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.

What is claimed is:  
     
         1 . A non-human animal having a liver that comprises human hepatocytes, wherein the animal has a normal immune system but has been rendered tolerant to human hepatocytes.  
     
     
         2 . The non-human animal of  claim 1 , wherein the animal carries a transgene that is directly toxic to hepatocytes of the non-human animal but not specifically toxic to human hepatocytes.  
     
     
         3 . The non-human animal of  claim 2 , where the transgene is a urokinase gene operably linked to a promoter selectively active in liver cells of the animal.  
     
     
         4 . The non-human animal of  claim 3 , where the promoter is the albumin promoter.  
     
     
         5 . The non-human animal of  claim 1 , wherein the animal carries a transgene that is indirectly toxic to hepatocytes of the non-human animal but not specifically toxic to human hepatocytes, and wherein the toxic effect is induced by administering an exogenous agent to the animal.  
     
     
         6 . The non-human animal of  claim 5 , where the transgene is a Herpes simplex virus thymidine kinase gene operably linked to a promoter selectively active in liver cells of the animal.  
     
     
         7 . The non-human animal of  claim 6 , where the exogenous agent is gancyclovir.  
     
     
         8 . The non-human animal of  claim 6 , where the promoter is the albumin promoter.  
     
     
         9 . A method of preparing a non-human animal having a liver comprising human hepatocytes, comprising the steps of: (i) inducing tolerance in a non-human host animal toward hepatocytes from a human donor and (ii) introducing hepatocytes from the human donor into the tolerized animal produced in step (i) such that at least some of the introduced human hepatocytes localize in the liver of the non-human animal.  
     
     
         10 . The method of  claim 9  where tolerance is induced by intraperitoneal injection of an antigen-containing composition selected from the group consisting of a human cell lysate and intact human cells.  
     
     
         11 . The method of  claim 9  where tolerance is induced by intrathymic injection of an antigen-containing composition selected from the group consisting of a human cell lysate and intact human cells.  
     
     
         12 . The method of  claim 9  further comprising the step of subjecting the animal to a selection pressure which favors the proliferation of human hepatocytes.  
     
     
         13 . The method of  claim 12  where the selection pressure is maturation of the animal which activates the expression of a hepatotoxic transgene in the hepatocytes of the non-human animal host but not the donor human hepatocytes.  
     
     
         14 . The method of  claim 12  where the selection pressure is provided by exposing the non-human animal to a toxin, wherein donor human hepatocytes are protected against the toxin by transfection of a transgene into the human hepatocytes, wherein the transgene protects the donor hepatocytes from the toxic effects of the toxin, and wherein hepatocytes of the non-human animal lack the transgene and are therefore not protected against the toxin.  
     
     
         15 . The method of  claim 14 , wherein the protective transgene encodes a product selected from the group consisting of an antisense RNA and a ribozyme that functionally inactivates a cytochrome selected from the group consisting of 2E1, 1A2 and 3A4, such that the transgene would prevent activation of acetaminophen in human donor hepatocytes but not hepatocytes of the non-human animal.  
     
     
         16 . The method of  claim 14 , wherein the protective transgene encodes a mutant RNA polymerase II that would render the human donor hepatocytes resistant to phalloidin.  
     
     
         17 . The method of  claim 13  where the hepatotoxic transgene is a urokinase gene operably linked to an albumin promoter.  
     
     
         18 . The method of  claim 12  where the selection pressure is the administration of a compound that is metabolized to a toxic substance in hepatocytes of the non-human animal host but not the donor human hepatocytes.  
     
     
         19 . The method of  claim 18  where the non-human animal host carries a Herpes simplex virus thymidine kinase gene that is selectively expressed in hepatocytes of the animal and the administered compound is gancyclovir.  
     
     
         20 . A method for identifying a toxic effect of a test agent, comprising administering the test agent to a non-human animal having a liver that comprises human hepatocytes and subsequently evaluating whether changes have occurred in at least one marker of bodily function of the animal.  
     
     
         21 . A method for identifying a toxic effect of a test agent, comprising administering the test agent to a non-human animal having a liver that comprises human hepatocytes and subsequently evaluating whether changes have occurred in the viability of human hepatocytes in the animal.  
     
     
         22 . A model system for a human liver disease comprising a non-human animal having a liver that comprises human hepatocytes in which an agent associated with causation of the disease in humans has been introduced.  
     
     
         23 . The model system of  claim 22  where the human liver disease is alcohol-associated liver disease and where the non-human animal has been administered an amount of alcohol effective in producing hepatocellular degenerative changes.  
     
     
         24 . A model system for a human liver disease caused by an infectious agent, comprising a non-human animal having a liver that comprises human hepatocytes infected by the infectious agent.  
     
     
         25 . The model system of  claim 24 , where the infectious agent is hepatitis C virus.  
     
     
         26 . The model system of  claim 24 , where the infectious agent is hepatitis B virus.  
     
     
         27 . The model system of  claim 24 , where the infectious agent is hepatitis A virus.  
     
     
         28 . The model system of  claim 24 , where the infectious agent is hepatitis D virus.  
     
     
         29 . The model system of  claim 24 , where the infectious agent is Yellow Fever virus.  
     
     
         30 . The model system of  claim 24 , where the infectious agent is hepatitis E virus.  
     
     
         31 . The model system of  claim 24 , where the infectious agent is malaria.  
     
     
         32 . The model system of  claim 24 , where the infectious agent is cytomegalovirus.  
     
     
         33 . The model system of  claim 24 , where the infectious agent is Epstein Barr virus.  
     
     
         34 . A model system for a human liver disease caused by a virus, comprising a non-human animal having a liver that comprises human hepatocytes containing a nucleic acid of the virus, where the nucleic acid is selected from the group consisting of a nucleic acid comprised in the genome of the virus and a nucleic acid transcript encoded by the genome of the virus.  
     
     
         35 . The model system of  claim 34 , where the virus is hepatitis C virus.  
     
     
         36 . The model system of  claim 34 , where the virus is hepatitis B virus.  
     
     
         37 . The model system of  claim 34 , where the virus is hepatitis A virus.  
     
     
         38 . The model system of  claim 34 , where the virus is hepatitis D virus.  
     
     
         39 . The model system of  claim 34 , where the virus is Yellow Fever virus.  
     
     
         40 . The model system of  claim 34 , where the virus is hepatitis E virus.  
     
     
         41 . The model system of  claim 34 , where the virus is cytomegalovirus.  
     
     
         42 . The model system of  claim 34 , where the virus is Epstein Barr virus.  
     
     
         43 . A method of treating a human subject having a genetic defect, comprising (i) propagating human hepatocytes that correct the genetic defect in a non-human animal, where the human hepatocytes are histocompatible with the human subject; (ii) collecting hepatocytes from the animal; (iii) separating human hepatocytes from non-human hepatocytes; and (iv) introducing the human hepatocytes into the subject.  
     
     
         44 . The method of  claim 43 , where the human hepatocytes are originally obtained from the subject and then engineered to correct the genetic defect prior to propagation in the non-human animal.  
     
     
         45 . A method of treating a human subject having a damaged liver, comprising (i) propagating human hepatocytes in a non-human animal, where the human hepatocytes are histocompatible with the human subject; (ii) collecting hepatocytes from the animal; (iii) separating human hepatocytes from non-human hepatocytes; and (iv) introducing the human hepatocytes into the subject.  
     
     
         46 . The method of  claim 45 , where the human hepatocytes are originally obtained from the subject.