Mouse having a humanized cluster of differentiation 47 gene

Patent metadata

Field	Value
Publication number	US-12389889-B2
Application number	US-202418413219-A
Country	US
Kind code	B2
Filing date	Jan 16, 2024
Priority date	Dec 5, 2014
Publication date	Aug 19, 2025
Grant date	Aug 19, 2025

Abstract

Non-human animals, and methods and compositions for making and using the same, are provided, wherein said non-human animals comprise a humanization of an endogenous cluster of differentiation (CD) gene, in particular a humanization of a CD47 gene. Said non-human animals may be described, in some embodiments, as having a genetic modification to an endogenous CD47 gene so that said non-human animals express a CD47 polypeptide that includes a human portion and a non-human portion (e.g., a murine portion).

First claim

We claim:

1. A targeting nucleic acid vector, comprising
a 5′ homology arm comprising a genomic sequence upstream of exon 2 of a mouse CD47 gene,
a genomic DNA fragment comprising exons 2-7 of a human CD47 gene,
a drug selection cassette, and
a 3′ homology arm comprising a genomic sequence downstream of exon 7 of the mouse CD47 gene,
wherein the genomic fragment comprising exons 2-7 of the human CD47 gene encodes a polypeptide comprising an amino acid sequence having at least 98% identity with the amino acid sequence as set forth in amino acids 16-292 of SEQ ID NO: 10,
wherein the genomic sequence upstream of exon 2 of the mouse CD47 gene comprises exon 1 of the mouse CD47 gene,
wherein the genomic sequence downstream of exon 7 of the mouse CD47 gene comprises the exons downstream of exon 7 of the mouse CD47 gene, and
wherein the 5′ and 3′ homology arms mediate integration of the genomic fragment comprising exons 2-7 of the human CD47 gene into a mouse CD47 locus to form a humanized CD47 gene.

2. The targeting nucleic acid vector of claim 1 , wherein the genomic fragment comprising exons 2-7 of the human CD47 gene encodes a polypeptide comprising the amino acid sequence as set forth in amino acids 16-292 of SEQ ID NO: 10.

3. The targeting nucleic acid vector of claim 1 , wherein the drug selection cassette is a self-deleting drug selection cassette.

4. A method of making a genetically modified mouse embryonic stem (ES) cell, comprising:
introducing the targeting nucleic acid vector of claim 1 into mouse ES cells, and
selecting a mouse ES cell in which the genomic fragment comprising exons 2-7 of the human CD47 gene has integrated into the mouse CD47 locus and replaced exons 2-7 of the endogenous mouse CD47 gene,
thereby obtaining the genetically modified mouse ES cell in which the genomic fragment comprising exons 2-7 of the human CD47 gene has integrated into the mouse CD47 locus and replaced exons 2-7 of the endogenous mouse CD47 gene.

5. The method of claim 4 , wherein the genomic fragment comprising exons 2-7 of the human CD47 gene encodes a polypeptide comprising the amino acid sequence as set forth in amino acids 16-292 of SEQ ID NO: 10.

6. The method of claim 4 , wherein the drug selection cassette is a self-deleting drug selection cassette.

7. A method of making a genetically modified mouse, comprising:
introducing the targeting nucleic acid vector of claim 1 into mouse ES cells,
selecting a genetically modified mouse ES cell in which the genomic fragment comprising exons 2-7 of the human CD47 gene has integrated into the mouse CD47 locus and replaced exons 2-7 of the endogenous mouse CD47 gene; and
producing the genetically modified mouse from the selected genetically modified mouse ES cell, wherein the genome of the genetically modified mouse comprises a replacement of a genomic fragment comprising exons 2-7 of a mouse CD47 gene at an endogenous mouse CD47 locus with the human genomic fragment comprising exons 2-7 of the human CD47 gene to form the humanized CD47 gene, wherein the exons of the humanized CD47 gene consist of exon 1 of the mouse CD47 gene, exons 2-7 of the human CD47 gene, and the remaining exons downstream of exon 7 of the mouse CD47 gene, wherein the humanized CD47 gene is under control of the endogenous mouse CD47 promoter at the endogenous mouse CD47 locus, and wherein the mouse expresses a humanized CD47 protein encoded by the humanized CD47 gene.

8. The method of claim 7 , wherein the genomic fragment comprising exons 2-7 of the human CD47 gene encodes a polypeptide comprising the amino acid sequence as set forth in amino acids 16-292 of SEQ ID NO: 10.

9. The method of claim 7 , wherein the drug selection cassette is a self-deleting drug selection cassette.

Assignees

• Regeneron Pharma

Inventors

• Gurer Cagan
• Ioffe Ella
• Mujica Alexander
• Thurston Gavin

Classifications

• A01K2207/15

  Humanized animals · CPC title

• A01K2207/12

  Animals modified by administration of exogenous cells · CPC title

• C12N15/8509

  for producing genetically modified animals, e.g. transgenic · CPC title

• C07K14/70503

  Immunoglobulin superfamily · CPC title

• C07K14/705

  Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} · CPC title