Human liver chimeric non-human animal with deficient p450 oxidoreductase and methods of using same

Abstract

The present disclosure provides a chimeric non-human animal comprising human hepatocytes, methods for preparing the chimeric non-human animal comprising human hepatocytes and methods of utilizing the chimeric non-human animal comprising human hepatocytes to screening and identifying metabolites for any type of drugs, typically small molecule drugs, which might affect human liver functions and any other bodily function.

What is claimed is: 
     
         1 . A method for preparing a chimeric non-human animal comprising human hepatocytes, the method comprising:
 (a) providing a non-human animal comprising a reduction or deletion of NADPH-P450 oxidoreductase (Por) gene resulting in reduced or absent expression of Por protein; and   (b) transplanting human hepatocytes into the non-human animal.   
     
     
         2 . The method of  claim 1 , wherein providing the non-human animal comprises reducing or deleting the Por gene resulting in reduced or absent expression of Por protein. 
     
     
         3 . The method of  claim 1 , wherein the reduced or deleted Por gene is a conditional knockdown or knockout of the Por gene. 
     
     
         4 . The method of  claim 1 , wherein the reduced or deleted Por gene is the result of a mutation, a transgene, treatment with an exogenous substance or somatic genome engineering. 
     
     
         5 . The method of  claim 4 , wherein the somatic genome engineering comprises Guide RNA (gRNA) and Caspase 9. 
     
     
         6 . The method of  claim 1 , wherein the non-human animal comprises a floxed allele of the Por gene, and wherein the non-human animal is provided with a Cre recombinase sufficient to produce a conditional knockout of the Por gene. 
     
     
         7 . The method of  claim 6 , wherein the non-human animal comprising the floxed allele of the Por gene is provided with at least a first dose of a virus that encodes Cre recombinase. 
     
     
         8 . The method of  claim 7 , wherein the non-human animal is provided with at least a second dose of a virus that encodes Cre recombinase. 
     
     
         9 . The method of  claim 1 , comprising
 (a) providing a non-human animal comprising a floxed allele of the Por gene with a first does of a virus that encodes Cre recombinase;   (b) transplanting human hepatocytes into the non-human animal; and   (c) providing the non-human animal with a second dose of a virus that encodes Cre recombinase.   
     
     
         10 . The method of  claim 9 , wherein steps (a) and (b) occur sequentially. 
     
     
         11 . The method of  claim 9 , wherein steps (a) and (b) occur simultaneously. 
     
     
         12 . The method of  claim 7 , wherein the non-human animal comprising the floxed allele of the Por gene is crossed with a transgenic non-human animal strain expressing Cre recombinase. 
     
     
         13 . The method of  claim 1 , wherein the non-human animal further comprises a reduction or deletion of at least one additional gene encoding an enzyme involved in drug metabolism. 
     
     
         14 . The method of  claim 13 , wherein the at least one additional enzyme is a phase II drug enzyme. 
     
     
         15 . The method of  claim 1 , wherein the non-human animal further comprises a reduction or deletion of UDP-glucose 6-dehydrogenase (UGDH) gene. 
     
     
         16 . The method of  claim 1 , wherein the non-human animal further comprises a reduction or deletion of Glutathione synthetase (GSS) gene. 
     
     
         17 . The method of  claim 1 , wherein the non-human animal is selected from the group consisting of primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep and pig. 
     
     
         18 . The method of  claim 1 , wherein the non-human animal is a mouse. 
     
     
         19 . The method of  claim 1 , wherein the non-human animal is selected from the group consisting of (i) the FRG (Fah −/− Rag2 −/− /Il2rg −/− ) non-human animal, (ii) a transgenic urokinase type plasminogen activator (uPA) non-human animal, which overexpress uPA under an inducible promoter, preferably a liver-restricted albumin promoter, (iii) the thymidine kinase-NOD/Shi-scid/IL-2Rγ null  (TK-NOG) non-human animal, which is a immunodeficient NOG non-human animal with transgenic expression of thymidine kinase under control of liver-restricted promoter, (iv) a non-human animal expressing an inducible Caspase 8 in the liver, and (v) a non-human animal expressing an inducible Caspase 9 in the liver. 
     
     
         20 . A chimeric non-human animal, offspring thereof, or a portion thereof, which has a chimeric liver comprising human hepatocytes, prepared by the method according to any one of  claims 1 - 19 . 
     
     
         21 . The chimeric non-human animal of  claim 20 , wherein the non-human animal is selected from the group consisting of primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep and pig. 
     
     
         22 . The chimeric non-human animal of  claim 20 , wherein the non-human animal is a mouse. 
     
     
         23 . The chimeric non-human animal of  claim 20 , wherein chimeric non-human animal substantially lacks autogenous hepatocytes. 
     
     
         24 . The chimeric non-human animal of  claim 20 , wherein human hepatocytes account for at least 60% of all hepatocytes in the chimeric liver. 
     
     
         25 . The chimeric non-human animal of  claim 20 , wherein human hepatocytes account for at least 70% of all hepatocytes in the chimeric liver. 
     
     
         26 . The chimeric non-human animal of  claim 20 , wherein human hepatocytes account for at least 80% of all hepatocytes in the chimeric liver. 
     
     
         27 . The chimeric non-human animal of  claim 20 , wherein human hepatocytes account for at least 90% of all hepatocytes in the chimeric liver. 
     
     
         28 . The chimeric non-human animal of  claim 20 , wherein the chimeric non-human animal is immunodeficient. 
     
     
         29 . A method for screening for a substance that affects human liver functions, comprising:
 (a) administering a test substance to the chimeric non-human animal of  claim 20 ;   (b) measuring one or more values in the chimeric non-human animal to which the test substance is administered in (a); and   (c) selecting a test substance that causes an increase or decrease in one or more values measured in (b), compared with the one or more values measured in a chimeric non-human animal to which no test substance is administered.   
     
     
         30 . The method of  claim 29 , wherein the one or more values is selected from the group consisting of a metabolite of the test substance, human albumin concentration, body weight curve, liver-weight-to-body-weight ratio, total albumin level, total protein level, Alanine Aminotransferase (ALT) level, Aspartate Aminotransferase (AST) level, and total bilirubin level, creatinine, Blood Urea Nitrogen (BUN), troponine, blood count, TSH and histological assessment for pathologies in the human and non-human organs. 
     
     
         31 . A method for evaluating the toxicity of a test substance against human hepatocytes and any other non-human organ in the non-human chimeric animal, comprising:
 (a) administering a test substance to the chimeric non-human animal of  claim 20 ;   (b) measuring one or more indicators in the chimeric non-human animal to which the test substance is administered in (a); and   (c) evaluating the effect of the test substance on human hepatocytes using, one or more indicators measured in (b), compared with the one or more indicators measured in a chimeric non-human animal to which no test substance is administered.   
     
     
         32 . The method of  claim 31 , wherein the one or more indicators is selected from the group consisting of an increase or a decrease in any one or more of a metabolite of the test substance, human albumin concentration, body weight curve, liver-weight-to-body-weight ratio, total albumin level, total protein level, ALT level, AST level, and total bilirubin level, creatinine, BUN, troponine, blood count and TSH as well as histological alterations including but not limited to necrosis and apoptosis in the human and non-human organs.