# US 2016/0249591 A1 · Fumarylacetoacetate hydrolase (fah)-deficient and immunodeficient rats and uses thereof

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- Number: US 2016/0249591 A1
- Document ID: US20160249591A1
- Status: Published application
- Kind: A1
- Application number: 15/159,200
- Filed: 2016-05-19
- Published: 2016-09-01
- Earliest claimed: 2011-08-26
- Assignees: Yecuris Corporation
- Applicants: Yecuris Corporation
- Inventors: John R. Bial; Elizabeth M. Wilson; Aron M. Geurts
- Web page: https://patentgrep.com/patent/US20160249591A1

## CPC

- A01K 67/0276: Knock-out vertebrates
- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- C07K 14/7155: {for interleukins [IL]}
- C12N 9/14: Hydrolases (3)
- C12N 9/6462: {u-Plasminogen activator (3.4.21.73), i.e. urokinase}
- C12Y 304/21073: u-Plasminogen activator (3.4.21.73), i.e. urokinase
- A01K 2207/12: Animals modified by administration of exogenous cells
- A01K 2207/15: Humanized animals
- A01K 2217/00: Genetically modified animals
- A01K 2217/075: inducing loss of function, i.e. knock out
- A01K 2227/105: Murine
- A01K 2267/03: Animal model, e.g. for test or diseases
- A01K 2267/0387: Animal model for diseases of the immune system
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12Y 307/01002: Fumarylacetoacetase (3.7.1.2)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Described herein are rats with a hepatic deficiency comprising decreased function, activity, or expression of an enzyme in the tyrosine catabolic pathway (such as fumarylacetoacetate hydrolase), and methods of using the same for in vivo engraftment and expansion of heterologous hepatocytes, such as human hepatocytes, analysis of human liver disease, and analysis of xenobiotics. Also disclosed is the use of immunodeficient rats for the engraftment and expansion of heterologous hepatocytes.

## Claims

### Claim 1

A genetically modified Fah-deficient rat whose genome is homozygous for a disruption in the Fah gene such that the disruption results in loss of expression of functional FAH protein and decreased liver function.

### Claim 2 (depends on 1)

The Fah-deficient rat of claim 1, wherein the rat is immunosuppressed.

### Claim 3 (depends on 2)

The Fah-deficient rat of claim 2, wherein the immunosuppression is the result of administration of one or more immunosuppressive agents.

### Claim 4 (depends on 3)

The Fah-deficient rat of claim 3, wherein the one or more immunosuppressive agents are selected from the group consisting of FK506, cyclosporin A, fludarabine, mycophenolate, prednisone, rapamycin, azathioprine, and combinations thereof.

### Claim 5 (depends on 2)

The Fah-deficient rat of claim 2, wherein the immunosuppression is the result of one or more genetic alterations that inhibit the development of functional immune cells.

### Claim 6 (depends on 5)

The Fah-deficient rat of claim 5, wherein the one or more genetic alterations is selected from the group consisting of Rag1 deficiency, Rag2 deficiency, Il2rg deficiency, SCID, Sirp-αhum/hum, nude, perforin knockouts, and combinations thereof.

### Claim 7 (depends on 1)

The Fah-deficient rat of claim 1, wherein the rat comprises transplanted heterologous hepatocytes.

### Claim 8 (depends on 7)

The Fah-deficient rat of claim 7, wherein the heterologous hepatocytes are mammalian hepatocytes from a mammal selected from the group consisting of dogs, pigs, horses, rabbits, mice, marmoset, woodchuck, non-human primates, and humans.

### Claim 9 (depends on 1)

The Fah-deficient rat of claim 1, wherein the rat comprises expanded human hepatocytes, wherein liver function is restored in the rat.

### Claim 10

A method of expanding human hepatocytes in vivo, comprising:
transplanting heterologous hepatocytes into a first rat, wherein said first rat is:
a genetically modified Fah-deficient rat whose genome is homozygous for a disruption in the Fah gene such that the disruption results in loss of expression of functional FAR protein and decreased liver function, or
an immunodeficient rat, wherein the immunodeficiency of the rat is due to a genetic alteration, immunosuppression, or a combination thereof, and
allowing the heterologous hepatocytes to expand, thereby expanding heterologous hepatocytes in vivo.

### Claim 11 (depends on 10)

The method of claim 10, wherein the heterologous hepatocytes transplanted into the first rat are isolated human hepatocytes.

### Claim 12 (depends on 10)

The method of claim 10, wherein the human hepatocytes are isolated from the liver of a humanized non-human mammal prior to transplanting into the first rat, wherein the non-human mammal is an FRG KO mouse.

### Claim 13 (depends on 10)

The method of claim 10, further comprising inducing acute liver damage in the first rat prior to transplanting the heterologous hepatocytes.

### Claim 14 (depends on 10)

The method of claim 10, further comprising administering a vector encoding urokinase to the first rat prior to transplanting the heterologous hepatocytes.

### Claim 15 (depends on 10)

The method of claim 10, wherein the first rat is an Fah-deficient rat, further comprising administering one or more immunosuppressive agents to the first Fah-deficient rat at least about 2 days prior to transplanting the heterologous hepatocytes.

### Claim 16 (depends on 10)

The method of claim 10, wherein the first rat is an immunodeficient rat, wherein the immunodeficiency is due to one or more genetic alterations that inhibit the development of functional immune cells, wherein the one or more genetic alterations is selected from the group consisting of selected from the group consisting of Rag1 deficiency, Rag2 deficiency, Il2rg deficiency, SCID, Sirp-αhum/hum, nude, perforin−/−, and combinations thereof.

### Claim 17 (depends on 10)

The method of claim 10, further comprising collecting the expanded heterologous hepatocytes from the first rat.

### Claim 18 (depends on 17)

The method of claim 17, further comprising expanding the collected heterologous hepatocytes by serial transplantation, wherein said serial transplantation comprises:
collecting expanded heterologous hepatocytes from the first rat;
transplanting the collected expanded heterologous hepatocytes from the first rat into a second rat wherein said second rat is:
a genetically modified Fah-deficient rat whose genome is homozygous for a disruption in the Fah gene such that the disruption results in loss of expression of functional FAH protein and decreased liver function, or
an immunodeficient rat, wherein the immunodeficiency of the rat is due to a genetic alteration, immunosuppression, or a combination thereof; and
allowing the heterologous hepatocytes to expand, thereby expanding heterologous hepatocytes in vivo.

### Claim 19

An immunodeficient rat having a liver comprising transplanted and expanded human hepatocytes, wherein the immunodeficiency of the rat is due to a genetic alteration, immunosuppression, or a combination thereof.

### Claim 20 (depends on 19)

The immunodeficient rat of claim 19, wherein the immunodeficiency is due to one or more genetic alterations selected from the group consisting of selected from the group consisting of Rag1 deficiency, Rag2 deficiency, Il2rg deficiency, SCID, Sirp-αhum/hum, nude, perforin−/−, and combinations thereof.