GENETICALLY MODIFIED MAJOR HISTOCOMPATIBILITY COMPLEX ANIMALS

• US 20180288986A1
• Filed: 06/21/2018
• Published: 10/11/2018
• Est. Priority Date: 10/28/2011
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Macdonald, Lynn, Murphy, Andrew J., Gurer, Cagan, McWhirter, John, Voronina, Vera, Harris, Faith, Stevens, Sean, Xue, Yingzi
• Granted Patent: US 10,779,520 B2
• Time in Patent Office: 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 2217/15 Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
• 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 2267/0387 Animal model for diseases of the immune system
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates

Abstract

The invention provides genetically modified non-human animals that express chimeric human/non-human MHC I polypeptide 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

1-27. -27. (canceled)

28. A non-human animal comprising two, three, four, five, or six nucleotide sequences, each at a different endogenous non-human animal MHC I locus and each encoding a different chimeric human/non-human MHC I polypeptide,

wherein a human portion of each chimeric polypeptide comprises an extracellular domain of a human MHC I polypeptide,

wherein a non-human portion of each chimeric polypeptide comprises transmembrane and cytoplasmic domain of a non-human MHC I polypeptide, and

wherein the animal expresses the two, three, four, five, or six different chimeric human/non-human MHC I polypeptides.

40. The non-human animal of claim 28, wherein the human portion of each chimeric polypeptide comprises α

1, α

2, and α

3 domains of a human MHC I polypeptide.

41. The non-human animal of claim 28, wherein the non-human portion of each chimeric polypeptide comprises transmembrane and cytoplasmic domains of an endogenous non-human MHC I polypeptide.

42. The non-human animal of claim 28, wherein the human MHC I polypeptide is selected from the group consisting of HLA-A, HLA-B, and HLA-C.

43. The non-human animal of claim 28, wherein the non-human animal does not express any classical endogenous MHC I polypeptides on a cell surface.

44. The non-human animal of claim 28, further comprising at its 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.

45. The non-human animal of claim 44, wherein the animal does not express a functional endogenous non-human β

2 microglobulin from its endogenous non-human locus.

46. The non-human animal of claim 44, wherein the nucleotide sequence encoding the human or humanized β

2 microglobulin polypeptide is operably linked to non-human β

2 microglobulin regulatory elements.

47. The non-human animal of claim 44, wherein the nucleotide sequence encoding the human or humanized β

2 microglobulin polypeptide comprises a nucleotide sequence set forth in exon 2, exon 3, and exon 4 of a human β

2 microglobulin gene.

48. The non-human animal of claim 44, wherein the nucleotide sequence encoding the human or humanized β

2 microglobulin polypeptide comprises a nucleotide sequence set forth in exon 2 to exon 4 of a human β

2 microglobulin gene.

49. The non-human animal of claim 28, wherein the animal is a rodent.

50. The non-human animal of claim 49, wherein the rodent is a rat or a mouse.

51. The non-human animal of claim 50, wherein the rodent is a mouse that comprises two or three different nucleotides sequences encoding two or three different chimeric human/mouse MHC I polypeptides on at least one of the two chromosomes 17 of the mouse.

52. The non-human animal of claim 51, wherein each chimeric human/mouse MHC I polypeptide comprises (a) α

1, α

2, and α

3 domains of a human MHC I polypeptide selected from the group consisting of HLA-A, HLA-B, and HLA-C and (b) transmembrane and cytoplasmic domains of an endogenous mouse MHC I polypeptide selected from the group consisting of H-2D, H-2K, and H-2L.

53. The mouse of claim 52, wherein the mouse does not express any classical endogenous mouse MHC I polypeptides on a cell surface.

54. The non-human animal of claim 52, wherein the mouse comprises two different nucleotide sequences encoding different chimeric human/mouse MHC I polypeptides.

55. The non-human animal of claim 54, wherein the two nucleotide sequences respectively encode chimeric HLA-A2/H-2K and HLA-B27/H-2D polypeptides, and wherein the mouse expresses the chimeric HLA-A2/H-2K and HLA-B27/H-2D polypeptides.

56. The mouse of claim 55, wherein the nucleotide sequence encoding chimeric HLA-A2/H-2K is located at an endogenous H-2K locus, and the nucleotide sequence encoding chimeric HLA-B27/H-2D is located at an endogenous H-2D locus.

57. The non-human animal of claim 56, further comprising at its 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.

58. The non-human animal of claim 57, wherein the nucleotide sequence encoding a human or humanized β

2 microglobulin polypeptide (a) is operably linked to endogenous mouse β

2 microglobulin regulatory elements, (b) comprises from 5′

to 3′

;

exon 2, exon 3 and exon 4 of a human β

2 microglobulin gene, and (c) replaces the endogenous mouse nucleotide sequence encoding exon 2, exon 3 and exon 4.

29-39. -39. (canceled)