Propagation of human hepatocytes in non-human animals

Abstract

The present invention relates to the preparation of non-human animals having chimeric livers, whereby some or substantially all of the hepatocytes present are human hepatocytes. It is based, at least in part, on the discovery that rats, tolerized in utero against human hepatocytes, were found to serve as long-term hosts for human hepatocytes introduced post-natally, and the introduced hepatocytes maintained their differentiated phenotype, as evidenced by continued production of human albumin. The present invention further relates to the use of such animals as models of various liver diseases, including viral invention. Such embodiments are based on the discovery that transplanted human hepatocytes in chimeric livers were found to be susceptible to Hepatitis B virus and Hepatitis C virus infection.

1 - 5 . (Canceled)  
     
     
         6 . A non-human mammal having a chimeric liver comprising human hepatoblastoma cells.  
     
     
         7 . The non-human mammal of  claim 6 , wherein the mammal has a transgene, and wherein the transgene is more toxic to hepatocytes of the mammal than the human hepatoblastoma cells.  
     
     
         8 . The non-human mammal of  claim 7 , wherein the transgene comprises a gene encoding a toxin, wherein the gene is operably linked to a promoter, and wherein the promoter is more active in hepatocytes of the mammal than in human hepatoblastoma cells.  
     
     
         9 . The non-human mammal of  claim 8 , wherein the promoter comprises a nucleotide sequence selected from the group consisting of an albumin promoter, PEPCK promoter, hepatitis B surface antigen promoter, and metallothionein promoter.  
     
     
         10 . The non-human mammal of  claim 8 , wherein the transgene comprises a gene encoding a product selected from the group consisting of thymidine kinase and urokinase.  
     
     
         11 . The non-human mammal of  claim 8 , wherein the promoter is upregulated by administering an exogenous agent to the mammal.  
     
     
         12 . The non-human mammal of  claim 7 , wherein the transgene encodes a product that converts an administered, exogenous agent that is not substantially toxic to hepatocytes of the mammal to a compound that is toxic to the hepatocytes.  
     
     
         13 . The non-human mammal of  claim 12 , wherein the agent comprises gancyclovir.  
     
     
         14 . A method of preparing a non-human mammal having a chimeric liver comprising human hepatoblastoma cells, the method comprising introducing human hepatoblastoma cells into the mammal, wherein the number of introduced cells is effective in colonizing the liver of the mammal.  
     
     
         15 . The method of  claim 14  further comprising subjecting the mammal to a selection pressure that favors proliferation of introduced cells.  
     
     
         16 . The method of  claim 14 , wherein the selection pressure is provided by maturation of the mammal, wherein the mammal has a transgene, wherein the transgene comprises a gene encoding a toxin, wherein maturation of the mammal upregulates expression of the toxin, and wherein exposure of the mammal to the toxin favors proliferation of human hepatoblastoma cells.  
     
     
         17 . The method of  claim 14 , wherein the selection pressure is provided by a toxin, wherein the human hepatoblastoma cells have a transgene, wherein the transgene comprises a gene encoding a product that protects against the toxin, and wherein exposure of the mammal to the toxin favors proliferation of human hepatoblastoma cells.  
     
     
         18 . The method of  claim 17 , wherein the product reduces acetaminophen toxicity.  
     
     
         19 . The method of  claim 17 , wherein the product inhibits cytochrome activity.  
     
     
         20 . The method of  claim 19 , wherein the cytochrome is selected from the group consisting of 2E1, 1A2, and 3A4.  
     
     
         21 . The method of  claim 17 , wherein the product is selected from the group consisting of an enzyme, antisense RNA, and ribozyme.  
     
     
         22 . The method of  claim 21 , wherein the enzyme is a mutant RNA polymerase II that renders the human hepatoblastoma cells resistant to phalloidin.  
     
     
         23 . The method of  claim 15 , wherein the selection pressure is provided by a compound, wherein the compound is metabolized to a toxin in hepatocytes of the mammal, wherein the compound is metabolized to a toxin at greater levels in hepatocytes of the mammal than in human hepatoblastoma cells, and wherein exposure of the mammal to the compound favors proliferation of human hepatoblastoma cells.  
     
     
         24 . The method of  claim 23 , wherein the compound is gancyclovir, wherein the mammal has a transgene, wherein the transgene comprises a gene encoding Herpes simplex virus thymidine kinase, wherein the gene is operably linked to a promoter, and wherein the promoter is more active in hepatocytes of the mammal than in human hepatoblastoma cells.  
     
     
         25 . A method for identifying a toxic effect of a test agent, the method comprising administering the test agent to a non-human mammal having a chimeric liver comprising human hepatoblastoma cells, and subsequently evaluating whether changes have occurred in the viability of the human hepatoblastoma cells.  
     
     
         26 . A method for determining a toxic effect of a test agent on a non-human mammal, the method comprising administering the test agent on the mammal, performing a toxicology test on the mammal, and determining whether the test agent has a toxic effect on the mammal, wherein a positive score on the test indicates a toxic effect of the test agent on the mammal, and wherein the mammal has a chimeric liver comprising human hepatoblastoma cells.  
     
     
         27 . A method for determining the metabolic fate of a test agent on a non-human mammal of one species, the method comprising administering the test agent on the mammal and performing an assay that determines a metabolite of the test agent, wherein the mammal has a chimeric liver comprising human hepatoblastoma cells, wherein the mammal has received a test agent, wherein the test agent passes through the liver, and wherein the metabolic fate of the test agent in the mammal substantially recapitulates the metabolic fate of the test agent in a normal mammal of the respective species.  
     
     
         28 . The method of claims  14 ,  25 ,  26 , or  27 , wherein the human hepatoblastoma cells are of the Huh7 cell line.  
     
     
         29 . A model system for a human liver disease comprising a non-human mammal having a chimeric liver comprising human hepatoblastoma cells, wherein a toxin or pathogen is introduced.  
     
     
         30 . A model system for alcohol-associated liver disease comprising a non-human mammal having a chimeric liver comprising human hepatoblastoma cells, wherein the mammal has been administered an amount of alcohol effective in producing hepatocellular degenerative changes.  
     
     
         31 . A toxicology model system comprising a non-human mammal having a chimeric liver comprising human hepatoblastoma cells, wherein the mammal has received a test agent.  
     
     
         32 . A model system comprising a non-human mammal of one species, wherein the mammal has a chimeric liver comprising human hepatoblastoma cells, wherein the mammal has received a test agent, wherein the test agent passes through the liver, and wherein the model system substantially recapitulates the metabolic fate of the test agent in a normal mammal of the respective species.  
     
     
         33 . The model system of claims  29 ,  30 ,  31 , or  32 , wherein the human hepatoblastoma cells are of the Huh7 cell line.