# US 11,109,578 B2 · Humanized IL-4 and IL-4Ra animals

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- Number: US 11,109,578 B2
- Document ID: US11109578B2
- Status: Granted
- Kind: B2
- Application number: 16/594,537
- Filed: 2019-10-07
- Published: 2020-01-23
- Granted: 2021-09-07
- Earliest claimed: 2014-05-07
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Li-Hsien Wang; Yingzi Xue; Andrew J. Murphy; Sean Stevens
- Web page: https://patentgrep.com/patent/US11109578B2

## CPC

- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- C07K 14/5406: {IL-4}
- C07K 14/7155: {for interleukins [IL]}
- A01K 2207/15: Humanized animals
- A01K 2217/072: maintaining or altering function, i.e. knock in
- A01K 2217/20: Animal model comprising regulated expression system
- A01K 2227/105: Murine
- A01K 2267/01: Animal expressing industrially exogenous proteins
- A01K 2267/0368: Animal model for inflammation
- A01K 2267/0393: Animal model comprising a reporter system for screening tests
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Non-human animals comprising a human or humanized IL-4 and/or IL-4Rα nucleic acid sequence are provided. Non-human animals that comprise a replacement of the endogenous IL-4 gene and/or IL-4Rα gene with a human IL-4 gene and/or IL-4Rα gene in whole or in part, and methods for making and using the non-human animals, are described. Non-human animals comprising a human or humanized IL-4 gene under control of non-human IL-4 regulatory elements is also provided, including non-human animals that have a replacement of non-human IL-4-encoding sequence with human IL-4-encoding sequence at an endogenous non-human IL-4 locus. Non-human animals comprising a human or humanized IL-4Rα gene under control of non-human IL-4Rα regulatory elements is also provided, including non-human animals that have a replacement of non-human IL-4Rα-encoding sequence with human or humanized IL-4Rα-encoding sequence at an endogenous non-human C IL-4Rα locus. Non-human animals comprising human or humanized IL-4 gene and/or IL-4Rα sequences, wherein the non-human animals are rodents, e.g., mice or rats, are provided.

## Claims

### Claim 1

A mouse embryonic stem (ES) cell, comprising a replacement of a genomic DNA of a mouse IL-4 gene at an endogenous mouse IL-4 locus with a nucleic acid sequence of a human IL-4 gene to form a modified IL-4 gene, wherein the modified IL-4 gene encodes a human IL-4 protein and comprises exon 1 starting from the ATG initiation codon through exon 4 of the human IL-4 gene, and wherein expression of the modified IL-4 gene is under control of mouse regulatory elements at the endogenous mouse IL-4 locus.

### Claim 2 (depends on 1)

The mouse ES cell of claim 1, wherein said replacement is a replacement of a genomic DNA comprising exon 1 starting from the ATG initiation codon through exon 4 of the endogenous mouse IL-4 gene with a genomic DNA comprising exon 1 starting from the ATG initiation codon through exon 4 of the human IL-4 gene.

### Claim 3 (depends on 1)

The mouse ES cell of claim 1, wherein the mouse ES cell is incapable of expressing a mouse IL-4 protein.

### Claim 4 (depends on 1)

The mouse ES of cell claim 1, wherein the mouse ES cell comprises a nucleic acid encoding a humanized IL-4Rα protein, and wherein the humanized IL-4Rα protein comprises the ectodomain of a human IL-4Rα protein.

### Claim 5 (depends on 4)

The mouse ES cell of claim 4, wherein the humanized IL-4Rα protein comprises the transmembrane domain and the cytoplasmic domain of a mouse IL-4Rα protein.

### Claim 6 (depends on 4)

The mouse ES cell of claim 4, wherein the humanized IL-4Rα protein is encoded by a humanized IL-4Rα gene formed by a replacement of a mouse IL-4Rα ectodomain-coding sequence at an endogenous mouse IL-4Rα locus with a human IL-4Rα ectodomain-coding sequence, and wherein the humanized IL-4Rα gene is under control of the endogenous mouse IL-4Rα regulatory elements at the endogenous mouse IL-4Rα locus.

### Claim 7 (depends on 1)

A mouse embryo, comprising the mouse ES cell of claim 1.

### Claim 8 (depends on 7)

The mouse embryo of claim 7, wherein said replacement is a replacement of a genomic DNA comprising exon 1 starting from the ATG initiation codon through exon 4 of the endogenous mouse IL-4 gene with a genomic DNA comprising exon 1 starting from the ATG initiation codon through exon 4 of the human IL-4 gene.

### Claim 9 (depends on 7)

The mouse embryo of claim 7, wherein the mouse ES cell is incapable of expressing a mouse IL-4 protein.

### Claim 10 (depends on 7)

The mouse embryo of claim 7, wherein the mouse ES cell comprises a nucleic acid encoding a humanized IL-4Rα protein, and wherein the humanized IL-4Rα protein comprises the ectodomain of a human IL-4Rα protein.

### Claim 11 (depends on 10)

The mouse embryo of claim 10, wherein the humanized IL-4Rα protein comprises the transmembrane domain and the cytoplasmic domain of a mouse IL-4Rα protein.

### Claim 12 (depends on 10)

The mouse embryo of claim 10, wherein the humanized IL-4Rα protein is encoded by a humanized IL-4Rα gene formed by a replacement of a mouse IL-4Rα ectodomain-coding sequence at an endogenous mouse IL-4Rα locus with a human IL-4Rα ectodomain-coding sequence, and wherein the humanized IL-4Rα gene is under control of the endogenous mouse IL-4Rα regulatory elements at the endogenous mouse IL-4Rα locus.

### Claim 13

A method of making a mouse, comprising:
(a) modifying a mouse ES cell by replacing a genomic DNA of a mouse IL-4 gene at an endogenous mouse IL-4 locus with a genomic DNA of a human IL-4 gene to form a modified IL-4 gene, wherein the genomic DNA of the human IL-4 gene comprises exon 1 starting from the ATG initiation codon through exon 4 of the human IL-4 gene, and wherein the modified IL-4 gene is operably linked to the mouse IL-4 promoter at the endogenous mouse IL-4 locus;
(b) obtaining a modified mouse ES cell from step (a); and
(c) making a mouse using the modified mouse ES cell obtained in step (b).

### Claim 14 (depends on 13)

The method of claim 13, wherein the genomic DNA of an endogenous mouse IL-4 gene being replaced comprises exon 1 starting from the ATG initiation codon through exon 4 of the mouse IL-4 gene.