# US 11,026,408 B2 · Genetically modified non-human animals and methods of use thereof

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- Number: US 11,026,408 B2
- Document ID: US11026408B2
- Status: Granted
- Kind: B2
- Application number: 16/586,573
- Filed: 2019-09-27
- Published: 2020-03-26
- Granted: 2021-06-08
- 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/US11026408B2

## 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 method for in vivo evaluation of a vaccine, the method comprising:
administering a vaccine to a 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 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 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 at a mouse GM-CSF gene locus,
a replacement of a mouse TPO gene with a nucleic acid encoding a human TPO polypeptide at a mouse TPO gene locus, and
an insertion of a nucleic acid encoding a human SIRP polypeptide,
wherein each of the nucleic acids encoding the human M-CSF polypeptide, the human IL-3 polypeptide, the human GM-CSF polypeptide, the human SIRP polypeptide, and the human TPO polypeptide is operably linked to a promoter,
wherein the mouse expresses the human M-CSF polypeptide, the human IL-3 polypeptide, the human GM-CSF polypeptide, the human SIRPA polypeptide, and the human TPO polypeptide, and
wherein the mouse is engrafted with human hematopoietic cells; and
assessing an immune response to the vaccine in the genetically modified mouse.

### Claim 2 (depends on 1)

The method of claim 1, wherein the human hematopoietic cells comprise hematopoietic stem cells (HSCs).

### Claim 3 (depends on 1)

The method of claim 1, wherein the human hematopoietic cells comprise hematopoietic stem and progenitor cells (HSPCs).

### Claim 4 (depends on 1)

The method of claim 1, wherein the mouse is not sub-lethally irradiated prior to the engraftment of the human hematopoietic cells.

### Claim 5

A method for in vivo evaluation of a vaccine, the method comprising:
administering a vaccine to a 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 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 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 at a mouse GM-CSF gene locus,
a replacement of a mouse TPO gene with a nucleic acid encoding a human TPO polypeptide at a mouse TPO gene locus, and
an insertion of a nucleic acid encoding a human SIRP polypeptide,
wherein each of the nucleic acids encoding the human M-CSF polypeptide, the human IL-3 polypeptide, the human GM-CSF polypeptide, the human SIRP polypeptide, and the human TPO polypeptide is operably linked to a promoter,
wherein the mouse expresses the human M-CSF polypeptide, the human IL-3 polypeptide, the human GM-CSF polypeptide, the human SIRPA polypeptide, and the human TPO polypeptide, and
wherein the mouse is engrafted with CD34+human hematopoietic cells and wherein the mouse exhibits a significantly higher proportion of human CD33+myeloid cells in blood and bone marrow relative to a genetically modified mouse engrafted with human CD34+cells which does not express the human M-CSF polypeptide, the human IL-3 polypeptide, the human GM-CSF polypeptide, the human SIRPA polypeptide, and the human TPO polypeptide; and
assessing an immune response to the vaccine in the genetically modified mouse.

### Claim 6 (depends on 5)

The method of claim 5, wherein the mouse is not sub-lethally irradiated prior to the engraftment of the CD34+human hematopoietic cells.

### Claim 7

A method for testing the effect of an agent that modulates cancer cell growth or survival, or for the in vivo evaluation of a cancer treatment involving the agent, the method comprising:
administering the agent to a 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 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 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 at a mouse GM-CSF gene locus,
a replacement of a mouse TPO gene with a nucleic acid encoding a human TPO polypeptide at a mouse TPO gene locus, and
an insertion of a nucleic acid encoding a human SIRP polypeptide,
wherein each of the nucleic acids encoding the human M-CSF polypeptide, the human IL-3 polypeptide, the human GM-CSF polypeptide, the human SIRP polypeptide, and the human TPO polypeptide is operably linked to a promoter,
wherein the mouse expresses the human M-CSF polypeptide, the human IL-3 polypeptide, the human GM-CSF polypeptide, the human SIRPA polypeptide, and the human TPO polypeptide, and
wherein the mouse is engrafted with human hematopoietic cells and wherein the mouse is engrafted with human cancer cells; and
assessing effect of the agent on cancer cell growth or survival in the genetically modified mouse.

### Claim 8 (depends on 7)

The method of claim 7, wherein the human hematopoietic cells comprise CD34+cells.

### Claim 9 (depends on 7)

The method of claim 7, wherein the human hematopoietic cells comprise hematopoietic stem cells (HSCs).

### Claim 10 (depends on 7)

The method of claim 7, wherein the human hematopoietic cells comprise hematopoietic stem and progenitor cells (HSPCs).

### Claim 11 (depends on 7)

The method of claim 7, wherein the mouse is not sub-lethally irradiated prior to the engraftment of the human hematopoietic cells.

### Claim 12 (depends on 7)

The method of claim 7, wherein the human cancer cells are primary human cancer cells isolated from a patient.

### Claim 13 (depends on 12)

The method of claim 12, wherein the human cancer cells and the human hematopoietic cells are isolated from the same patient.

### Claim 14 (depends on 7)

The method of claim 7, wherein the human cancer cells are from a cancer cell line.

### Claim 15 (depends on 7)

The method of claim 7, wherein the human cancer cells are selected from leukemia cells, breast cancer cells, lung cancer cells, and melanoma cells.

### Claim 16 (depends on 7)

The method of claim 7, wherein the human cancer cells are leukemia cells.

### Claim 17 (depends on 7)

The method of claim 7, wherein the human cancer cells are melanoma cells.