAS, TRAIL and TNFR, by the complex of Ad E3- 10.4/14.5K; inhibition of signal transduction pathways such as NFkappaB and AP-1 by Ad E3-10.4/14.5K; and prevention of chemokine (MCP-1, IL-8, IP-10) synthesis in response to TNFalpha. In addition, we will attempt to dissect whether Ad E3 acts only on the hepatocyte by intrinsic mechanisms or also alters the effector arm of the immune system by tolerizing by clonal deletion or induction of suppressor cells. The experiments proposed will use both hepatocytes conditionally immortalized with SV40 T antigen (Tag) and primary hepatocytes. Hepatocytes present new challenges and opportunities, because unlike pancreatic islets, the host liver can be manipulated by controlled destruction of endogenous hepatocytes to permit extensive repopulation of the liver with the progeny of the transplanted cells. We will use a conditionally immortalized Fisher rat hepatocyte line to insert Ad E3 genes in new combinations expeditiously by transfection or lentivirus infection. The lentivirus technology also will allow us to use primary hepatocytes to assess their efficacy in reconstituting normal bilirubin processing in the liver.