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A2AR gene humanized non-human animal and construction method and application thereof

A non-human animal, construction method technology, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as genetic complexity

Inactive Publication Date: 2021-12-10
BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the differences in physiology and pathology between animals and humans, coupled with the complexity of genes (i.e., genetic factors), how to construct an "effective" humanized animal model for new drug development is still the biggest challenge ( Scheer N, Snaith M, Wolf CR, Seibler J. Generation and utility of genetically humanized mouse models, Drug Discov Today; 18(23-24):1200-11, 2013)

Method used

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  • A2AR gene humanized non-human animal and construction method and application thereof
  • A2AR gene humanized non-human animal and construction method and application thereof
  • A2AR gene humanized non-human animal and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0197] Example 1 Preparation of A2AR Gene Humanized Mice

[0198]Mouse A2AR gene (NCBI Gene ID: 11540, Primary source: MGI: 99402, UniProt ID: Q60613, located at positions 75316877 to 75334792 of chromosome 10 NC_000076.6, based on transcript NM_009630.3 and its encoded protein NP_033760.2 (SEQ ID NO: 1)) and human A2AR gene (NCBI Gene ID: 135, Primary source: HGNC: 263, UniProt ID: P29274, located at positions 24423597 to 24442360 of chromosome 22 NC_000022.11, based on transcript The comparison diagram of NM_001278497.1 and its encoded protein NP_001265426.1 (SEQ ID NO: 2)) is as follows figure 1 shown.

[0199] In order to achieve the purpose of the present invention, the gene sequence encoding human A2AR protein can be introduced into the mouse endogenous A2AR locus, so that the mouse expresses human or humanized A2AR protein. Specifically, the corresponding sequence of the mouse can be replaced by the nucleotide sequence (such as DNA sequence, cDNA sequence, etc.) The ...

Embodiment 2

[0225] Example 2 In vivo efficacy verification

[0226] A2AR humanized mice prepared by this method can be used to evaluate the efficacy of modulators targeting human A2AR. For example, A2AR humanized mouse homozygotes were subcutaneously inoculated with mouse colon cancer cells MC38, and the tumor volume grew to about 100 mm. 3 Afterwards, they were divided into control group or treatment group according to tumor volume. The treatment group randomly selected compounds X, Y, Z, etc. targeting human A2AR, and the control group was injected with an equal volume of normal saline. Regular measurement of tumor volume and weighing of mice can effectively assess the in vivo safety and efficacy of compounds by comparing changes in mouse body weight and tumor size.

Embodiment 3

[0227] Example 3 Dual-gene or multi-gene humanized mice

[0228] Using this method or the prepared A2AR gene humanized mouse, a double-gene modified or multi-gene modified mouse model can also be prepared. For example, in the aforementioned Example 1, embryonic stem cells used for blastocyst microinjection can be selected from mice containing PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, CD73 and other genetic modifications, or, alternatively, On the basis of humanized A2AR mice, the mouse ES embryonic stem cell isolation and gene recombination targeting technology can be used to obtain A2AR and other gene-modified double-gene or multi-gene-modified mouse models. The A2AR mouse homozygote or heterozygote obtained by this method can also be mated with other gene-modified homozygous or heterozygous mice, and the offspring are screened. Gene-modified double-gene or poly-gene-modified heterozygous mice, and then the heterozygotes can be mated with each other to obtain double-gene or poly...

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Abstract

The invention provides an A2AR gene humanized non-human animal, a construction method thereof, a humanized A2AR protein, a humanized A2AR gene, a targeting vector of the A2AR gene and an application of the targeting vector in the field of biological medicine. A nucleotide sequence for coding the human A2AR protein is introduced into a non-human animal genome in a homologous recombination mode; and the human or humanized A2AR protein can be normally expressed in an animal body, and the human or humanized A2AR protein can be used as an animal model for human A2AR signal mechanism research and screening of medicines for cardiovascular and cerebrovascular diseases, neurological diseases, tumors and autoimmune diseases, and has important application value in research and development of new medicines for immune targets, nerve targets and targets for preventing and treating cardiovascular and cerebrovascular diseases.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering and genetic modification, and in particular relates to a non-human animal with humanized A2AR gene, its construction method and its application in the field of biomedicine. Background technique [0002] The full name of A2AR is Adenosine A2a Receptor, also known as ADORA2A. It belongs to the G protein-coupled receptor family and is widely distributed in the body. It is expressed in the central nervous system, peripheral, heart, liver, lung, kidney and other tissues; at the cellular level, A2AR is not only expressed in γ-amino acid (GCBA) neurons, dopaminergic neurons, and parenchymal cells of peripheral tissues, but also in T cells, B cells, neutrophils, microglia, monocytes and macrophages, platelets, endothelial cells, etc. Cells, fibroblasts, NK cells and other immune cells are expressed at a high level, and participate in the regulation of various physiological and pathological proces...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/12C12N5/10C07K14/705C12Q1/02A01K67/027A61K49/00
CPCC12N15/8509C07K14/70571G01N33/5011G01N33/5008A01K67/0278A61K49/0008A01K2207/15A01K2217/072A01K2227/105A01K2267/03G01N2500/10
Inventor 沈月雷白阳姚佳维郭雅南尚诚彰
Owner BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD
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