Genetically modified major histocompatibility complex mice

• US 10,314,296 B2
• Filed: 12/12/2017
• Issued: 06/11/2019
• Est. Priority Date: 02/22/2013
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Voronina, Vera, Gurer, Cagan, Murphy, Andrew J., Macdonald, Lynn, Xue, Yingzi
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US15/839,655
• Publication Number:
  US 20180116190A1
• Time in Patent Office: 546 Days
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C07K 14/70514 CD4
• CPC Class Codes: C07K 14/70517 CD8
• CPC Class Codes: C07K 14/70539 MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic

Abstract

The invention provides genetically modified non-human animals that express chimeric human/non-human MHC I and MHC II polypeptides and/or human or humanized β2 microglobulin polypeptide, as well as embryos, cells, and tissues comprising the same. Also provided are constructs for making said genetically modified animals and methods of making the same. Methods of using the genetically modified animals to study various aspects of human immune system are provided.

Claims

1. A genetically modified mouse whose genome comprises at an endogenous MHC locus:

a first nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide that comprises α

1, α

2 and α

3 domains of a human MHC I polypeptide operably linked to transmembrane and cytoplasmic domains of a mouse MHC I polypeptide,

a second nucleotide sequence encoding a chimeric human/mouse MHC II α

polypeptide that comprises α

1 and α

2 domains of a human MHC II α

polypeptide operably linked to transmembrane and cytoplasmic domains of a mouse MHC II α

polypeptide, and

a third nucleotide sequence encoding a chimeric human/mouse MHC II β

polypeptide that comprises β

1 and β

2 domains of a human MHC II β

polypeptide operably linked to transmembrane and cytoplasmic domains of a mouse MHC II β

polypeptide,

wherein the mouse expresses the chimeric human/mouse MHC I, MHC II α and

MHC II β

polypeptides from the endogenous MHC locus, and

wherein all genes encoding classical endogenous mouse MHC I and MHC II proteins that are expressed on a cell surface are functionally inactivated.

2. The mouse of claim 1, wherein the first nucleotide sequence is located at an endogenous mouse MHC I locus, the second nucleotide sequence is located at an endogenous mouse MHC II α

locus, and the third nucleotide sequence is located at an endogenous mouse MHC II β

locus.

3. The mouse of claim 1, wherein the first, second and/or third nucleotide sequence(s) are operably linked to endogenous mouse regulatory elements.

4. The mouse of claim 1, wherein the human MHC I polypeptide is selected from the group consisting of HLA-A, HLA-B, and HLA-C.

5. The mouse of claim 1, further comprising at an endogenous mouse β

2 microglobulin locus a nucleotide sequence encoding a human or humanized β

2 microglobulin polypeptide, wherein the mouse expresses the human or humanized β

2 microglobulin polypeptide.

6. The mouse of claim 1, wherein the first nucleotide sequence is operably linked to endogenous mouse MHC I promoter and regulatory elements, the second nucleotide sequence is operably linked to endogenous mouse MHC II α

promoter and regulatory elements, and the third nucleotide sequence is operably linked to endogenous mouse MHC II β

promoter and regulatory elements.

7. The mouse of claim 1, wherein the human MHC II α

and β

polypeptides are α and

β

polypeptides, respectively, of a human HLA class II protein selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP.

8. The mouse of claim 7, wherein the human HLA class II protein is a human HLA-DR protein.

9. The mouse of claim 1, wherein the mouse MHC I polypeptide is H-2K, H-2D, or H-2L.

10. The mouse of claim 9, wherein the mouse MHC I polypeptide is H-2K.

11. The mouse of claim 7, wherein the mouse MHC II α

and β

polypeptides are α and

β

polypeptides, respectively, of a mouse MHC II protein selected from H-2E and H-2A.

12. The mouse of claim 11, wherein the mouse MHC II protein is H-2E.

13. The mouse of claim 12, wherein the mouse endogenous gene encoding a H-2A polypeptide is functionally inactivated.

14. The mouse of claim 1, wherein the first nucleotide sequence encodes a chimeric HLA-A/H-2K polypeptide, the second nucleotide sequence encodes an α

chain of a chimeric HLA-DR/H-2E polypeptide, and the third nucleotide sequence encodes a β

chain of a chimeric HLA-DR/H-2E polypeptide, and wherein the mouse expresses HLA-A/H-2K and HLA-DR/H-2E proteins.

15. The mouse of claim 14, wherein the first nucleotide sequence is located at an endogenous mouse H-2K locus and wherein the mouse lacks all or part of an endogenous H-2D gene and/or all or part of an endogenous mouse H-2L gene.

16. The mouse of claim 14, wherein the second nucleotide sequence is located at an endogenous H-2E α

locus and the third nucleotide sequence is located at an endogenous H-2E β

locus gene, and wherein the mouse lacks all or part of an endogenous mouse H-2A gene.

17. The mouse of claim 15, wherein the second nucleotide sequence is located at an endogenous H-2E α

locus and the third nucleotide sequence is located at an endogenous H-2E β

locus, and wherein the mouse lacks all or part of an endogenous mouse H-2A gene.

18. The mouse of claim 1, wherein the classical endogenous mouse MHC I proteins that are expressed on the cell surface are H-2K, H-2D, and H-2L, and wherein the classical endogenous mouse MHC II proteins that are expressed on the cell surface are H-2A and H-2E.