Genetically modified non-human animal with human or chimeric OX40

Patent metadata

Field	Value
Publication number	US-11279948-B2
Application number	US-201716329269-A
Country	US
Kind code	B2
Filing date	Aug 30, 2017
Priority date	Aug 31, 2016
Publication date	Mar 22, 2022
Grant date	Mar 22, 2022

Abstract

The present disclosure relates to the genetically modified non-human animals that express a human or chimeric OX40, and methods of use thereof.

First claim

What is claimed is:

1. A method for making a genetically-modified mouse, comprising:
(1) replacing in a fertilized egg or an embryonic stem cell, at an endogenous OX40 gene locus, a nucleic acid sequence encoding a portion of the extracellular region of an endogenous OX40 protein with a nucleic acid sequence encoding a portion of an extracellular region of a human OX40 protein, wherein the replaced nucleic acid sequence comprises a portion of exon 1, exons 2-4 and a portion of exon 5 of the endogenous OX40 gene;
(2) transplanting the fertilized egg obtained in step (1) into the oviduct of a pseudopregnant female mouse or transplanting the embryonic stem cell obtained in step (1) into a blastocyst which is then transplanted into the oviduct of a pseudopregnant female mouse to obtain a child mouse; and
(3) mating the child mouse obtained in step (2) to obtain a homozygote mouse,
wherein the homozygote mouse detectably expresses a humanized OX40 protein comprising a humanized OX40 extracellular region and an endogenous OX40 cytoplasmic region on the surface of activated T cells, wherein the humanized OX40 protein comprises SEQ ID NO: 32.

2. The method of claim 1 , wherein the sequence encoding the humanized OX40 protein is operably linked to an endogenous regulatory element at the endogenous OX40 gene locus.

3. The method of claim 1 , wherein the genetically-modified mouse does not express the endogenous OX40 protein.

4. The method of claim 1 , wherein an anti-human OX40 antibody can bind to the humanized OX40 protein expressed in the mouse and increase immune response in the mouse.

5. The method of claim 1 , wherein the mouse can be used to test effectiveness of an anti-human OX40 antibody for treating cancer.

6. A method for making a genetically-modified mouse, comprising:
(1) providing a plasmid comprising a human OX40 gene fragment, flanked by a 5′ homology arm and a 3′ homology arm, wherein the 5′ and 3′ homology arms target an endogenous OX40 gene;
(2) providing two small guide RNAs (sgRNAs) that target the endogenous OX40 gene;
(3) modifying genome of a fertilized egg or an embryonic stem cell by using the plasmid of step (1), the sgRNAs of step (2), and Cas9; and
(4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudopregnant female mouse or transplanting the embryonic stem cell obtained in step (3) into a blastocyst which is then transplanted into the oviduct of a pseudopregnant female mouse to produce a child mouse that functionally expresses a humanized OX40 protein,
(5) mating the child mouse obtained in step (2) to obtain a homozygote mouse,
wherein the fertilized egg is modified by CRISPR with sgRNAs that target a 5′-terminal targeting site selected from the group consisting of SEQ ID NOS: 1-7 and a 3′-terminal targeting site selected from the group consisting of SEQ ID NOS: 8-15, and the humanized OX40 protein comprises SEQ ID NO: 32.

7. The method of claim 6 , wherein the 5′-terminal targeting site is SEQ ID NO: 1 and the 3′-terminal targeting site is SEQ ID NO: 11.

8. The method of claim 6 , wherein the sequence encoding the humanized OX40 protein is operably linked to an endogenous regulatory element at the endogenous OX40 gene locus.

9. The method of claim 6 , wherein the genetically-modified mouse does not express an endogenous OX40 protein.

10. The method of claim 6 , wherein an anti-human OX40 antibody can bind to the humanized OX40 protein expressed in the mouse and increase immune response in the mouse.

11. The method of claim 6 , wherein the mouse can be used to test effectiveness of an anti-human OX40 antibody for treating cancer.

Assignees

• Biocytogen Pharmaceuticals Beijing Co Ltd

Inventors

• Shen Yuelei
• Guo Chaoshe
• Bai Yang
• Yao Jiawei
• Zhang Meiling
• Zhou Xiaofei

Classifications

• A01K67/0278

  Knock-in vertebrates, e.g. humanised vertebrates · CPC title

• A01K2227/105

  Murine · CPC title

• A01K2267/0393

  Animal model comprising a reporter system for screening tests · CPC title

• A01K2207/15

  Humanized animals · CPC title

• C12N2800/107

  for mammalian · CPC title