A kind of construction method of fcrn gene humanized animal model

A humanized and genetic technology, applied in the field of animal genetic engineering and genetic modification, can solve the problems of clinical differences in antibody half-life, achieve the effect of increasing the content of drug evaluation and solving clinical differences

Active Publication Date: 2022-06-28
广东药康生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This humanized mouse is used to study the drug PK of human Fc fusion protein, solving the problem of clinical differences in the evaluation of antibody half-life

Method used

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  • A kind of construction method of fcrn gene humanized animal model
  • A kind of construction method of fcrn gene humanized animal model
  • A kind of construction method of fcrn gene humanized animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: FcRn humanization targeting homologous DNA donor construction

[0042] Homologous recombination was used to replace the coding region of the extracellular region of the human FCRN gene with the coding region of the extracellular region of the C57BL / 6 mouse FcRn gene, and the UTR sequence of the C57BL / 6 mouse was retained to construct a targeting vector containing the C57BL / 6 background. Sequence shown in SEQ ID NO: 1: wherein 1-782bp is the C57BL / 6 mouse-derived 5'UTR coding region and homology arm sequence, including exon1, intron1, and part of exon2 in turn; 783-2838bp is the human FCRN gene part, including part in turn Sequences of Exon2, Intron2, Exon3, Intron3, Exon4, Exon5, Intron5, and part of Exon6; the 2839-3727bp part is the C57BL / 6 mouse FcRn region, 3'UTR and homology arm sequences, including parts of exon 6, intron6, and exon7 in turn .

[0043] SEQ ID NO: 1

[0044]ggctggacagaggcccaaggggtgtgcttaggagctagtgggtggagttggatgccctcagagttctccagtcctaact...

Embodiment 2

[0045] Example 2 FcRn humanized mouse sgRNA design

[0046] Determine the approximate region of sgRNA based on the replacement fragments. Use the sgRNA evaluation software to select the sequences with the lowest off-target rate for the target fragment, and the following sequences are used as the candidate sgRNAs. Design and synthesis recognize the 5' end target site and 3' end target site, and construct the sgRNA expression vector. The sgRNA recognition sites at both ends are located on the first and fifth exons of the mouse Tnfrsf18 gene, respectively.

[0047] sgRNA transcription preparation method: using PrimerStar or PrimerStar Max system, sgRNA-F and sgRNA-R as primers, and the puc57-sgRNA plasmid (1:30 dilution) with the correct sequencing as the template to carry out PCR, the PCR product is purified, and the sgRNA transcription preparation template is prepared . Transcription of sgRNA was performed using the T7-ShortScript in vitro transcription kit (AM1354).

[004...

Embodiment 3F

[0052] Example 3 Establishment of FcRn humanized mouse model

[0053] The sgRNA designed in Example 2 and the FcRn humanized targeting homologous DNA donor designed in Example 1 were mixed with Cas9 protein, injected into 0.5-day-old mouse fertilized eggs, and transplanted into 0.5-day-old pseudopregnant female mice. , After the mice were born, the target mice (F0) were screened out by genetic identification.

[0054] Genotype identification of the F0 generation of humanized mice: The obtained mouse tail genomic DNA of F0 mice was identified by PCR on both ends of the target after hitting the target, primers 710181-FCRN-5tF1 / 710181-hFCRN-5tR1 respectively It is located outside the 5' homology arm and within the human fragment of the targeting vector. If the pair of primers is amplified to produce a PCR product, it means that the target vector has been effectively inserted at the 5' of the mouse genome; 710181-hFCRN-3tF1 / 710181-FCRN- 3tR1 is located in the human segment of the t...

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Abstract

The invention provides a method for constructing an FcRn gene humanized animal model. The humanized animal can be used to study the drug PK of human Fc fusion protein, and can also be used for the half-life difference between different antibodies and different mutants of the same antibody Evaluation, or evaluation of human Fc fusion protein maximum half-life data, estimates the minimum dose to achieve therapeutic serum concentrations, thereby reducing the need for potentially dangerous dose-escalation therapy during clinical trials.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering and genetic modification, and in particular relates to a method for constructing an FcRn gene-modified humanized animal model based on CRISPR / Cas9 technology and its application in biomedicine. Background technique [0002] Therapeutic monoclonal antibodies (mAbs) have emerged as an important class of drugs, with more than 40 antibody-based therapies approved by the U.S. FDA, covering a range of diseases including cancer, autoimmune diseases, infectious diseases, neurodegenerative diseases, macular degeneration, osteoporosis and transplant rejection, and more in clinical trials. Since the breakthrough in hybridoma technology and monoclonal antibody (mAb) development in 1975, antibody engineering scholars have rapidly advanced mAbs from the first generation of highly immunogenic murine and chimeric antibodies to better tolerated Humanized and fully human mAbs. More recently, various anti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/85C12N15/90C12N15/55C12N15/113A01K67/027A61K49/00
CPCC07K14/70535C12N15/8509C12N15/907C12N9/22C12N15/1138A01K67/0278A61K49/0008C12N2800/107C12N2310/20A01K2207/15A01K2217/072A01K2227/105A01K2267/03
Inventor 琚存祥王韬张宇曦蒋余亭陈嘉怡蓝婷英
Owner 广东药康生物科技有限公司
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