Genetically modified non-human animals expressing human EPO

Patent metadata

Field	Value
Publication number	US-10463028-B2
Application number	US-201514715446-A
Country	US
Kind code	B2
Filing date	May 18, 2015
Priority date	May 19, 2014
Publication date	Nov 5, 2019
Grant date	Nov 5, 2019

Abstract

Genetically modified non-human animals expressing human EPO from the animal genome are provided. Also provided are methods for making non-human animals expressing human EPO from the non-human animal genome, and methods for using non-human animals expressing human EPO from the non-human animal genome. These animals and methods find many uses in the art, including, for example, in modeling human erythropoiesis and erythrocyte function; in modeling human pathogen infection of erythrocytes; in in vivo screens for agents that modulate erythropoiesis and/or erythrocyte function, e.g. in a healthy or a diseased state; in in vivo screens for agents that are toxic to erythrocytes or erythrocyte progenitors; in in vivo screens for agents that prevent against, mitigate, or reverse the toxic effects of toxic agents on erythrocytes or erythrocyte progenitors; in in vivo screens of erythrocytes or erythrocyte progenitors from an individual to predict the responsiveness of an individual to a disease therapy.

First claim

That which is claimed is:

1. A genetically modified mouse, comprising:
a nucleic acid sequence that encodes a human erythropoietin (hEPO) protein operably linked to an endogenous mouse erythropoietin (EPO) gene promoter at the mouse EPO gene locus in the genome of the genetically modified mouse, wherein the mouse expresses the hEPO protein;
a nucleic acid sequence that encodes a human macrophage colony stimulating factor 1 (hM-CSF) protein operably linked to an endogenous macrophage colony stimulating factor (M-CSF) gene promoter at the mouse M-CSF gene locus in the genome of the genetically modified mouse, wherein the mouse expresses the hM-CSF protein;
a nucleic acid sequence that encodes a human interleukin 3 (hIL3) protein operably linked to an endogenous interleukin 3 (IL-3) gene promoter at the mouse IL-3 gene locus in the genome of the genetically modified mouse, wherein the mouse expresses the hIL3 protein;
a nucleic acid sequence that encodes a human granulocyte-macrophage colony stimulating factor 2 (hGM-CSF) protein operably linked to an endogenous granulocyte-macrophage colony stimulating factor (GM-CSF) gene promoter at the mouse GM-CSF gene locus in the genome of the genetically modified mouse, wherein the mouse expresses the hGM-CSF protein;
a nucleic acid sequence that encodes a human thrombopoietin (hTPO) protein operably linked to an endogenous thrombopoietin (TPO) gene promoter at the mouse TPO gene locus in the genome of the genetically modified mouse, wherein the mouse expresses the hTPO protein; and
a SIRPa transgene that encodes a human signal-regulatory protein alpha (hSirpa) protein, wherein the mouse expresses the hSirpa protein, wherein the genetically modified mouse is a Rag2 −/− Il2rg null Tpo h/h Mcsf h/h Il3 h/h Gmcsf h/h Epo h/h SIRPα-tg+ mouse, and wherein, when engrafted with human hematopoietic stem cells (HSC), the genetically modified mouse has an increased percentage of human erythroid cells in the bone marrow of the genetically modified mouse as compared to a Rag2 −/− Il2rg null Tpo h/h IL3 h/h Gmcsf h/h Mcsf h/h mouse that does not express hEPO and hSirpa.

2. The mouse according to claim 1 , wherein the nucleic acid sequence that encodes the hEPO comprises human EPO genomic coding and non-coding sequence.

3. The mouse according to claim 2 , wherein the nucleic acid sequence that encodes the hEPO comprises human EPO cDNA sequence.

4. The mouse according to claim 1 , wherein the mouse further comprises an engraftment of human hematopoietic cells.

5. The mouse according to claim 4 , wherein the human hematopoietic cells comprise one or more cells selected from the group consisting of a human CD34-positive cell, a human hematopoietic stem cell, a human myeloid precursor cell, a human erythroid precursor cell, a human myeloid cell, a human dendritic cell, a human monocyte, a human granulocyte, a human erythrocyte, a human neutrophil, a human mast cell, a human thymocyte, and a human B lymphocyte.

6. The mouse according to claim 5 , wherein the mouse is treated with clodronate.

7. The mouse according to claim 6 , wherein the mouse further comprises an infection with a pathogen that targets human cells of the erythroid lineage.

8. The mouse according to claim 7 , wherein the pathogen is selected from a Plasmodium sp., Babesia sp., and a Theileri sp.

9. A method for identifying an agent that prevents an infection by a pathogen that targets human cells of the erythroid lineage, the method comprising:
a. contacting the genetically modified mouse of claim 1 with clodronate, wherein the genetically modified mouse comprises
an engraftment of human hematopoietic cells,

b. administering a candidate agent to the genetically modified mouse,
c. injecting the genetically modified mouse with parasitized reticulocytes or erythrocytes, and
d. determining whether the agent prevents the infection of the human reticulocytes and/or erythrocytes of the mouse.

10. The mouse according to claim 1 , wherein the hSirpa protein is a humanized Sirpα protein.

11. A genetically modified mouse, comprising:
a nucleic acid sequence that encodes a human erythropoietin (hEPO) protein operably linked to an endogenous mouse erythropoietin (EPO) gene promoter at the mouse EPO gene locus in the genome of the genetically modified mouse, wherein the mouse expresses the hEPO protein;
a nucleic acid sequence that encodes a human macrophage colony stimulating factor 1 (hM-CSF) protein operably linked to an endogenous macrophage colony stimulating factor (M-CSF) gene promoter at the mouse M-CSF gene locus in the genome of the genetically modified mouse, wherein the mouse expresses the hM-CSF protein;
a nucleic acid sequence that encodes a human interleukin 3 (hIL3) protein operably linked to an endogenous interleukin 3 (IL-3) gene promoter at the mouse IL-3 gene locus in the genome of the genetically modified mouse, wherein the mouse expresses the hIL3 protein;
a nucleic acid sequence that encodes a human granulocyte-macrophage colony stimulating factor 2 (hGM-CSF) protein operably linked to an endogenous granulocyte-macrophage colony stimulating factor (GM-CSF) gene promoter at the mouse GM-CSF gene locus in the genome of the genetically modified mouse, wherein the mouse expresses the hGM-CSF protein;
a nucleic acid sequence that encodes a human thrombopoietin (hTPO) protein operably linked to an endogenous thrombopoietin (TPO) gene promoter at the mouse TPO gene locus in the genome of the genetically modified mouse, wherein the mouse expresses the hTPO protein; and
a nucleic acid sequence that encodes a human signal-regulatory protein alpha (hSirpa) protein operably linked to an endogenous signal-regulatory protein alpha (SIRPα) gene promoter in the genome of the genetically modified mouse, wherein the mouse expresses the hSirpa protein, wherein the genetically modified mouse is a Rag2 −/− Il2rg null Tpo h/h Mcsf h/h Il3 h/h Gmcsf h/h Epo h/h SIRP h/h mouse, and wherein, when engrafted with human hematopoietic stem cells (HSC), the genetically modified mouse has an increased percentage of human erythroid cells in the bone marrow of the genetically modified mouse as compared to a Rag2 −/− Il2rg null Tpo h/h IL3 h/h Gmcsf h/h Mcsf h/h mouse that does not express hEPO and hSirpa.

12. The mouse according to claim 11 , wherein the nucleic acid sequence that encodes the hEPO comprises human EPO genomic coding and non-coding sequence.

13. The mouse according to claim 11 , wherein the nucleic acid sequence that encodes the hEPO comprises human EPO cDNA sequence.

14. The mouse according to claim 11 , wherein the mouse further comprises an engraftment of human hematopoietic cells.

15. The mouse according to claim 14 , wherein the human hematopoietic cells comprise one or more cells selected from the group consisting of a human CD34-positive cell, a human hematopoietic stem cell, a human myeloid precursor cell, a human erythroid precursor cell, a human myeloid cell, a human dendritic cell, a human monocyte, a human granulocyte, a human erythrocyte, a human neutrophil, a human mast cell, a human thymocyte, and a human B lymphocyte.

16. The mouse according to claim 15 , wherein the mouse is treated with clodronate.

17. The mouse according to claim 16 , wherein the mouse further comprises an infection with a pathogen that targets human cells of the erythroid lineage.

18. The mouse according to claim 17 , wherein the pathogen is selected from a Plasmodium sp., Babesia sp., and a Theileri sp.

19. A method for identifying an agent that pr

Assignees

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

Inventors

• Murphy Andrew J
• Stevens Sean
• Flavell Richard
• Manz Markus Gabriel
• Shan Liang

Classifications

• G01N33/5088

  of vertebrates · CPC title

• C12Q1/18

  Testing for antimicrobial activity of a material · CPC title

• C12N9/00

  Enzymes; Proenzymes; Compositions thereof (preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; {enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113}); Processes for preparing, activating, inhibiting, separating or purifying enzymes (preparation of malt C12C1/00) · CPC title

• C07K14/7155

  for interleukins [IL] · CPC title

• C07K14/5437

  IL-13 · CPC title