Genetically modified mice expressing humanized CD47
The present disclosure relates to genetically modified non-human animals that express a human or chimeric (e.g., humanized) CD47, and methods of use thereof.
1. A genetically modified mouse whose genome comprises a nucleic acid sequence encoding a chimeric CD47 protein comprising the amino acid sequence of SEQ ID NO: 74, 75, 76, 77, 78, 79 or 80, operably linked to a promoter of the mouse CD47 gene, wherein the mouse functionally expresses the chimeric CD47.
2. The genetically modified mouse of claim 1 , wherein the mouse does not express endogenous CD47.
3. The genetically modified mouse of claim 1 , wherein the mouse is homozygous with respect to the nucleic acid sequence encoding the chimeric CD47 protein.
4. The genetically modified mouse of claim 1 , wherein the genome of the mouse further comprises a nucleic acid sequence encoding a humanized signal regulatory protein alpha (SIRPα).
5. The genetically modified mouse of claim 1 , wherein the nucleic acid sequence encodes a chimeric CD47 protein comprising the amino acid sequence of SEQ ID NO: 74.
6. The genetically modified mouse of claim 1 , wherein the nucleic acid sequence 9 encodes a chimeric CD47 protein comprising the amino acid sequence of SEQ ID NO: 75.
7. The genetically modified mouse of claim 1 , wherein the nucleic acid sequence encodes a chimeric CD47 protein comprising the amino acid sequence of SEQ ID NO: 76.
8. The genetically modified mouse of claim 1 , wherein the nucleic acid sequence encodes a chimeric CD47 protein comprising the amino acid sequence of SEQ ID NO: 77.
9. The genetically modified mouse of claim 1 , wherein the nucleic acid sequence encodes a chimeric CD47 protein comprising the amino acid sequence of SEQ ID NO: 78.
10. The genetically modified mouse of claim 1 , wherein the nucleic acid sequence encodes a chimeric CD47 protein comprising the amino acid sequence of SEQ ID NO: 79.
11. The genetically modified mouse of claim 1 , wherein the nucleic acid sequence encodes a chimeric CD47 protein comprising the amino acid sequence of SEQ ID NO: 80.
12. A method of evaluating a drug for the treatment of cancer, the method comprising:
a) administering the drug to a genetically modified mouse that has a tumor comprising human cancer cells, and
b) determining whether the drug inhibits the tumor,
wherein the genetically modified mouse has a genome comprising a nucleic acid sequence encoding a chimeric CD47 protein comprising the amino acid sequence of SEQ ID NO: 74, 75, 76, 77, 78, 79 or 80 operably linked to a promoter of the mouse CD47 gene, and wherein the mouse functionally expresses the chimeric CD47.
13. The method of claim 12 , wherein the drug is an anti-CD47 antibody.
14. A method of preparing a genetically modified mouse, the method comprising:
1) providing a plasmid comprising a human CD47 gene fragment from exon 2 of the human CD47 gene, a 5′ homology arm, and a 3′ homology arm, wherein the 5′ and 3′ homology arms target exon 2 of a mouse CD47 gene;
2) providing a first small guide RNA (sgRNA) that targets a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8;
3) providing a second sgRNA that targets a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17;
4) modifying the genome of a mouse embryo using the plasmid of step 1), the first sgRNA of step 2), the second sgRNA of step 3), and Cas9; and
5) transplanting the embryo obtained in step 4) into a recipient mouse such that a transgenic mouse is obtained, wherein the transgenic mouse has a genome comprising a nucleic acid sequence encoding a chimeric CD47 protein comprising the amino acid sequence of SEQ ID NO: 74, 75, 76, 77, 78, 79 or 80, operably linked to a promoter of the mouse CD47 gene, wherein the mouse functionally expresses the chimeric CD47.
15. The genetically modified mouse of claim 14 , wherein the first sgRNA targets SEQ ID NO: 6 and the second sgRNA targets SEQ ID NO: 9.