GENETICALLY MODIFIED NON-HUMAN ANIMAL WITH HUMAN OR CHIMERIC MHC PROTEIN COMPLEX

Abstract

The present disclosure relates to genetically modified non-human animals that express a human or chimeric (e.g., humanized) major histocompatibility complex (MHC) protein complex, and methods of use thereof.

Claims

1. A genetically-modified non-human animal expressing a fusion protein comprising β2 microglobulin (B2M) and a human or humanized major histocompatibility complex (MHC) α chain.

2. The animal of claim 1, wherein the genome of the animal comprises at least one chromosome comprising a sequence encoding the fusion protein.

3. The animal of claim 1 or 2, wherein the fusion protein comprises a human or humanized B2M protein.

4. The animal of any one of claims 1-3, wherein the MHC α chain is a MHC class I α chain.

5. The animal of any one of claims 1-4, wherein the MHC α chain is a human HLA-A protein.

6. The animal of any one of claims 1-4, wherein the MHC α chain is a chimeric MHC α chain.

7. The animal of any one of claims 1-4, wherein the MHC α chain is a human HLA-A/mouse H2-D1 chimeric molecule.

8. The animal of claim 1, wherein the fusion protein comprises a human B2M protein and a chimeric MHC α chain comprising human HLA-A α1 and α2 domains.

9. The animal of claim 8, wherein the chimeric MHC α chain further comprises a mouse H2-D1 α3 domain.

10. The animal of claim 1, wherein the fusion protein comprises a human B2M protein and a human HLA-A protein.

11. The animal of any one of claims 2-10, wherein the sequence encoding the fusion protein is operably linked to an endogenous regulatory element (e.g., a promoter) at the endogenous β2 microglobulin (B2M) gene locus in the at least one chromosome.

12. The animal of any one of claims 2-10, wherein the sequence encoding the fusion protein is operably linked to an endogenous regulatory element (e.g., a promoter) at the endogenous MHC gene locus in the at least one chromosome.

13. The animal of any one of claims 2-10, wherein the animal is a mouse, and the sequence encoding the fusion protein is operably linked to an endogenous regulatory element at the mouse H2-D1 gene locus in the at least one chromosome.

14. The animal of any one of claims 5-13, wherein the human HLA-A is human HLA-A2.1.

15. The animal of any one of claims 5-13, wherein the human HLA-A is human HLA-A1*0101.

16. The animal of any one of claims 1-4, wherein the fusion protein comprises (a) a human B2M; and (b) a human HLA-A.

17. The animal of claim 16, wherein the human B2M and the human HLA-A are linked via a linker peptide sequence.

18. The animal of claim 16 or 17, wherein the human B2M comprises or consists of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 4 or amino acids 21-119 of SEQ ID NO: 4.

19. The animal of any one of claims 16-18, wherein the human HLA-A is HLA-A2.1.

20. The animal of any one of claims 16-19, wherein the human HLA-A comprises or consists of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8, amino acids 25-365 of SEQ ID NO: 8, SEQ ID NO: 59, or amino acids 22-362 of SEQ ID NO: 59.

21. The animal of any one of claims 16-20, wherein the fusion protein comprises or consists of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 62.

22. The animal of any one of claims 1-4, wherein the fusion protein comprises (a) a human B2M; and (b) a chimeric MHC α chain.

23. The animal of claim 22, wherein the human B2M and the chimeric MHC α chain are linked via a linker peptide sequence.

24. The animal of claim 22 or 23, wherein the human B2M comprises or consists of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 4 or amino acids 21-119 of SEQ ID NO: 4.

25. The animal of any one of claims 22-24, wherein the chimeric MHC α chain comprises human HLA-A α1 and α2 domains.

26. The animal of claim 25, wherein the chimeric MHC α chain further comprises a human HLA-A α3 domain.

27. The animal of claim 25, wherein the chimeric MHC α chain further comprises an endogenous MHC α3 domain and/or an endogenous MHC cytoplasmic region.

28. The animal of any one of claims 22-27, wherein the chimeric MHC α chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8, amino acids 25-206 of SEQ ID NO: 8, SEQ ID NO: 59, or amino acids 22-203 of SEQ ID NO: 59.

29. The animal of any one of claims 22-28, wherein the chimeric MHC α chain comprises a α3 domain, a connecting peptide, a transmembrane region, and a cytoplasmic region of an endogenous MHC.

30. The animal of any one of claims 22-28, wherein the animal is a mouse, and the chimeric MHC α chain comprises a α3 domain, a connecting peptide, a transmembrane region, and a cytoplasmic region of mouse H2-D1.

31. The animal of any one of claims 22-30, wherein the chimeric MHC α chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to amino acids 207-362 of SEQ ID NO: 6.

32. The animal of any one of claims 22-31, wherein the chimeric MHC α chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 61 or SEQ ID NO: 63.

33. The animal of any one of claims 1-32, wherein the fusion protein further comprises a signal peptide of human HLA-A2.1 (e.g., at the N-terminus of the fusion protein).

34. The animal of claim 33, wherein the signal peptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to amino acids 1-21 of SEQ ID NO: 59.

35. The animal of any one of claims 1-34, wherein the animal is heterozygous with respect to the sequence encoding the fusion protein.

36. The animal of any one of claims 1-34, wherein the animal is homozygous with respect to the sequence encoding the fusion protein.

37. A genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a chimeric MHC α chain comprising a human HLA-A α1 domain, a human HLA-A α2 domain and an endogenous MHC α3 domain.

38. The animal of claim 37, wherein the sequence encoding the chimeric MHC α chain is operably linked to an endogenous regulatory element at the endogenous MHC α chain gene locus in the at least one chromosome.

39. The animal of claim 37 or 38, wherein the genome of the animal further comprises a sequence encoding a human B2M, wherein the human B2M and the chimeric MHC α chain can associate with each other, forming a functional MHC protein complex in the animal.

40. The animal of claim 39, wherein the sequence encoding the human B2M is operably linked to an endogenous regulatory element (e.g., a promoter) at the endogenous B2M gene locus.

41. The animal of any one of claims 37-40, wherein the animal is a mouse, and the sequence encoding the chimeric MHC α chain is operably linked to an endogenous regulatory element (e.g., a promoter) at the mouse H2-D1 gene locus.

42. The animal of any one of claims 37-41, wherein the human HLA-A is human HLA-A2.1.

43. A genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human HLA-A.

44. The animal of claim 43, wherein the sequence encoding the human HLA-A is operably linked to an endogenous regulatory element at the endogenous MHC α chain gene locus in the at least one chromosome.

45. The animal of claim 43 or 44, wherein the genome of the animal further comprises a sequence encoding a human B2M, wherein the human B2M and the human HLA-A can associate with each other, forming a functional MHC protein complex in the animal.

46. The animal of claim 45, wherein the sequence encoding the human B2M is operably linked to an endogenous regulatory element (e.g., a promoter) at the endogenous B2M gene locus.

47. The animal of any one of claims 43-46, wherein the animal is a mouse, and the sequence encoding the human HLA-A is operably linked to an endogenous regulatory element (e.g., a promoter) at the mouse H2-D1 gene locus.

48. The animal of any one of claims 43-47, wherein the human HLA-A is human HLA-A2.1.

49. The animal of any one of claims 1-48, wherein the animal does not express endogenous B2M.

50. The animal of any one of claims 1-49, wherein the animal does not express endogenous MHC α chain.

51. The animal of any one of claims 1-50, wherein B2M and the MHC α chain can associate with each other, forming a functional MHC protein complex, wherein the protein complex can present a non-self antigen to the surface of one or more cells.

52. The animal of claim 51, wherein a human T cell (e.g., a cytotoxic T cell) can recognize the presented non-self antigen and initiate immune response.

53. The animal of claim 51, wherein an endogenous T cells (e.g., a cytotoxic T cell) can recognize the presented non-self antigen and initiate immune response.

54. The animal of any one claims 1-53, wherein the animal is a mammal, e.g., a monkey, a rodent or a mouse.

55. The animal of claim 54, wherein the animal is a mouse (e.g., with a C57BL/6 background).

56. The animal of any one of claims 1-55, wherein the animal is an immunodeficient mouse.

57. The animal of any one of claims 1-56, wherein the genome of the animal comprises a disruption in the animal's endogenous CD132 gene.

58. The animal of any one of claims 1-57, wherein the animal is a NOD/scid mouse, a NOD/scid nude mouse, or a B-NDG mouse.

59. The animal of any one of claims 1-58, wherein the animal further comprises a sequence encoding an additional human or chimeric protein.

60. The animal of claim 59, wherein the additional human or chimeric protein is programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Lymphocyte Activating 3 (LAG-3), B And T Lymphocyte Associated (BTLA), Programmed Cell Death 1 Ligand 1 (PD-L1), CD27, CD28, SIRPα, CD47, THPO, CD137, CD154, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT), T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3), Glucocorticoid-Induced TNFR-Related Protein (GITR), Signal regulatory protein α(SIRPα) or TNF Receptor Superfamily Member 4 (OX40).

61. A method for making a genetically-modified, non-human animal, comprising: replacing in at least one cell of the animal, at an endogenous B2M gene locus, a sequence encoding a region of endogenous B2M with a sequence encoding a human B2M or a sequence encoding a fusion protein comprising a human B2M and a human or humanized MHC α chain.

62. The method of claim 61, wherein the sequence encoding the region of endogenous B2M comprises all or a part of exon 1, exon 2, and exon 3 of endogenous B2M gene.

63. A method for making a genetically-modified, non-human animal, comprising: replacing in at least one cell of the animal, at an endogenous MHC gene locus, a sequence encoding a region of endogenous MHC α chain with a sequence encoding a human MHC α chain or a sequence encoding a fusion protein comprising a human B2M and a human or humanized MHC α chain.

64. The method of claim 63, wherein the sequence encoding the region of endogenous MHC molecule comprises all or a part of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 of endogenous MHC gene.

65. The method of claim 63, wherein the animal is mouse, and the sequence encoding the region of endogenous MHC comprises all or a part of exon 1, exon 2, exon 3 of mouse H2-D1 gene.

66. The method of any one of claims 61-65, wherein the sequence encoding the fusion protein comprises the following elements: (a) exon 1, exon 2, and/or exon 3 of human B2M; (b) an optional sequence encoding a linker peptide sequence; and (c) exon 2 and/or exon 3 of human HLA-A2.1.

67. The method of claim 66, wherein the sequence encoding the fusion protein further comprises exon 4, exon 5, exon 6, exon 7, and/or exon 8 of the endogenous MHC molecule gene that is downstream of element (c).

68. The method of claim 66 or 67, wherein the animal is mouse, and the sequence encoding the fusion protein further comprises the 3′ UTR of mouse H2-D1 gene.

69. The method of any one of claims 61-68, wherein the fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 64.

70. A method of determining effectiveness of an agent or a combination of agents for the treatment of cancer, comprising: engrafting tumor cells to the animal of any one of claims 1-60, thereby forming one or more tumors in the animal; administering the agent or the combination of agents to the animal; and determining the inhibitory effects on the tumors.

71. The method of claim 70, wherein before engrafting the tumor cells to the animal, human peripheral blood cells (hPBMC) or human hematopoietic stem cells are injected to the animal.

72. The method of claim 70, wherein the tumor cells are from cancer cell lines.

73. The method of claim 70, wherein the tumor cells are from a tumor sample obtained from a human patient.

74. The method of claim 70, wherein the inhibitory effects are determined by measuring the tumor volume in the animal.

75. The method of claim 70, wherein the tumor cells are melanoma cells, lung cancer cells, primary lung carcinoma cells, non-small cell lung carcinoma (NSCLC) cells, small cell lung cancer (SCLC) cells, primary gastric carcinoma cells, bladder cancer cells, breast cancer cells, and/or prostate cancer cells.

76. A method of producing an animal comprising a human hemato-lymphoid system, the method comprising: engrafting a population of cells comprising human hematopoietic cells or human peripheral blood cells into the animal of any one of claims 1-60.

77. The method of claim 76, wherein the human hemato-lymphoid system comprises human cells selected from the group consisting of hematopoietic stem cells, myeloid precursor cells, myeloid cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, lymphocytes, and platelets.

78. The method of claim 76 or 77, further comprising: irradiating the animal prior to the engrafting.

79. A fusion protein comprising β2 microglobulin (B2M) and a human or humanized MHC α chain.

80. A nucleic acid encoding the fusion protein of claim 79.

81. A protein comprising an amino acid sequence, wherein the amino acid sequence is one of the following: (f) an amino acid sequence set forth in SEQ ID NO: 4, 8, 59, 61, 62, 63, or 64; (g) an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, 8, 59, 61, 62, 63, or 64; (h) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4, 8, 59, 61, 62, 63, or 64; (i) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 4, 8, 59, 61, 62, 63, or 64 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and (j) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 4, 8, 59, 61, 62, 63, or 64.

82. A nucleic acid comprising a nucleotide sequence, wherein the nucleotide sequence is one of the following: (e) a sequence that encodes the protein of claim 81; (f) SEQ ID NO: 9, 10, 13, 14, 15, 16, 52, 54, or 65; (g) a sequence that is at least 90% identical to SEQ ID NO: 9, 10, 13, 14, 15, 16, 52, 54, or 65; and (h) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9, 10, 13, 14, 15, 16, 52, 54, or 65.

83. A cell comprising the protein of claim 81 and/or the nucleic acid of claim 82.

84. An animal comprising the protein of claim 81 and/or the nucleic acid of claim 82.

Description

DESCRIPTION OF DRAWINGS

[0335] FIG. 1 is a 3D schematic structure of HLA-A.

[0336] FIG. 2 are schematic diagrams showing mouse B2M gene locus and human B2M gene locus.

[0337] FIG. 3 are schematic diagrams showing mouse H2-D1 gene locus and human HLA-A gene locus.

[0338] FIG. 4 is a schematic diagram showing humanized mouse B2M gene locus. Mouse B2M gene coding region is replaced with a nucleic acid sequence encoding human B2M protein, a portion of human HLA-A2.1 protein, and a portion of mouse H2-D1 protein.

[0339] FIG. 5 is a schematic diagram showing humanized mouse B2M gene locus. Mouse B2M gene coding region is replaced with the coding region of human B2M gene.

[0340] FIG. 6 is a schematic diagram showing humanized mouse H2-D1 gene locus. A portion of mouse H2-D1 gene is replaced with a nucleic acid sequence encoding a portion of human HLA-A2.1 protein.

[0341] FIG. 7 is a schematic diagram showing humanized mouse H2-D1 gene locus. A portion of mouse H2-D1 gene is replaced with a nucleic acid sequence encoding human B2M protein and a portion of human HLA-A2.1 protein.

[0342] FIG. 8 is a schematic diagram showing humanized mouse B2M gene locus. Mouse B2M gene coding region was replaced with a nucleic acid sequence encoding the signal peptide of human HLA-A2.1, human B2M protein, a portion of human HLA-A2.1 protein, and a portion of mouse H2-D1 protein.

[0343] FIG. 9 shows a schematic diagram of a targeting strategy at mouse B2M gene locus.

[0344] FIG. 10A shows activity testing results for sgRNA1-sgRNA7 (sg1-sg7). PC is positive control. Con. is negative control. Blank is blank control.

[0345] FIG. 10B shows activity testing results for sgRNA9-sgRNA15 (sg9-sg15). PC is positive control. Con. is negative control. Blank is blank control.

[0346] FIG. 11A shows 5′ end PCR detection result of F0 generation mice by primers L-GT-F and L-GT-R. M is marker. H.sub.2O is water control. WT is wildtype control. PC is positive control. F0-01, F0-02, F0-03, F0-04, F0-05, F0-06, F0-07, F0-08, F0-09, and F0-10 are mouse numbers.

[0347] FIG. 11B shows 3′ end PCR detection result of F0 generation mice by primers R-GT-F and R-GT-R. M is marker. H.sub.2O is water control. WT is wildtype control. PC is positive control. F0-01, F0-02, F0-03, F0-04, F0-05, F0-06, F0-07, F0-08, F0-09, and F0-10 are mouse numbers.

[0348] FIG. 12A shows 5′ end PCR detection result of F1 generation mice by primers L-GT-F and L-GT-R. M is marker. H.sub.2O is water control. WT is wildtype control. PC is positive control. F1-01, F1-02, F1-03, F1-04, F1-05, F1-06 and F1-07 are positive mouse numbers.

[0349] FIG. 12B shows 3′ end PCR detection result of F1 generation mice by primers R-GT-F and R-GT-R. M is marker. H.sub.2O is water control. WT is wildtype control. PC is positive control. F1-01, F1-02, F1-03, F1-04, F1-05, F1-06, and F1-07 are positive mouse numbers.

[0350] FIG. 13 shows Southern Blot analysis result of F1 generation mice by P1 or P2 probe. M is marker. WT is wildtype control. F1-01, F1-02, F1-03, F1-04, F1-05, F1-06, and F1-07 are mouse numbers.

[0351] FIG. 14A shows a flow cytometry result of spleen cells from unstimulated wildtype C57BL/6 mouse. The spleen cells were stained with anti-mouse B2M antibody mβ2M PE and anti-mouse CD45 antibody mCD45 APC.

[0352] FIG. 14B shows a flow cytometry result of spleen cells from unstimulated MHC humanized homozygous mouse (H/H). The spleen cells were stained with anti-mouse B2M antibody mβ2M PE and anti-mouse CD45 antibody mCD45 APC.

[0353] FIG. 14C shows a flow cytometry result of spleen cells from wildtype C57BL/6 mouse stimulated by anti-mouse CD3 antibody. The spleen cells were stained with anti-mouse B2M antibody mβ2M PE and anti-mouse CD45 antibody mCD45 APC.

[0354] FIG. 14D shows a flow cytometry result of spleen cells from MHC humanized homozygous mouse (H/H) stimulated by anti-mouse CD3 antibody. The spleen cells were stained with anti-mouse B2M antibody mβ2M PE and anti-mouse CD45 antibody mCD45 APC.

[0355] FIG. 14E shows a flow cytometry result of spleen cells from unstimulated wildtype C57BL/6 mouse. The spleen cells were stained with anti-human B2M antibody hβ2M PE and anti-mouse CD45 antibody mCD45 APC.

[0356] FIG. 14F shows a flow cytometry result of spleen cells from unstimulated MHC humanized homozygous mouse (H/H). The spleen cells were stained with anti-human B2M antibody hβ2M PE and anti-mouse CD45 antibody mCD45 APC.

[0357] FIG. 14G shows a flow cytometry result of spleen cells from wildtype C57BL/6 mouse stimulated by anti-mouse CD3 antibody. The spleen cells were stained with anti-human B2M antibody hβ2M PE and anti-mouse CD45 antibody mCD45 APC.

[0358] FIG. 14H shows a flow cytometry result of spleen cells from MHC humanized homozygous mouse (H/H) stimulated by anti-mouse CD3 antibody. The spleen cells were stained with anti-human B2M antibody hβ2M PE and anti-mouse CD45 antibody mCD45 APC.

[0359] FIG. 14I shows a flow cytometry result of spleen cells from unstimulated wildtype C57BL/6 mouse. The spleen cells were stained with anti-mouse H-2Kb/H-2Db antibody and anti-mouse CD45 antibody mCD45 APC.

[0360] FIG. 14J shows a flow cytometry result of spleen cells from unstimulated MHC humanized homozygous mouse (H/H). The spleen cells were stained with anti-mouse H-2Kb/H-2Db antibody and anti-mouse CD45 antibody mCD45 APC.

[0361] FIG. 14K shows a flow cytometry result of spleen cells from wildtype C57BL/6 mouse stimulated by anti-mouse CD3 antibody. The spleen cells were stained with anti-mouse H-2Kb/H-2Db antibody and anti-mouse CD45 antibody mCD45 APC.

[0362] FIG. 14L shows a flow cytometry result of spleen cells from MHC humanized homozygous mouse (H/H) stimulated by anti-mouse CD3 antibody. The spleen cells were stained with anti-mouse H-2Kb/H-2Db antibody and anti-mouse CD45 antibody mCD45 APC.

[0363] FIG. 14M shows a flow cytometry result of spleen cells from unstimulated wildtype C57BL/6 mouse. The spleen cells were stained with anti-human HLA-A2 antibody hHLA-A2 PE and anti-mouse CD45 antibody mCD45 APC.

[0364] FIG. 14N shows a flow cytometry result of spleen cells from unstimulated MHC humanized homozygous mouse (H/H). The spleen cells were stained with anti-human HLA-A2 antibody hHLA-A2 PE and anti-mouse CD45 antibody mCD45 APC.

[0365] FIG. 14O shows a flow cytometry result of spleen cells from wildtype C57BL/6 mouse stimulated by anti-mouse CD3 antibody. The spleen cells were stained with anti-human HLA-A2 antibody hHLA-A2 PE and anti-mouse CD45 antibody mCD45 APC.

[0366] FIG. 14P shows a flow cytometry result of spleen cells from MHC humanized homozygous mouse (H/H) stimulated by anti-mouse CD3 antibody. The spleen cells were stained with anti-human HLA-A2 antibody hHLA-A2 PE and anti-mouse CD45 antibody mCD45 APC.

[0367] FIG. 15A shows a flow cytometry result of leukocytes in wildtype C57BL/6 mouse spleen cells. The spleen cells were stained with anti-mouse CD45 antibody mCD45 APC. The ratio of leukocytes was 88.6%.

[0368] FIG. 15B shows a flow cytometry result of leukocytes in MHC humanized homozygous mouse (H/H) spleen cells. The spleen cells were stained with anti-mouse CD45 antibody mCD45 APC. The ratio of leukocytes was 96.5%.

[0369] FIG. 15C shows a flow cytometry result of T cells and B cells in wildtype C57BL/6 mouse leukocytes. The T cells and B cells were stained with mouse T cell surface antibody mTCRB-APC-Cy7 and anti-mouse CD19 antibody mCD19-PE, respectively.

[0370] FIG. 15D shows a flow cytometry result of T cells and B cells in MHC humanized homozygous mouse (H/H) leukocytes. The T cells and B cells were stained with mouse T cell surface antibody mTCRB-APC-Cy7 and anti-mouse CD19 antibody mCD19-PE, respectively.

[0371] FIG. 15E shows a flow cytometry result of CD4+ T cells and CD8+ T cells in wildtype C57BL/6 mouse T cells. The CD4+ T cells and CD8+ T cells were stained with anti-mouse CD4 antibody mCD4-BV421 and the anti-mouse mCD8a antibody mCD8a-BV711, respectively.

[0372] FIG. 15F shows a flow cytometry result of CD4+ T cells and CD8+ T cells in MHC humanized homozygous mouse (H/H) T cells. The CD4+ T cells and CD8+ T cells were stained with anti-mouse CD4 antibody mCD4-BV421 and the anti-mouse mCD8a antibody mCD8a-BV711, respectively.

[0373] FIG. 16 shows PCR results from SIRPα knockout mice. M is Marker. WT is wildtype control. H.sub.2O is water control. Numbers 1-10 are positive mouse numbers.

[0374] FIG. 17 is a schematic diagram showing humanized mouse B2M gene locus. Mouse B2M gene coding region was replaced with a nucleic acid sequence encoding the signal peptide of human HLA-A2.1, human B2M protein, and human HLA-A2.1 protein.

[0375] FIG. 18 shows a schematic diagram of a targeting strategy at mouse B2M gene locus.

[0376] FIG. 19A shows 5′ end PCR detection result of F0 generation mice by primers L-GT-F and BNDG-L-GT-R. M is marker. H.sub.2O is water control. WT is wildtype control. PC is positive control. BNDG-F0-01, BNDG-F0-02, BNDG-F0-03, BNDG-F0-04, BNDG-F0-05, and BNDG-F0-06 are mouse numbers.

[0377] FIG. 19B shows 3′ end PCR detection result of F0 generation mice by primers BNDG-R-GT-F and R-GT-R. M is marker. H.sub.2O is water control. WT is wildtype control. PC is positive control. BNDG-F0-01, BNDG-F0-02, BNDG-F0-03, BNDG-F0-04, BNDG-F0-05, and BNDG-F0-06 are mouse numbers.

[0378] FIG. 20A shows 5′ end PCR detection result of F1 generation mice by primers L-GT-F and BNDG-L-GT-R. M is marker. H.sub.2O is water control. WT is wildtype control. PC is positive control. BNDG-F0-01 and BNDG-F0-02 are mouse numbers.

[0379] FIG. 20B shows 3′ end PCR detection result of F1 generation mice by primers BNDG-R-GT-F and R-GT-R. M is marker. H.sub.2O is water control. WT is wildtype control. PC is positive control. BNDG-F0-01 and BNDG-F0-02 are mouse numbers.

[0380] FIG. 21 shows Southern Blot analysis result of F1 generation mice by BNDG-P1 or BNDG-P2 probe. M is marker. WT is wildtype control. BNDG-F0-01 and BNDG-F0-02 are mouse numbers.

[0381] FIG. 22A shows a flow cytometry result of spleen cells from B-NDG mouse. The spleen cells were stained with anti-mouse B2M antibody mβ2M PE and anti-mouse CD45 antibody mCD45 APC.

[0382] FIG. 22B shows a flow cytometry result of spleen cells from B-NDG background MHC humanized heterozygous mouse (H/+). The spleen cells were stained with anti-mouse B2M antibody mβ2M PE and anti-mouse CD45 antibody mCD45 APC.

[0383] FIG. 22C shows a flow cytometry result of spleen cells from B-NDG mouse. The spleen cells were stained with anti-human B2M antibody hβ2M PE and anti-mouse CD45 antibody mCD45 APC.

[0384] FIG. 22D shows a flow cytometry result of spleen cells from B-NDG background MHC humanized heterozygous mouse (H/+). The spleen cells were stained with anti-human B2M antibody hβ2M PE and anti-mouse CD45 antibody mCD45 APC.

[0385] FIG. 22E shows a flow cytometry result of spleen cells from B-NDG mouse. The spleen cells were stained with anti-mouse H-2Kb/H-2Db antibody and anti-mouse CD45 antibody mCD45 APC.

[0386] FIG. 22F shows a flow cytometry result of spleen cells from B-NDG background MHC humanized heterozygous mouse (H/+). The spleen cells were stained with anti-mouse H-2Kb/H-2Db antibody and anti-mouse CD45 antibody mCD45 APC.

[0387] FIG. 22G shows a flow cytometry result of spleen cells from B-NDG mouse. The spleen cells were stained with anti-human HLA-A2 antibody hHLA-A2 PE and anti-mouse CD45 antibody mCD45 APC.

[0388] FIG. 22H shows a flow cytometry result of spleen cells from B-NDG background MHC humanized heterozygous mouse (H/+). The spleen cells were stained with anti-human HLA-A2 antibody hHLA-A2 PE and anti-mouse CD45 antibody mCD45 APC.

[0389] FIG. 23 shows the alignment between mouse B2M amino acid sequence (NP_033865.2; SEQ ID NO: 2) and human B2M amino acid sequence (NP_004039.1; SEQ ID NO: 4).

[0390] FIG. 24 shows the alignment between rat B2M amino acid sequence (NP_036644.1; SEQ ID NO: 66) and human B2M amino acid sequence (NP_004039.1; SEQ ID NO: 4).

[0391] FIG. 25 shows the alignment between mouse H2-D1 amino acid sequence (NP_034510.3; SEQ ID NO: 6) and human HLA-A2.1 amino acid sequence (AAC24825.1; SEQ ID NO: 59).

[0392] FIG. 26 shows the alignment between mouse H2-D1 amino acid sequence (NP_034510.3; SEQ ID NO: 6) and human HLA-A*0101 amino acid sequence (NP_001229687.1; SEQ ID NO: 8).

EXAMPLES

[0393] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Materials and Methods

[0394] The following materials were used in the following examples.

[0395] NOD-Prkdc.sup.scid IL-2rg.sup.null (B-NDG) mice were obtained from Beijing Biocytogen Co., Ltd. The catalog number is B-CM-001 or B-CM-002.

[0396] UCA kit was obtained from Beijing Biocytogen Co., Ltd. The catalog number is BCG-DX-001.

[0397] Ambion™ in vitro transcription kit was purchased from Ambion, Inc. The catalog number is AM1354.

[0398] Cas9 mRNA was obtained from SIGMA. The catalog number is CAS9MRNA-1EA.

[0399] PE anti-human β2-microglobulin Antibody (hβ2M PE) was purchased from BioLegend. The catalog number is 316305.

[0400] PE anti-mouse β2-microglobulin Antibody (mβ2M PE) was purchased from BioLegend. The catalog number is 154503.

[0401] PE anti-human HLA-A2 Antibody (hHLA-A2 PE) was purchased from BioLegend. The catalog number is 343305.

[0402] PE anti-mouse H-2K.sup.b/H-2D.sup.b Antibody (H-2Kb/H-2Db PE) was purchased from BioLegend. The catalog number is 114607.

[0403] FITC anti-mouse CD19 Antibody (mCD19FITC) was purchased from BioLegend. The catalog number is 115506.

[0404] Purified anti-mouse CD16/32 Antibody was purchased from BioLegend. The catalog number is 101302.

[0405] APC anti-mCD45 (mCD45APC) was purchased from BioLegend. The catalog number is 559864.

[0406] APC/Cy7 anti-mouse TCR R chain Antibody (mTCRβ APC/Cy7) was purchased from BioLegend. The catalog number is 109220.

[0407] Alexa Fluor® 488 anti-mouse CD3 Antibody (mCD3 Alexa Flour 488) was purchased from Biolegend. The catalog number is 100210.

[0408] PE anti-mouse CD19 Antibody (mCD19 PE) was purchased from Biolegend. The catalog number is 115508.

[0409] Brilliant Violet 421™ anti-mouse CD4 Antibody (mCD4 BV421) was purchased from BioLegend. The catalog number is 100438.

[0410] Brilliant Violet 711™ anti-mouse CD8a Antibody (mCD8a BV711) was purchased from BioLegend. The catalog number is 100747.

[0411] BamHI, BglII, EcoNI, and SspI restriction enzymes were purchased from NEB. The catalog numbers are R3136, R0144, R0521, and R3132, respectively.

Example 1: Generation of MHC Humanized Mice

[0412] The genome of a non-human animal (e.g., a mouse) can be modified to include a nucleic acid sequence encoding all or a part of a human B2M and HLA-A2.1 proteins, such that the genetically modified non-human animal can express human or humanized B2M and HLA-A2.1 proteins. The mouse B2M gene (NCBI Gene ID: 12010, Primary source: MGI: 88127, UniProt ID: P01887) is located in chromosome 2 of the mouse genome (from 122,147,686 to 122,153,083 of NC_000068.7). The transcript sequence NM_009735.3 is set forth in SEQ ID NO: 1, and the corresponding protein sequence NP_033865.2 is set forth in SEQ ID NO: 2. The human B2M gene (NCBI Gene ID: 567, Primary source: HGNC: 914, UniProt ID: P61769) is located in chromosome 15 of the human genome (from 44,711,487 to 44,718,877 of NC_000015.10). The transcript sequence NM_004048.3 is set forth in SEQ ID NO: 3, and the corresponding protein sequence NP_004039.1 is set forth in SEQ ID NO: 4. Mouse and human B2M gene loci are shown in FIG. 2.

[0413] The mouse H2-D1 gene (NCBI Gene ID: 14964, Primary source: MGI: 95896, UniProt ID: P01899) is located in chromosome 17 of the mouse genome. The transcript sequence NM_010380.3 is set forth in SEQ ID NO: 5, and the corresponding protein sequence NP_034510.3 is set forth in SEQ ID NO: 6. The human HLA-A gene (NCBI Gene ID: 3105, Primary source: HGNC: 4931, UniProt ID: P04439) is located in chromosome 6 of the human genome. The transcript sequence NM_001242758.1 is set forth in SEQ ID NO: 7, and the corresponding protein sequence NP_001229687.1 is set forth in SEQ ID NO: 8. Mouse H2-D1 gene locus and human HLA-A gene locus are shown in FIG. 3.

[0414] To obtain a transgenic mouse expressing a human or humanized MHC molecule, various strategies can be used. For example, the mouse endogenous B2M gene and endogenous H2-D1 gene can be inactivated, and a nucleic acid sequence encoding a polypeptide sequence including: human B2M; the signal peptide and a portion of the extracellular region of human HLA-A2.1 (e.g., amino acids 1-203 of SEQ ID NO: 59); a portion of the extracellular region (Alpha-3 region and connecting peptide), the transmembrane region and the cytoplasmic region of mouse H2-D1 (e.g., amino acids 207-362 of SEQ ID NO: 6) can be knocked into mouse genome.

[0415] Different modifications and combinations can also be used on the mouse B2M and/or mouse H2-D1 gene loci.

[0416] For example, the mouse endogenous B2M gene locus can be humanized as follows. As shown in FIG. 4, within exon 1 of the mouse endogenous B2M gene, a nucleic acid sequence encoding a polypeptide including: human B2M; the signal peptide, Alpha-1, and Alpha-2 regions of human HLA-A2.1 (e.g., amino acids 1-203 of AAC24825.1 (SEQ ID NO: 59) encoded by human HLA-A2.1 exons 1-3); the Alpha-3 region, connecting peptide, transmembrane region, and the cytoplasmic region of mouse H2-D1 (e.g., amino acids 207-362 of NP_034510.3 (SEQ ID NO: 6) encoded by mouse H2-D1 exons 4-8) can be used to replace a sequence spanning exons 1-3 the mouse endogenous B2M gene.

[0417] As a different strategy, the mouse endogenous B2M gene can be directly humanized. As shown in FIG. 5, the coding region of mouse B2M gene can be replaced with the coding region of human B2M gene. Meanwhile, a sequence encoding a polypeptide (SEQ ID NO: 64) including: the signal peptide, Alpha-1, and Alpha-2 regions of human HLA-A2.1; the Alpha-3 region, connecting peptide, transmembrane region, and the cytoplasmic region of mouse H2-D1 can be knocked into mouse genome by transgenic techniques. Alternatively, as shown in FIG. 6, at mouse endogenous H2-D1 gene locus, a sequence encoding a polypeptide including the signal peptide, Alpha-1, and Alpha-2 regions of human HLA-A2.1 can be used to replace a corresponding sequence of mouse H2-D1 gene encoding the signal peptide, Alpha-1 and Alpha-2 regions. When the mouse B2M gene and H2-D1 gene are respectively humanized, double-gene humanized mice can be prepared by one-step or multistep targeting strategies. It is also possible to prepare single-gene humanized mice separately, and obtain double-gene humanized mice through methods such as breeding. The obtained double-gene humanized mice can simultaneously express human B2M protein and humanized MHC α chain protein in vivo.

[0418] As a different strategy, the mouse endogenous B2M gene can be knocked out. Meanwhile, as shown in FIG. 7, at mouse endogenous H2-D1 gene locus, a sequence encoding a polypeptide (SEQ ID NO: 63) including human B2M; the signal peptide, Alpha-1, and Alpha-2 regions of human HLA-A2.1 can be used to replace a sequence encoding the signal peptide, Alpha-1 and Alpha-2 regions of mouse H2-D1.

[0419] In the following experiment, the humanization method shown in FIG. 4 was used to generate transgenic mice with humanized MHC molecules. Gene editing technology can be used to modify mouse cells. The endogenous mouse B2M gene locus can be knocked into a sequence encoding human B2M protein, a portion of human HLA-A2.1 protein, and a portion of mouse H2-D1 protein, which can also disrupt the mouse B2M gene coding region. The generated humanized mice can express humanized MHC molecules in vivo, which contains: human B2M protein; the Alpha-1 and Alpha-2 regions of human HLA-A2.1 protein (NCBI reference sequence: AAC24825.1, SEQ ID NO: 59); the Alpha-3 region, connecting peptide, the transmembrane region, and the cytoplasmic region of mouse H2-D1 protein. The human HLA-A2.1 protein portion is directly connected to the mouse H2-D1 protein portion, and the humanized mice does not express endogenous B2M protein. Further, in order to ensure correct expression of human HLA-A2.1 protein, a sequence encoding the signal peptide of human HLA-A2.1 protein can also be inserted before the human B2M coding region. The schematic diagram of the humanized mouse B2M locus is shown in FIG. 8. The mRNA sequence transcribed from the humanized B2M, HLA-A2.1 and H2-D1 genes is shown in SEQ ID NO: 9, and the DNA sequence of the humanized B2M locus (only the modified part) is shown in SEQ ID NO: 10.

[0420] Given that human B2M have multiple isoforms or transcripts, the methods described herein can be applied to other isoforms or transcripts.

[0421] The CRISPR/Cas system was applied for gene editing, and the targeting strategy is shown in FIG. 9. A targeting vector was designed, containing homologous arm sequences upstream and downstream of mouse B2M gene, and an “A fragment” encoding human B2M protein, a portion of human HLA-A2.1 protein, and a portion of mouse H2-D1 protein. The upstream homologous arm sequence (5′ homologous arm, SEQ ID NO: 11) is identical to nucleic acids 122146329-122147737 of NCBI reference sequence NC_000068.7. The downstream homologous arm sequence (3′ homologous arm, SEQ ID NO: 12) is identical to nucleic acids 122152171-122153513 of NCBI reference sequence NC_000068.7. The “A fragment” contains sequences from 5′ end to 3′ end that encode the following polypeptides: the signal peptide of human HLA-A2.1; human B2M; a flexible linker polypeptide sequence; a portion of the human HLA-A2.1 protein; and a portion of the mouse H2-D1 protein. Specifically, the sequence encoding the signal peptide of human HLA-A2.1 (SEQ ID NO: 13) is identical to nucleic acids 99567-99638 of GenBank reference sequence AF055066.1. The sequence encoding human B2M (SEQ ID NO: 14) is identical to nucleic acids 91-387 of NCBI reference sequence NM_004048.3. The flexible linker polypeptide sequence is the (GGGGS).sub.3 (SEQ ID NO: 67) linker that is encoded by a 45 bp sequence 5′-GGAGGTGGCGGATCCGGCGGAGGCGGCTCGGGTGGCGGCGGCTCT-3′ (SEQ ID NO: 51). The portion of human HLA-A2.1 protein is encoded by a sequence (SEQ ID NO: 15) that is identical to nucleic acids 98606-99435 of GenBank reference sequence AF055066.1. The portion of mouse H2-D1 protein is encoded by a sequence (SEQ ID NO: 16) that is identical to nucleic acids 35266871-35267765 of NCBI reference sequence NC_000083.6. The protein expressed in the transgenic mice is shown in SEQ ID NO: 61. However, due to the existence of the flexible linker polypeptide sequence, the protein can have the functional domains of human B2M and HLA-A2.1 protein.

[0422] The targeting vector was constructed, e.g., by restriction enzyme digestion/ligation, or gene synthesis. The constructed targeting vector sequence was preliminarily verified by restriction enzyme digestion, then verified by sequencing. The verified targeting vector was used for subsequent experiments.

[0423] The target sequences are important for the targeting specificity of sgRNAs and the efficiency of Cas9-induced cleavage. Specific sgRNA sequences were designed and synthesized that recognize the 5′ end targeting site (sgRNA1-sgRNA7) and 3′ end targeting site (sgRNA8-sgRNA15). The 5′ end targeting site is located within the first exon or the first intron of the mouse B2M gene. The 3′ end targeting site is located on the third exon or third intron of the mouse B2M gene. The targeting site sequence of each sgRNA on the B2M gene locus is as follows:

TABLE-US-00005 sgRNA1 targeting site (SEQ ID NO: 17): 5′-CCTGGCCAATCCCGTCGGGAAGG-3′ sgRNA2 targeting site (SEQ ID NO: 18): 5′-CCGTCAGCACACTCGCAAACAGG-3′ sgRNA3 targeting site (SEQ ID NO: 19): 5′-GTTCTCCTTCCCGACGGGATTGG-3′ sgRNA4 targeting site (SEQ ID NO: 20): 5′-ACTCTGGATAGCATACAGGCCGG-3′ sgRNA5 targeting site (SEQ ID NO: 21): 5′-CTGGTGCTTGTCTCACTGACCGG-3′ sgRNA6 targeting site (SEQ ID NO: 22): 5′-GGGGAAAGAGGCACTCACTCTGG-3′ sgRNA7 targeting site (SEQ ID NO: 23): 5′-GACAAGCACCAGAAAGACCAGGG-3′ sgRNA8 targeting site (SEQ ID NO: 24): 5′-CTGGAGGCTTCCGGACACTCAGG-3′ sgRNA9 targeting site (SEQ ID NO: 25):  5′-TGATCAAGCATCATGATGGTAGG-3′ sgRNA10 targeting site (SEQ ID NO: 26): 5′-AGGAGCGTGAGAGGGAACGTGGG-3′ sgRNA11 targeting site (SEQ ID NO: 27): 5′-GAGGAACGTAGCCATGTCACTGG-3′ sgRNA12 targeting site (SEQ ID NO: 28): 5′-CATGTCACTGGCCCTCTAAAGGG-3′ sgRNA13 targeting site (SEQ ID NO: 29): 5′-CATGTGATCAAGCATCATGATGG-3′ sgRNA14 targeting site (SEQ ID NO: 30): 5′-ACCCGCAGAGCTCTGTCACTCGG-3′ sgRNA15 targeting site (SEQ ID NO: 31): 5′-CTCTGTCACTCGGCTCCTCTGGG-3′

[0424] The UCA kit was used to detect the activities of sgRNAs. The results showed that the sgRNAs had different activities. The results are shown in Table 5 and FIGS. 10A-10B. sgRNA3 and sgRNA14 were selected for subsequent experiments. Oligonucleotides were added to the 5′ end and a complementary strand to obtain a forward oligonucleotide and a reverse oligonucleotide (see Table 6 for the sequences). After annealing, the products were ligated to the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI), respectively, to obtain expression vectors PT7-B2M-HLA-A2.1-3 and pT7-B2M-HLA-A2.1-14.

TABLE-US-00006 TABLE 5 UCA test results showing sgRNA activity 5′ end targeting site 3′ end targeting site detection result detection result Con.  1.00 ± 0.06 Con.  1.00 ± 0.03 PC 73.04 ± 1.51 PC 55.01 ± 2.95 sgRNA1 99.08 ± 5.37 sgRNA8 143.48 ± 8.70  sgRNA2 18.89 ± 9.70 sgRNA9 41.19 ± 2.51 sgRNA3 118.85 ± 5.15  sgRNA10 120.32 ± 7.44  sgRNA4 64.87 ± 5.75 sgRNA11 75.28 ± 6.24 sgRNA5 49.91 ± 3.03 sgRNA12 41.31 ± 1.12 sgRNA6 53.58 ± 3.78 sgRNA13 44.85 ± 2.75 sgRNA7 71.85 ± 2.74 sgRNA14 122.80 ± 9.26  — − sgRNA15 15.83 ± 0.39

TABLE-US-00007 TABLE 6 sgRNA3 and sgRNA14 sequence list sgRNA3 sequences SEQ ID NO: 32 Upstream: 5′-GTTCTCCTTCCCGACGGGAT-3′ SEQ ID NO: 33 Upstream: 5′-TAGGTTCTCCTTCCCGACGGGAT-3′ (forward oligonucleotide) SEQ ID NO: 34 Downstream: 5′-ATCCCGTCGGGAAGGAGAA-3′ SEQ ID NO: 35 Downstream: 5′-AAACATCCCGTCGGGAAGGAGAA-3′ (reverse oligonucleotide) sgRNA14 sequences SEQ ID NO: 36 Upstream: 5′-ACCCGCAGAGCTCTGTCACT-3′ SEQ ID NO: 37 Upstream: 5′-TAGGACCCGCAGAGCTCTGTCACT-3′ (forward oligonucleotide) SEQ ID NO: 38 Downstream: 5′-AGTGACAGACTCTGCGGGT-3′ SEQ ID NO: 39 Downstream: 5-AAACAGTGACAGACTCTGCGGGT-3′ (reverse oligonucleotide)

[0425] T7 promoter and sgRNA scaffold (SEQ TD NO: 40), and was ligated to the backbone vector (Takara, Catalog number: 3299) after restriction enzyme digestion (EcoRI and BamHI). The resulting plasmid was confirmed by sequencing.

[0426] The pre-mixed Cas9 mRNA, the targeting vector, in vitro transcription products of the pT7-B2M-HLA-A2.1-3 and pT7-B2M-HLA-A2.1-14 plasmids (using Ambion in vitro transcription kit to carry out the transcription according to the method provided in the product instruction) were injected into the cytoplasm or nucleus of C57BL/6 mouse fertilized eggs with a microinjection instrument. The embryo microinjection was carried out according to the method described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition),” Cold Spring Harbor Laboratory Press, 2003. The injected fertilized eggs were then transferred to a culture medium to culture for a short time and then was transplanted into the oviduct of the recipient mouse to produce the genetically modified mice (F0 generation). The mouse population was further expanded by cross-breeding and self-breeding to establish stable mouse lines with human or humanized B2M and HLA-A2.1 gene.

[0427] Experiments were performed to identify somatic cell genotype of the F0 generation mice. For example, PCR analysis was performed using mouse tail genomic DNA of the F0 generation mice. The PCR analysis results for some of the F0 mice are shown in FIGS. 11A-11B. In view of the 5′ end primer detection result and the 3′ end primer detection result, the 10 mice numbered from F0-01 to F0-10 were all positive mice.

[0428] The following primers were used in the PCR:

TABLE-US-00008 5′ end primers: L-GT-F (SEQ ID NO: 41): 5′-GAATGTGTGCCTCCTCTCAGTTTCC-3′ L-GT-R (SEQ ID NO: 42): 5′-TCCTTCCCGTTCTCCAGGTATCTGC-3′ 3′ end primers: R-GT-F (SEQ ID NO: 43): 5′-GCGGCTACTACAACCAGAGCGAG-3′ R-GT-R (SEQ ID NO: 44): 5′-TCCAGCAATAAGAACCAGTCCCTAGCT-3′

[0429] The primer L-GT-F is located on the left side of the 5′ homologous arm. R-GT-R is located on the right side of the 3′ homologous arm. Both L-GT-R and R-GT-F are located on the human sequence.

[0430] The positive F0 generation MHC humanized mice were bred with wildtype mice to generate F1 generation mice. The same method (e.g., PCR) was used for genotypic identification of the F1 generation mice. As shown in FIGS. 12A-12B, 7 mice numbered F1-01, F1-02, F1-03, F1-04, F1-05, F1-06, and F1-07 were identified as positive mice. The 7 positive F1 generation mice were further analyzed by Southern Blot, to confirm if random insertions were introduced. Specifically, mouse tail genomic DNA was extracted, digested with BamHI or BglII restriction enzyme, transferred to a membrane, and then hybridized with probes. Probes P1 and P2 are located on the upstream region of the 5′ homologous arm and on the 3′ homologous arm, respectively. The probes used in Southern Blot assays are listed in the table below.

TABLE-US-00009 TABLE 7 Restriction enzyme Probe WT size Targeted size BamHI P1 9.7 kb 5.9 kb BglII P2 4.0 kb 2.9 kb

[0431] The probes were synthesized using the following primers:

TABLE-US-00010 P1-F (SEQ ID NO: 45): 5′-ATGAGGTCTTTTTGTGGGCAGAGCA-3′ P1-R (SEQ ID NO: 46): 5′-CTCCCTACGGCCACATCACCATTAC-3′ P2-F (SEQ ID NO: 47): 5′-TAACTTCATGTAAGGCACCGTCAC-3′ P2-R (SEQ ID NO: 48): 5′-TCCAGACCTCACCATCAAATGAG-3′

[0432] The detection result of Southern Blot is shown in FIG. 13. In view of the hybridization results by P1 and P2 probes, the seven F1 generation mice were confirmed to be positive heterozygotes and no random insertions were detected. This indicates that the method described above can be used to generate genetically-modified MHC gene humanized mice that can be stably passaged without random insertions.

[0433] The heterozygous mice identified as positive in the F1 generation can be bred with each other to obtain the F2 generation MHC humanized homozygous mouse (H/H).

[0434] The expression of human B2M protein and HLA-A2.1 protein in positive mice was confirmed by ELISA. Specifically, one wildtype C57BL/6 female mouse (6-week old) and one MHC humanized female homozygous mouse (6-week old) prepared by the method described herein were selected, and each mouse was injected intraperitoneally with 7.5 μg (volume: 200 l) anti-mouse CD3 antibody. After 24 hours, the mice were sacrificed and then spleen cells were collected. Anti-mouse B2M antibody mβ2M PE, anti-mouse H-2Kb/H-2Db antibody, anti-human B2M antibody hβ2M PE, or anti-human HLA-A2 antibody hHLA-A2 PE; together with anti-mouse CD45 antibody mCD45 APC were used for spleen cell staining. The stained cells were subjected to flow cytometry analysis with results shown in FIGS. 14A-14P. Regardless of whether the cells were stimulated by anti-mouse CD3 antibody, only mouse B2M-expressing cells were detected in C57BL/6 mice (FIGS. 14A and 14C), human or humanized B2M-expressing cells were not detected in C57BL/6 mice (FIGS. 14E and 14G). Cells expressing human B2M (FIGS. 14F and 14H), and cells expressing HLA-A2.1 (FIGS. 14N and 14P) were only detected in MHC humanized homozygous mice. However, cells expressing mouse B2M were not detected in MHC humanized homozygous mice (FIGS. 14B and 14D). In addition, because the mouse H2-D1 coding sequence was not knocked out, a small number of cells expressing mouse H2-D1 were detected in MHC humanized homozygous mice (FIGS. 14G and 14L).

[0435] To confirm whether the differentiation of B cells and T cells in F2 generation MHC humanized homozygous mice was consistent with that of wildtype mice, the mouse lymphocyte subsets were analyzed by flow cytometry. Specifically, one wildtype C57BL/6 male mouse (16-week old) and one MHC humanized homozygous male mouse (13-week old) were selected respectively. The spleen cells were collected, and anti-mouse CD45 antibody mCD45 APC was used for cell staining and flow cytometry detection. As shown in FIGS. 15A-15B, the ratios of leukocytes in wildtype C57BL/6 mice and MHC humanized homozygous mice were 88.6% and 96.5%, respectively. Subsequently, mouse T cell surface antibody mTCRB-APC-Cy7 and anti-mouse CD19 antibody mCD19-PE were used to stain T cells and B cells, respectively, for flow cytometry analysis. The results showed that the T cells and B cells in the wildtype C57BL/6 mice were 23.8% and 61.4%, respectively (FIG. 15C); whereas the T cells and B cells in the humanized MHC homozygous mice were 27.7% and 61.8%, respectively (FIG. 15D). Further, anti-mouse CD4 antibody mCD4-BV421 and the anti-mouse mCD8a antibody mCD8a-BV711 were used for cell staining, and the stained cells were subjected to flow cytometry analysis. As shown in FIGS. 15E-15F, the ratios of CD4+ T cells and CD8+ T cells in wildtype C57BL/6 mice and MHC humanized homozygous mice were comparable.

[0436] In summary, the above results showed that the expression of lymphocyte subsets in MHC humanized mice was similar to that of wildtype C57BL/6 mice. The results further indicated that the differentiation of T cells and B cells in MHC humanized mice was not affected by humanization of B2M and HLA-A2.1 genes.

[0437] Since the cleavage of Cas9 results in DNA double strand break, and the homologous recombination repair may result in insertion/deletion mutations, it is possible to obtain B2M gene knockout mice using the method described herein. A pair of primers was designed to detect the gene knockout mice. Wildtype mice should have no PCR bands, and knockout mice should have one PCR band at about 682 bp. As shown in FIG. 16, mice numbered 1-10 were identified as B2M gene knockout mice. One PCR primer was located on the left side of the 5′ targeting site, and the other PCR primer was located on the right side of the 3′ targeting site. The primers are shown below:

TABLE-US-00011 SEQ ID NO: 49: 5′-GAATAAATGAAGGCGGTCCCAGGCT-3′ SEQ ID NO: 50: 5′-AGGTGAGTTCTGGCTCCACCATTTG-3′

Example 2. MHC Humanized Mice with Severe Immunodeficiency

[0438] In addition to the humanization strategy as described in Example 1, mice with a higher degree of humanization of MHC molecules can also be designed. Furthermore, immunodeficient mice with humanized MHC molecules can be designed to provide effective experimental animal models for the research of pathogenesis mechanisms of immune system diseases, such as diabetes and transplant rejection, and drug development. In this example, B-NDG background mice were used to carry out a higher degree of humanization of MHC molecules. Specifically, gene editing technology was used to modify B-NDG mice. The endogenous mouse B2M gene locus was knocked into a sequence encoding human B2M protein and HLA-A2.1 protein, which also disrupted the mouse B2M gene coding region. The generated humanized mice can express humanized MHC molecules in vivo, containing human B2M protein and human HLA-A2.1 protein. The humanized mice did not express endogenous B2M protein. Further, in order to ensure correct expression of human HLA-A2.1 protein, a sequence encoding the signal peptide of human HLA-A2.1 protein was inserted before the human B2M coding region. The schematic diagram of the humanized mouse B2M locus is shown in FIG. 17. The mRNA sequence transcribed from the humanized B2M and HLA-A2.1 genes is shown in SEQ ID NO:52. The protein expressed by the transgenic mice is shown in SEQ ID NO: 62. However, due to the existence of the flexible linker polypeptide sequence, the protein can have the functional domains of human B2M protein and human HLA-A2.1 protein.

[0439] Given that human B2M gene have multiple isoforms or transcripts, the methods described herein can be applied to other isoforms or transcripts.

[0440] Further, a schematic diagram of the targeting strategy is shown in FIG. 18. The targeting vector is similar to the targeting vector used in Example 1, except that: the 5′ homologous arm (SEQ ID NO: 60) has 99% homology with nucleic acids 122146329-122147737 of NCBI reference number NC_000068.7, with mutations at position 122147015 (from G to T), position 122147108 (from C to T), and position 122147591 (from C to T); the 3′ homologous arm (SEQ ID NO: 53) has 99% homology with nucleic acids 122152171-122153513 of NCBI reference number NC_000068.7, with mutations at position 122152258 (from G to A), position 122152391 (from G to A), position 122152771 (from A to G), position 122153104 (from T to C), and position 122153148 (from A to C); and deletion at position 122152788 (deletion of A). the BNDG-A fragment (SEQ ID NO: 65) is similar to the “A fragment” in Example 1, but contains a sequence encoding the full-length human HLA-A2.1 protein. The sequence (SEQ ID NO: 54) encoding the full-length human HLA-A2.1 protein is identical to nucleic acids 95493-99436 of GenBank reference sequence AF055066.1. The BNDG-A fragment does not contain any sequence encoding moues H2-D1 protein.

[0441] The targeting vector was constructed, e.g., by restriction enzyme digestion/ligation, or gene synthesis. The constructed targeting vector sequence was preliminarily verified by restriction enzyme digestion, then verified by sequencing. The verified targeting vector was used for subsequent experiments (e.g., microinjection). The sgRNAs used was the same as the sgRNAs used in Example 1.

[0442] Specifically, the pre-mixed Cas9 mRNA, the targeting vector, in vitro transcription products of the pT7-B2M-HLA-A2.1-3 and pT7-B2M-HLA-A2.1-14 plasmids (using Ambion in vitro transcription kit to carry out the transcription according to the method provided in the product instruction) were injected into the cytoplasm or nucleus of B-NDG mouse fertilized eggs with a microinjection instrument. The embryo microinjection was carried out according to the method described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition),” Cold Spring Harbor Laboratory Press, 2003. The injected fertilized eggs were then transferred to a culture medium to culture for a short time and then was transplanted into the oviduct of the recipient mouse to produce the genetically modified mice (F0 generation). The mouse population was further expanded by cross-breeding and self-breeding to establish stable B-NDG background mouse lines with human B2M and HLA-A2.1 genes.

[0443] Experiments were performed to identify somatic cell genotype of the F0 generation mice with B-NDG background. For example, PCR analysis was performed using mouse tail genomic DNA of the F0 generation mice. The PCR analysis results for some of the F0 mice are shown in FIGS. 19A-19B. In view of the 5′ end primer detection result and the 3′ end primer detection result, the 6 mice numbered from BNDG-F0-01 to BNDG-F0-06 were all positive mice.

[0444] The following primers were used in the PCR:

TABLE-US-00012 5′ end primers: L-GT-F (SEQ ID NO: 41): 5′-GAATGTGTGCCTCCTCTCAGTTTCC-3′ BNDG-L-GT-R (SEQ ID NO: 55): 5′-CAGCTCCAAAGAGAACCAGGCCAG-3′ 3′ end primers: BNDG-R-GT-F (SEQ ID NO: 56): 5′-TACCCTGCGGAGATCACACTGACC-3′ R-GT-R (SEQ ID NO: 44): 5′-TCCAGCAATAAGAACCAGTCCCTAGCT-3′

[0445] The positive F0 generation MHC humanized mice were bred with wildtype mice to generate F1 generation mice. The same method (e.g., PCR) was used for genotypic identification of the F1 generation mice. As shown in FIGS. 20A-20B, 2 mice numbered BNDG-F1-01 and BNDG-F1-02 were identified as positive mice. The 2 positive F1 generation mice were further analyzed by Southern Blot, to confirm if random insertions were introduced. Specifically, mouse tail genomic DNA was extracted, digested with EcoNI or SspI restriction enzyme, transferred to a membrane, and then hybridized with probes. Probes BNDG-P1 and BNDG-P2 are located on the upstream region of the 5′ homologous arm and on the 3′ homologous arm, respectively. The probes used in Southern Blot assays are listed in the table below.

TABLE-US-00013 TABLE 8 Restriction enzyme Probe WT size Targeted size EcoNI BNDG-P1 8.8 kb 5.3 kb SspI BNDG-P2 12.1 kb 7.6 kb

[0446] The probes were synthesized using the following primers:

TABLE-US-00014 BNDG-P1-F (SEQ ID NO: 57): 5′-TTCTGATGCTCCTTCCTTCCGTGC-3′ BNDG-P1-R (SEQ ID NO: 58): 5′-TTCTCTGTGCTCAGTGTTCCCTGC-3′ BNDG-P2-F (SEQ ID NO: 47): 5′-TAACTTCATGTAAGGCACCGTCAC-3′ BNDG-P2-R (SEQ ID NO: 48): 5′-TCCAGACCTCACCATCAAATGAG-3′

[0447] The detection result of Southern Blot is shown in FIG. 21. In view of the hybridization results by BNDG-P1 and BNDG-P2 probes, the two F1 generation mice numbered BNDG-F1-01 and BNDG-F1-02 were confirmed to be positive heterozygotes and no random insertions were detected. This indicates that the method described above can be used to generate genetically-modified MHC gene humanized mice with B-NDG background that can be stably passaged without random insertions.

[0448] The expression of human B2M protein and HLA-A2.1 protein in positive mice (with B-NDG background) was confirmed by flow cytometry. Specifically, one B-NDG female mouse (6-week old) and one MHC humanized heterozygous female mouse (6-week old) prepared by the method described herein were sacrificed and then spleen cells were collected. Anti-mouse B2M antibody mβ2M PE, anti-mouse H-2Kb/H-2Db antibody, anti-human B2M antibody hβ2M PE, or anti-human HLA-A2 antibody hHLA-A2 PE; together with anti-mouse CD45 antibody mCD45 APC were used for spleen cell staining. The stained cells were subjected to flow cytometry analysis with results shown in FIGS. 22A-22H. The results showed that in B-NDG mice and B-NDG background MHC humanized heterozygous mice, cells expressing mouse B2M (FIGS. 22A and 22B) and cells expressing mouse H2-D1 (FIGS. 22E and 22F) were detected. However, cells expressing human B2M protein (FIG. 22D) and cells expressing human HLA-A2 protein (FIG. 22H) were only detected in humanized MHC mice with B-NDG background. Cells expressing human B2M protein (FIG. 22C) and cells expressing human HLA-A2 protein (FIG. 22G) were not detected in B-NDG mice.

Example 3. Method Based on Embryonic Stem Cells

[0449] The non-human mammals can also be prepared through other gene editing systems and approaches, which includes, but is not limited to, gene homologous recombination techniques based on embryonic stem cells (ES), zinc finger nuclease (ZFN) techniques, transcriptional activator-like effector factor nuclease (TALEN) technique, homing endonuclease (megakable base ribozyme), or other molecular biology techniques. In this example, the conventional ES cell gene homologous recombination technique is used as an example to describe how to obtain a MHC humanized mouse by other methods.

[0450] According to the gene editing strategy of the methods described herein and the modified B2M gene locus in MHC humanized mice (FIGS. 8-9), a targeting strategy can be designed with different targeting vector. In view of the fact that one of the objects is to disrupt the coding region of the mouse B2M gene coding region, and to knock in a nucleic acid sequence encoding human B2M protein, a portion of human HLA-A2.1 protein, and a portion of mouse H2-D1 protein at the mouse B2M gene locus, a targeting vector that contains a 5′ homologous arm, a 3′ homologous arm, and a humanized gene fragment is designed. The vector can also contain a resistance gene for positive clone screening, such as neomycin phosphotransferase coding sequence Neo. On both sides of the resistance gene, two site-specific recombination systems in the same orientation, such as Frt or LoxP, can be added. Furthermore, a coding gene with a negative screening marker, such as the diphtheria toxin A subunit coding gene (DTA), can be constructed downstream of the recombinant vector 3′ homologous arm. Vector construction can be carried out using methods known in the art, such as restriction enzyme digestion and ligation. The recombinant vector with correct sequence can then be transfected into mouse embryonic stem cells, and then the recombinant vector can be screened by the positive clone screening gene. The cells transfected with the recombinant vector are next screened by using the positive clone marker gene, and Southern Blot can be used for DNA recombination identification. For the selected correct positive clones, the positive clonal cells (black mice) are injected into the isolated blastocysts (white mice) by microinjection according to the method described in the book A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition),” Cold Spring Harbor Laboratory Press, 2003. The resulting chimeric blastocysts formed following the injection are transferred to the culture medium for a short time culture and then transplanted into the fallopian tubes of the recipient mice (white mice) to produce F0 generation chimeric mice (black and white). The F0 generation chimeric mice with correct gene recombination are then selected by extracting the mouse tail genomic DNA and PCR analysis for subsequent breeding and identification. The F1 generation mice are obtained by mating the F0 generation chimeric mice with wild-type mice. By extracting tail genomic DNA and PCR analysis, positive F1 generation heterozygous mice that can be stably passed are selected. Next, the F1 heterozygous mice are bred to each other to obtain genetically recombinant positive F2 generation homozygous mice. In addition, the F1 heterozygous mice can also be bred with Flp or Cre mice to remove the positive clone screening marker gene (Neo, etc.), and then the humanized homozygous mice can be obtained by breeding these mice with each other. The methods of genotyping and phenotypic detection of the obtained F1 heterozygous mice or F2 homozygous mice are similar to those used in the examples described above.

OTHER EMBODIMENTS

[0451] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.