# US 8,569,573 B2 · Method of expanding human hepatocytes in vivo

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- Number: US 8,569,573 B2
- Document ID: US8569573B2
- Status: Granted
- Kind: B2
- Application number: 12/663,219
- Filed: 2008-06-05
- Published: 2010-12-23
- Granted: 2013-10-29
- Earliest claimed: 2007-06-05
- Assignees: Oregon Health & Science University; The Board of Trustees of the Leland Stanford Junior University
- Applicants: Markus Grompe; Hisaya Azuma; Muhsen Al-Dhalimy; Mark A. Kay
- Inventors: Markus Grompe; Hisaya Azuma; Muhsen Al-Dhalimy; Mark A. Kay
- Web page: https://patentgrep.com/patent/US8569573B2

## CPC

- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- A01K 67/0271: Chimeric vertebrates, e.g. comprising exogenous cells
- A01K 67/0276: Knock-out vertebrates
- C12N 5/067: {Hepatocytes}
- A01K 2207/15: Humanized animals
- A01K 2217/00: Genetically modified animals
- A01K 2217/05: Animals comprising random inserted nucleic acids (transgenic)
- A01K 2217/075: inducing loss of function, i.e. knock out
- A01K 2227/105: Murine
- A01K 2267/02: Animal zootechnically ameliorated
- A01K 2267/025: Animal producing cells or organs for transplantation
- C12N 2799/022: where the vector is derived from an adenovirus
- C12N 2799/025: where the vector is derived from a parvovirus
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS

## Abstract

Described herein is a method of expanding human hepatocytes in vivo using an immunodeficient mouse which is further deficient in fumarylacetoacetate hydrolase (Fah). The method comprises transplanting human hepatocytes into the immunodeficient and Fah-deficient mice, allowing the hepatocytes to expand and collecting the expanded human hepatocytes. The method also allows serial transplantation of the human hepatocytes into secondary, tertiary, quaternary or additional mice. Also provided are mutant mice comprising homozygous deletions or point mutations in the Fah, Rag2 and Il2rg genes.

## Claims

### Claim 1

A method of expanding human hepatocytes in vivo, comprising:
i) transplanting human hepatocytes into a Rag2−/−/Il2rg−/− mouse, wherein the mouse is deficient for expression of Fah; and
ii) allowing the human hepatocytes to expand for at least about two weeks, thereby expanding the human hepatocytes.

### Claim 2 (depends on 1)

The method of claim 1, wherein the mouse is a Fah−/−/Rag2−/−/Il2rg−/− (FRG) mouse.

### Claim 3 (depends on 1)

The method of claim 1, wherein the mouse is a Fahpm/Rag2−/−/Il2rg−/− (FpmRG) mouse.

### Claim 4 (depends on 1)

The method of claim 1, wherein a vector encoding human urokinase is administered to the mouse prior to transplanting the human hepatocytes.

### Claim 5 (depends on 4)

The method of claim 4, wherein the urokinase is a secreted form of urokinase.

### Claim 6 (depends on 4)

The method of claim 4, wherein the urokinase is a non-secreted form of urokinase.

### Claim 7 (depends on 1)

The method of claim 1, wherein the mouse is administered 2-(2-nitro-4-trifluoro-methyl-benzoyl)-1,3 cyclohexanedione (NTBC) prior to hepatocyte transplantation.

### Claim 8 (depends on 7)

The method of claim 7, wherein the NTBC is administered at a dose of about 0.05 mg/kg/day to about 0.10 mg/kg/day.

### Claim 9 (depends on 1)

The method of claim 1, wherein transplanting the human hepatocytes comprises injecting the human hepatocytes into the spleen or portal vein of the mouse.

### Claim 10 (depends on 1)

The method of claim 1, wherein the transplanted human hepatocytes were isolated from the liver of an organ donor, isolated from a surgical resection, or derived from a stem cell, monocyte or amniocyte.

### Claim 11 (depends on 1)

The method of claim 1, wherein the transplanted human hepatocytes were cryopreserved prior to transplantation.

### Claim 12 (depends on 2)

The method of claim 2, further comprising depleting macrophages from the FRG mouse prior to hepatocyte transplantation.

### Claim 13

A genetically modified mouse whose genome is homozygous for deletions or one or more point mutations in the Fah, Rag2 and Il2rg genes such that the deletions or point mutations result in loss of expression of functional FAH, RAG-2 and IL-2Rγproteins, wherein the mouse is immunodeficient and exhibits decreased liver function, and wherein human hepatocytes can be expanded in the mouse.

### Claim 14 (depends on 13)

The mouse of claim 13, wherein the deletions or point mutations result in the complete loss of B cells, T cells and NK cells in the mouse.

### Claim 15 (depends on 13)

The mouse of claim 13, wherein the mouse is a FRG mouse.

### Claim 16 (depends on 13)

The mouse of claim 13, wherein the mouse is a FpmRG mouse.

### Claim 17 (depends on 13)

The mouse of claim 13, wherein the mouse expresses human urokinase.

### Claim 18 (depends on 17)

The mouse of claim 17, wherein the urokinase is a secreted form of urokinase.

### Claim 19 (depends on 17)

The mouse of claim 17, wherein the urokinase is a non-secreted form of urokinase.

### Claim 20 (depends on 17)

The mouse of claim 17, wherein expression of human urokinase results from incorporation of a transgene encoding human urokinase into the genome of the mouse.

### Claim 21 (depends on 17)

The mouse of claim 17, wherein expression of human urokinase results from administration of a vector encoding human urokinase.

### Claim 22 (depends on 1)

The method of claim 1, wherein the human hepatocytes are isolated human hepatocytes.

### Claim 23 (depends on 1)

The method of claim 1, further comprising collecting human hepatocytes from the mouse.

### Claim 24 (depends on 23)

The method of claim 23, wherein the human hepatocytes are collected from the liver of the mouse.

### Claim 25 (depends on 23)

The method of claim 23, further comprising expanding the collected human hepatocytes by serial transplantation.

### Claim 26 (depends on 1)

The method of claim 1, further comprising collecting a biological sample from the mouse.

### Claim 27 (depends on 26)

The method of claim 26, wherein the biological sample is a blood, urine, cell or tissue sample.