# US 2018/0020647 A1 · Genetically Modified Non-Human Animals And Methods Of Use Thereof

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- Number: US 2018/0020647 A1
- Document ID: US20180020647A1
- Status: Published application
- Kind: A1
- Application number: 15/598,080
- Filed: 2017-05-17
- Published: 2018-01-25
- Earliest claimed: 2012-09-07
- Assignees: Regeneron Pharmaceuticals, Inc.; Yale University; Institute for Research in Biomedicine (IRB)
- Applicants: Regeneron Pharmaceuticals, Inc.; Yale University; Institute for Research in Biomedicine (IRB)
- Inventors: Richard Flavell; Markus Manz; Anthony Rongvaux; Till Strowig; Tim Willinger; Andrew J. Murphy; Sean Stevens; George Yancopoulos
- Web page: https://patentgrep.com/patent/US20180020647A1

## CPC

- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- A01K 67/0271: Chimeric vertebrates, e.g. comprising exogenous cells
- C07K 14/4703: {Inhibitors; Suppressors}
- C07K 14/4705: {stimulating, promoting or activating activity}
- C07K 14/524: {Thrombopoietin, i.e. C-MPL ligand}
- C07K 14/53: Colony-stimulating factor [CSF]
- C07K 14/535: Granulocyte CSF; Granulocyte-macrophage CSF
- C07K 14/5403: {IL-3}
- C07K 14/70503: {Immunoglobulin superfamily}
- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- A01K 2207/12: Animals modified by administration of exogenous cells
- A01K 2207/15: Humanized animals
- A01K 2217/072: maintaining or altering function, i.e. knock in
- A01K 2217/15: Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
- A01K 2227/105: Murine
- A01K 2267/0331: Animal model for proliferative diseases
- A01K 2267/0381: Animal model for diseases of the hematopoietic system
- A01K 2267/0387: Animal model for diseases of the immune system
- 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)

## Abstract

The invention relates generally to genetically modified non-human animals expressing human polypeptides and their methods of use.

## Claims

### Claim 1

A genetically modified mouse comprising in its genome:
a recombination activating gene 2 (Rag-2) gene knock-out;
an IL2 receptor gamma chain (IL2rg) gene knock-out;
a replacement of a mouse M-CSF gene with a nucleic acid encoding a human M-CSF polypeptide, wherein the nucleic acid encoding the human M-CSF polypeptide is operably linked to a M-CSF promoter at a mouse M-CSF gene locus;
a replacement of a mouse IL-3 gene with a nucleic acid encoding a human IL-3 polypeptide, wherein the nucleic acid encoding the human IL-3 polypeptide is operably linked to a IL-3 promoter at a mouse IL-3 gene locus;
a replacement of a mouse GM-CSF gene with a nucleic acid encoding a human GM-CSF polypeptide, wherein the nucleic acid encoding the human GM-CSF polypeptide is operably linked to a GM-CSF promoter at a mouse GM-CSF gene locus;
a replacement of a mouse-SIRPA gene with a nucleic acid encoding a fusion protein comprising a biologically active fragment of a full length human SIRPA polypeptide, wherein the nucleic acid encoding the fusion protein is operably linked to a SIRPA promoter; and
a replacement of a mouse TPO gene with a nucleic acid encoding a human TPO polypeptide, wherein the nucleic acid encoding the human TPO polypeptide is operably linked to a TPO promoter at a mouse TPO gene locus, wherein the mouse expresses the human M-CSF polypeptide, the human IL-3 polypeptide, the human GM-CSF polypeptide, the fusion protein, and the human TPO polypeptide.

### Claim at position 2

-8. (canceled)

### Claim 9 (depends on 1)

The genetically modified mouse of claim 1, further comprising human hematopoietic cells.

### Claim 10 (depends on 1)

The genetically modified mouse of claim 1, further comprising a human cancer cell.

### Claim 11 (depends on 10)

The genetically modified mouse of claim 10, wherein the human cancer cell is a leukemia cell or a melanoma cell.

### Claim 12

A method of hematopoietic stem and progenitor cell (HSPC) engraftment in a genetically modified mouse, the method comprising the step of: administering at least one HSPC to the genetically modified mouse, wherein the genetically modified mouse comprises in its genome:
a recombination activating gene 2 (Rag-2) gene knock-out;
an IL2 receptor gamma chain (IL2rg) gene knock-out;
a replacement of a mouse M-CSF gene with a nucleic acid encoding a human M-CSF polypeptide, wherein the nucleic acid encoding the human M-CSF polypeptide is operably linked to a M-CSF promoter at a mouse M-CSF gene locus;
a replacement of a mouse IL-3 gene with a nucleic acid encoding a human IL-3 polypeptide, wherein the nucleic acid encoding the human IL-3 polypeptide is operably linked to a IL-3 promoter at a mouse IL-3 gene locus;
a replacement of a mouse GM-CSF gene with a nucleic acid encoding a human GM-CSF polypeptide, wherein the nucleic acid encoding the human GM-CSF polypeptide is operably linked to a GM-CSF promoter at a mouse GM-CSF gene locus;
a replacement of a mouse-SIRPA gene with a nucleic acid encoding a fusion protein comprising a biologically active fragment of a full length human SIRPA polypeptide, wherein the nucleic acid encoding the fusion protein is operably linked to a SIRPA promoter; and
a replacement of a mouse TPO gene with a nucleic acid encoding a human TPO polypeptide, wherein the nucleic acid encoding the human TPO polypeptide is operably linked to a TPO promoter at a mouse TPO gene locus, wherein the mouse expresses the human M-CSF polypeptide, the human IL-3 polypeptide, the human GM-CSF polypeptide, the fusion protein, and the human TPO polypeptide.

### Claim at position 7

-18. (canceled)

### Claim 19 (depends on 12)

The method of claim 12, wherein the genetically modified mouse comprises a human cancer cell.

### Claim 20 (depends on 19)

The method of claim 19, wherein the human cancer cell is a leukemia cell or a melanoma cell.

### Claim at position 10

-27. (canceled)

### Claim 28 (depends on 12)

The method of claim 12, wherein the administering step is not preceded by preconditioning irradiation of the genetically modified mouse or administration of a radiomimetic drug to the genetically modified mouse.

### Claim 29 (depends on 12)

The method of claim 12, further comprising administering a human cancer cell to the genetically modified mouse.

### Claim 30 (depends on 29)

The method of claim 29, wherein the human cancer cell is a leukemia cell or a melanoma cell.

### Claim 31 (depends on 29)

The method of claim 29, wherein the at least one HSPC and the human cancer cell are both isolated from the same patient.