Propagation of human hepatocytes in non-human animals

• US 6,995,299 B2
• Filed: 08/15/2001
• Issued: 02/07/2006
• Est. Priority Date: 11/02/1999
• Status: Expired due to Fees

• Current Assignee: University of Connecticut (State of Connecticut)
• Original Assignee: University of Connecticut (State of Connecticut)
• Inventors: Wu, George Y., Wu, Catherine H.
• Primary Examiner(s): FALK, ANNE MARIE
• Application Number:
  US09/930,781
• Publication Number:
  US 20020157121A1
• Time in Patent Office: 1,637 Days
• Field of Search: 800/8, 800/9, 435/325, 435/370, 424/93.1, 424/93.7
• US Class Current: 800/9
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A61K 2035/122 for inducing tolerance or supression of immune responses
• CPC Class Codes: A61K 39/001 Preparations to induce tolerance to non-self, e.g. prior to transplantation
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 9/1247 DNA-directed RNA polymerase (2.7.7.6)
• CPC Class Codes: C12N 9/6462 u-Plasminogen activator (3.4.21.73), i.e. urokinase
• CPC Class Codes: C12Y 304/21073 u-Plasminogen activator (3.4.21.73), i.e. urokinase
• CPC Class Codes: G01N 2333/02 Hepadnaviridae, e.g. hepatitis B virus
• CPC Class Codes: G01N 2333/18 Togaviridae; Flaviviridae

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.

Claims

1. A model system for Hepatitis C virus infection In humans, comprising a non-human mammal, wherein the mammal is immunocompetent but has been rendered inmunologically tolerant to human hepatocytes by fetal tolerization and subsequently transplanted with human hepatocytes and infected with Hepatitis C virus, whereby replication of Hepatitis C virus occurs in the model system.

2. The model system of claim 1, wherein the human hepatocytes are cells of the Huh7 cell line.

3. A method of preparing a non-human fetal mammal to receive a human hepatocyte trasplant, comprising the steps of:

(i) administering to the fetal mammal an effective amount of human heatoeytes, in a form selected from the group consisting of whole cells and a cell lysate, wherein the hepatocytes render the mammnal immunologically tolerant to human hepatocytes; and

(ii) administering to the mammal an effective amount of an agent, wherein the agent is metabolized by hepatocytes to produce a cytotoxin.

4. The method of claim 3, wherein the agent is retrorsine.

5. The method of claim 3, further comprising, after step ii, and after the mammal has been born, the step of introducing human hepatocytes into the mammal, wherein the number of introduced hepatocytes is effective in colonizing the liver of the mammal.

6. The method of claim 5, wherein the human hepatocytes are cells of the Huh7 cell line.