Genetically modified non-human animals and methods of use thereof

Patent metadata

Field	Value
Publication number	US-9820476-B2
Application number	US-201314420318-A
Country	US
Kind code	B2
Filing date	Sep 6, 2013
Priority date	Sep 7, 2012
Publication date	Nov 21, 2017
Grant date	Nov 21, 2017

Abstract

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

First claim

The invention claimed is:

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 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, an insertion of a nucleic acid encoding a human SIRPA polypeptide, and a replacement of a mouse TPO gene with a nucleic acid encoding a human TPO polypeptide at a mouse TPO gene locus, wherein each of the nucleic acids encoding the human M-CSF polypeptide, the human IL-3 polypeptide, the human GM-CSF polypeptide, the human SIRPA polypeptide, and the human TPO polypeptide is operably linked to a promoter, and 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.

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

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

4. The genetically modified mouse of claim 3 , wherein the human cancer cell is a leukemia cell or a melanoma cell.

5. A method of hematopoietic stem and progenitor cell (HSPC) engraftment comprising a step of administering at least one HSPC to the genetically modified mouse of claim 1 .

6. The method of claim 5 , wherein the genetically modified mouse comprises a human cancer cell.

7. The method of claim 6 , wherein the human cancer cell is a leukemia cell or a melanoma cell.

8. 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 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,
an insertion of a nucleic acid encoding a human SIRPA polypeptide, and
a replacement of a mouse TPO gene with a nucleic acid encoding a human TPO polypeptide at a mouse TPO gene locus,
wherein each of the nucleic acids encoding the human M-CSF polypeptide, the human IL-3 polypeptide, the human GM-CSF polypeptide, the human SIRPA 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, when engrafted with human CD34+ cells, 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.

9. The genetically modified mouse of claim 8 , wherein the mouse is engrafted with human CD34+ cells.

10. The method of claim 5 , wherein the administering step is not preceded by preconditioning irradiation of the genetically modified mouse.

11. 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 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,
an insertion of a nucleic acid encoding a human SIRPA polypeptide, and
a replacement of a mouse TPO gene with a nucleic acid encoding a human TPO polypeptide at a mouse TPO gene locus,
wherein each of the nucleic acids encoding the human M-CSF polypeptide, the human IL-3 polypeptide, the human GM-CSF polypeptide, the human SIRPA 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, when engrafted with human CD34+ cells and a human tumor, the mouse exhibits increased human myeloid cell infiltration of the engrafted tumor relative to a genetically modified mouse engrafted with human CD34+ cells and a human tumor, 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.

12. The genetically modified mouse of claim 11 , wherein the mouse is engrafted with human CD34+ cells and a human tumor.

13. The genetically modified mouse of claim 1 , wherein the human SIRPA polypeptide is a biologically active fragment of a full-length human SIRPA polypeptide.

14. The genetically modified mouse of claim 8 , wherein the human SIRPA polypeptide is a biologically active fragment of a full-length human SIRPA polypeptide.

15. The genetically modified mouse of claim 11 , wherein the human SIRPA polypeptide is a biologically active fragment of a full-length human SIRPA polypeptide.

Assignees

• Regeneron Pharma
• Univ Yale
• Institute For Res In Biomedicine (Irb)

Inventors

• Flavell Richard
• Manz Markus
• Rongvaux Anthony
• Strowig Till
• Willinger Tim
• Murphy Andrew J
• Stevens Sean
• Yancopoulos George

Classifications

• A01K67/0271

  Chimeric vertebrates, e.g. comprising exogenous cells · CPC title

• A01K2207/12

  Animals modified by administration of exogenous cells · CPC title

• A01K67/0278 Primary

  Knock-in vertebrates, e.g. humanised vertebrates · CPC title

• A01K2217/072

  maintaining or altering function, i.e. knock in · CPC title

• C12N15/87

  Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation · CPC title