Propagation of human hepatocytes in non-human mammals

• US 6,525,242 B1
• Filed: 11/02/1999
• Issued: 02/25/2003
• 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:
  US09/431,901
• Time in Patent Office: 1,211 Days
• Field of Search: 424/93.1, 424/93.2, 424/189.1, 424/422, 424/423, 435/325, 435/370, 435/6, 435/375, 536/24.5, 800/3, 800/8, 800/21, 800/9, 800/13, 800/18
• US Class Current: 800/8
• 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
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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.

Claims

1. A non-human mammal having a liver that comprises human hepatocytes, wherein the mammal has a normal immune system but (i) has been rendered tolerant to human hepatocytes by a method selected from the group consisting of intraperitoneal injection and intrathymic injection, of an effective amount of a composition selected from the group consisting of human hepatocytes and a human hepatocyte lysate, in a suitable carrier, during a time period selected from the group consisting of the period prior to birth of the mammal and the period after birth when the mammal is a neonate, and (ii) has subsequently received a transplant comprising human hepatocytes.

2. The non-human mammal of claim 1, where the mammal is a mouse which carries a transgene which is a urokinase gene operably linked to an albumin promoter.

3. A method of preparing a non-human mammal having a liver comprising human hepatocytes, comprising the steps of:

(i) inducing tolerance in a non-human host mammal toward hepatocytes from a human donor by a method selected from the group consisting of intraperitoneally injecting and intrathymically injecting an effective amount of a composition selected from the group consisting of human hepatocytes and a human hepatocyte lysate, in a suitable carrier, during a time period selected from the group consisting of the period prior to birth of the mammal and the period after birth when the mammal is a neonate, and

(ii) introducing hepatocytes from the human donor into the tolerized mammal produced in step (i) where the number of hepatocytes introduced are effective in colonizing the liver of the non-human mammal.

4. The method of claim 3 where the mammal is a mouse which carries a hepatotoxic transgene which is a urokinase gene operably linked to an albumin promoter.

5. The method of claim 3 where tolerance is induced by intraperitoneal injection of said composition.

6. The method of claim 3 where tolerance is induced by intrathymic injection of said composition.

7. A method for identifying a toxic effect of a test agent, comprising the steps of:

(a) administering the test agent to a non-human mammal having a liver that comprises human hepatocytes, wherein the mammal has a normal immune system but (i) has been rendered tolerant to human hepatocytes by a method selected from the group consisting of intraperitoneal injection and intrathymic injection, of an effective amount of a composition selected from the group consisting of human hepatocytes and a human hepatocyte lysate, in a suitable carrier, during a time period selected from the group consisting of the period prior to birth of the mammal and the period after birth when the mammal is a neonate, and (ii) has received a transplant comprising human hepatocytes; and

(b) subsequently evaluating whether changes have occurred in the viability of human hepatocytes in the mammal.

8. A model system for alcohol-associated liver disease comprising a non-human mammal having a liver that comprises human hepatocytes, wherein the mammal has a normal immune system but (i) has been rendered tolerant to human hepatocytes by a method selected from the group consisting of intraperitoneal injection and intrathymic injection, of an effective amount of a composition selected from the group consisting of human hepatocytes and a human hepatocyte lysate, in a suitable carrier, during a time period selected from the group consisting of the period prior to birth of the mammal and the period after birth when the mammal is a neonate, and (ii) has received a transplant comprising human hepatocytes, where the non-human mammal has been administered an amount of alcohol effective in producing hepatocellular degenerative changes.

9. A model system for a liver disease caused by Hepatitis B virus, comprising a non-human mammal having a liver that comprises human hepatocytes infected by Hepatitis B virus, wherein the mammal has a normal immune system but (i) has been rendered tolerant to human hepatocytes by a method selected from the group consisting of intraperitoneal injection and intrathymic injection, of an effective amount of a composition selected from the group consisting of human hepatocytes and a human hepatocyte lysate, in a suitable carrier, during a time period selected from the group consisting of the period prior to birth of the mammal and the period after birth when the mammal is a neonate, and (ii) has received a transplant comprising human hepatocytes, wherein Hepatitis B virus infection of the hepatocytes occurs before or after transplantation.