Preparation method and application of humanized LAG-3 (lymphocyte activation gene 3) modification animal model

A LAG-3, genetic modification technology, applied in the application field of biomedicine, can solve the problems of poor repeatability of test results, time-consuming, capital and scientific research effort, and inability to detect and evaluate drug efficacy.

Active Publication Date: 2020-06-19
BIOCYTOGEN JIANGSU CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that it generally takes more than 10 generations of backcrossing to obtain homozygotes, and backcrossing often takes 2 years, which consumes time, money and scientific research energy; on the other hand, different times of backcrossing will also cause poor repeatability of test results
[0006] Since the current model mice have the above-mentioned defects and cannot be used for in vivo drug efficacy testing and evaluation of anti-human LAG-3 antibody drugs, considering that the LAG-3 gene has great application value in the field of tumor immunotherapy, in order to make the preclinical The test is more effective, and the present invention provides a new method for establishing a humanized LAG-3 gene modified animal model, and obtains a LAG-3 gene humanized animal model

Method used

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  • Preparation method and application of humanized LAG-3 (lymphocyte activation gene 3) modification animal model
  • Preparation method and application of humanized LAG-3 (lymphocyte activation gene 3) modification animal model
  • Preparation method and application of humanized LAG-3 (lymphocyte activation gene 3) modification animal model

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Experimental program
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Effect test

Embodiment 1

[0189] Embodiment 1 LAG-3 gene humanized mouse

[0190] Mouse LAG-3 gene (NCBI Gene ID: 16768, Primary source: MGI: 106588, UniProtID: Q61790, located at positions 124904359 to 124912434 of chromosome 6 NC_000072.6, based on transcript NM_008479.2 (SEQ ID NO: 1 ) and its encoded protein NP_032505.1 (SEQ ID NO: 2)) and human LAG-3 gene (NCBIGene ID: 3902, Primary source: HGNC: 6476, UniProt ID: P18627, located at No. 6772483 of chromosome 12 NC_000012.12 To position 6778455, based on the comparison of the transcript NM_002286.5 (SEQ ID NO: 3) and its encoded protein NP_002277.4 (SEQ ID NO: 4)) as shown in figure 1 shown.

[0191]In order to achieve the purpose of the present invention, the gene sequence encoding human LAG-3 protein can be introduced into the extracellular region of the endogenous mouse LAG-3 locus, so that the mouse expresses human or humanized LAG-3 protein. Modify mouse cells with gene editing technology, replace specific mouse LAG-3 gene sequences with cer...

Embodiment 2

[0277] Example 2 In vivo efficacy verification of LAG-3 gene humanized mouse model

[0278] The tumor model constructed by using the humanized mouse prepared by the method can be used to test the drug targeting human LAG3. In one experiment, the LAG-3(s) gene humanized homozygous mice (4-6 weeks) prepared in Example 1 were subcutaneously inoculated with mouse colon cancer cells MC38 until the tumor volume was about 100mm 3 Then they were randomly divided into control group or treatment group (n=8 / group). The treatment group randomly selected an anti-human LAG-3 monoclonal antibody (AB1, obtained by immunizing mice with conventional methods, see

[0279] Janeway's Immunobiology

[0280] (9thEdition)), the dosage was 10mg / kg, and the control group was injected with normal saline. Dosing method: intraperitoneal injection, 2 times a week, 6 times in total. The tumor volume was measured twice a week, and the tumor volume of a single mouse reached 3000mm after inoculation 3 Per...

Embodiment 3

[0286] Example 3 Preparation and identification of double humanized or multiple humanized mice

[0287] Double-humanized or multiple-humanized mouse models can also be prepared by using the method or the prepared LAG-3 mice. For example, in the aforementioned Example 1, the fertilized egg cells or embryonic stem cells used in the microinjection and embryo transfer process can be selected from mice containing other genetic modifications, or can also be selected from fertilized LAG-3 humanized mice Gene editing of egg cells can further generate a double-gene or polygene-modified mouse model of LAG-3 humanization and other genetic modifications. The homozygous or heterozygous LAG-3 mice obtained by this method can also be mated with other genetically modified homozygous or heterozygous mice, and their offspring can be screened. According to the law of Mendelian inheritance, there is a certain probability of obtaining LAG-3 Humanized and other gene-modified double-gene or polygen...

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Abstract

The invention relates to a gene modification non-human animal of a humanized gene, in particular to a gene modification rodent, especially a gene modification mouse. The invention particularly relatesto a construction method for a humanized LAG-3 (lymphocyte activation gene 3) modification animal model, and application of the humanized LAG-3gene modification animal model in the field of biological medicines.

Description

technical field [0001] This application relates to the establishment method and application of a humanized genetically modified animal model, in particular, to a construction method based on a humanized LAG-3 genetically modified animal model and its application in biomedicine. Background technique [0002] Immunotherapy, which activates the immune system to attack and kill cancer cells, is an important area of ​​cancer research and has been used in clinical treatment for nearly a decade. Studies have shown that targeting the inhibitory receptors of T cells has a significant therapeutic effect and is currently the most successful field of targeted gene therapy. Among them, monoclonal antibodies targeting CTLA-4 and PD-1 / PD-L1 have achieved definite curative effects, but the response rate of patients is low and cannot fully meet the clinical needs. More drugs targeting other inhibitory receptors New drugs are or have entered into clinical research. [0003] Lymphocyte activ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N9/22C12N15/113C12N15/62A01K67/027A61K49/00
CPCC12N15/8509C12N15/907C12N9/22C07K14/70503C12N15/1138A01K67/0278A61K49/0008C12N2800/107C12N2310/20C07K2319/00A01K2207/15A01K2217/072A01K2217/075A01K2227/105A01K2267/0325C07K2319/02C12N2320/11C12N15/11A01K2267/01A01K2217/077A01K2267/0331
Inventor 沈月雷姚佳维郭朝设郭雅南白阳黄蕤赵磊张美玲
Owner BIOCYTOGEN JIANGSU CO LTD
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