GENETICALLY MODIFIED MAJOR HISTOCOMPATIBILITY COMPLEX MICE

• US 20140245467A1
• Filed: 02/20/2014
• Published: 08/28/2014
• Est. Priority Date: 02/22/2013
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: MACDONALD, Lynn, MURPHY, Andrew J., VORONINA, Vera, GURER, Cagan
• Granted Patent: US 10,154,658 B2
• Time in Patent Office: Days
• US Class Current: 800/18
• 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 2267/03 Animal model, e.g. for test or diseases
• 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 non-human animal comprising at an endogenous MHC locus:

a first nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide, wherein a human portion of the chimeric MHC I polypeptide comprises an extracellular domain of a human MHC I polypeptide,

a second nucleotide sequence encoding a chimeric human/non-human MHC II α

polypeptide, wherein a human portion of the chimeric human/non-human MHC II α

polypeptide comprises an extracellular domain of a human MHC II α

polypeptide, and

a third nucleotide sequence encoding a chimeric human/non-human MHC II β

polypeptide, wherein a human portion of the chimeric human/non-human MHC II β

polypeptide comprises an extracellular domain of a human MHC II β

polypeptide,

wherein the non-human animal expresses chimeric human/non-human MHC I and MHC II proteins from its endogenous non-human MHC locus.

2. The animal of claim 1, wherein the animal does not express functional endogenous MHC I, II α

, and/or II β

polypeptides from the endogenous non-human MHC locus.

3. The animal of claim 1, wherein the first nucleotide sequence is located at the endogenous non-human MHC I locus, the second nucleotide sequence is located at the endogenous non-human MHC II α

locus, and the third nucleotide sequence is located at the endogenous non-human MHC II β

locus.

4. The animal of claim 1, wherein the first, second and/or third nucleotide sequence(s) are operably linked to endogenous non-human regulatory elements.

5. The animal of claim 1, wherein the human portion of the chimeric MHC I polypeptide comprises α

1, α

2, and α

3 domains of the human MHC I polypeptide.

6. The animal of claim 1, wherein a non-human portion of the chimeric MHC I polypeptide comprises transmembrane and cytoplasmic domains of an endogenous non-human MHC I polypeptide.

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

8. The animal of claim 1, further comprising at an endogenous non-human β

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

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

2 microglobulin polypeptide.

9. The animal of claim 1, wherein the human MHC II α

extracellular domain comprises human MHC II α

1 and α

2 domains.

10. The animal of claim 1, wherein the human MHC II β

extracellular domain comprises human MHC II β

1 and β

2 domains.

11. The animal of claim 1, wherein the first nucleotide sequence is operably linked to endogenous non-human MHC I promoter and regulatory elements, the second nucleotide sequence is operably linked to endogenous non-human MHC II α

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

promoter and regulatory elements.

12. The animal of claim 1, wherein a non-human portion of the chimeric human/non-human MHC II α

polypeptide comprises transmembrane and cytoplasmic domains of an endogenous non-human MHC II α

polypeptide.

13. The animal of claim 1, wherein a non-human portion of the chimeric human/non-human MHC II β

polypeptide comprises transmembrane and cytoplasmic domains of an endogenous non-human MHC II β

polypeptide.

14. The animal of claim 1, wherein the human portions of the chimeric human/mouse MHC II α

and β

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

15. The animal of claim 14, wherein the human portions of the chimeric human/non-human MHC II α

and β

polypeptides are derived from a human HLA-DR protein.

16. The animal of claim 1, wherein the animal is a rodent.

17. The rodent of claim 16, wherein the rodent is a mouse or a rat.

18. The rodent of claim 17, wherein the rodent is a mouse.

19. The mouse of claim 18, wherein the first nucleotide sequence encodes a chimeric human/mouse MHC I polypeptide, and wherein a mouse portion of the chimeric MHC I polypeptide is derived from H-2K, H-2D, or H-2L.

20. The mouse of claim 19, wherein the mouse portion of the chimeric MHC I polypeptide is derived from H-2K.

21. The mouse of claim 18, wherein the second nucleotide sequence encodes a chimeric human/mouse MHC II α

polypeptide, the third nucleotide sequence encodes a chimeric human/mouse MHC II β

polypeptide, and mouse portions of the chimeric MHC II α and

β

polypeptides are derived from H-2E or H-2A.

22. The mouse of claim 21, wherein the mouse portions of the chimeric MHC II polypeptides are derived from H-2E.

23. A mouse comprising at an endogenous MHC locus:

a first nucleotide sequence encoding a chimeric human/mouse MHC I polypeptide, wherein a human portion of the chimeric MHC I polypeptide comprises an extracellular domain of a human MHC I polypeptide,

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

polypeptide, wherein a human portion of the chimeric human/non-human MHC II α

polypeptide comprises an extracellular domain of a human MHC II α

polypeptide, and

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

polypeptide, wherein a human portion of the chimeric human/non-human MHC II β

polypeptide comprises an extracellular domain of a human MHC II β

polypeptide,

wherein the mouse expresses chimeric human/mouse MHC I and MHC II proteins from its endogenous mouse MHC locus.

24. The mouse of claim 24, 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.

25. The mouse of claim 24, wherein the mouse further comprises at an endogenous β

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

2 microglobulin polypeptide.

26. The mouse of claim 24, wherein the mouse does not express functional endogenous MHC polypeptides from its endogenous MHC locus.

27. A method of generating a genetically modified non-human animal comprising:

replacing at an endogenous non-human MHC II locus a nucleotide sequence encoding a non-human MHC II complex with a nucleotide sequence encoding a chimeric human/non-human MHC II complex to generate a first non-human animal; and

replacing at an endogenous non-human MHC I locus a nucleotide sequence encoding a non-human MHC I polypeptide with a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide to generate a second non-human animal.

28. The method of claim 27, wherein the steps of replacing nucleotide sequences comprise homologous recombination in non-human ES cells, and wherein second non-human animal is generated by homologous recombination in ES cells bearing nucleotide sequences encoding chimeric human/non-human MHC II complex.

29. The method of claim 27, wherein the steps of replacing nucleotide sequences comprise homologous recombination in non-human ES cells, and wherein first non-human animal is generated by homologous recombination in ES cells bearing nucleotide sequences encoding chimeric human/non-human MHC I polypeptide.

30. The method of claim 27, wherein the animal is a mouse.