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WO2023093005A1 - Site capable d'exprimer de manière stable une protéine dans le gène de cellule cho nw_003613756,1 et son utilisation - Google Patents

Site capable d'exprimer de manière stable une protéine dans le gène de cellule cho nw_003613756,1 et son utilisation Download PDF

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WO2023093005A1
WO2023093005A1 PCT/CN2022/099460 CN2022099460W WO2023093005A1 WO 2023093005 A1 WO2023093005 A1 WO 2023093005A1 CN 2022099460 W CN2022099460 W CN 2022099460W WO 2023093005 A1 WO2023093005 A1 WO 2023093005A1
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protein
site
gene
expression
cho
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陈蕴
金坚
丁学峰
瞿丽丽
李华钟
蔡燕飞
朱景宇
杨兆琪
鲁晨
王恩铭
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/60—Vectors containing traps for, e.g. exons, promoters

Definitions

  • the invention relates to a site for stably expressing protein in CHO cell gene NW_003613756.1 and its application, belonging to the field of gene technology.
  • CHO cells Chinese hamster ovary (CHO) cells were established in Dr. Theodore T. Puck's laboratory in 1957. They are immortalized non-secreting cells that rarely secrete endogenous proteins; Post-translational modifications closer to human natural proteins, less susceptible to human virus infection, and large-scale culture in serum-free media with defined chemical components, etc., are widely used in the field of biopharmaceuticals, and more than 70% of proteins are produced drug.
  • mammalian cells CHO cells have a long culture cycle and high culture costs.
  • the demand for recombinant products such as monoclonal antibodies continues to increase. The continuous growth of demand means that the specific productivity still needs to be optimized.
  • the expression level can be increased by increasing the copy number of the gene, developing a new strong promoter, finding a suitable enhancer, etc.
  • the expression level of most CHO cells is unstable during long-term culture, which directly affects the supervision of the product by the regulatory authorities. Approval and listing issues. Therefore, it is very important to construct a CHO expression strain that stably and highly expresses the target gene for the development and marketing of protein drugs.
  • Random integration is the most mature traditional method for constructing protein expression systems, but obtaining stable high-expression cell lines through random integration requires multiple screenings, which is time-consuming and expensive.
  • the loss of expression stability of the cells in the later stage of culture is completely unpredictable: this instability may not occur at all; it may also gradually appear after numerous cell divisions; It is also possible that apparent instability occurs after only a few generations of cell division.
  • the stability issue will not only affect the time to market of the product, but will also conflict with drug regulatory management.
  • the information of random integration sites is not clear, and the site effect of exogenous gene integration will also lead to a significant decrease in the expression level of the target gene. According to existing literature reports, this instability of recombinant CHO cell lines occurs in all recombinant CHO cell lines, making the problem of unstable expression an extremely common problem.
  • the present invention provides a site for stably expressing proteins in the genome of CHO cells.
  • the screening process and time in the cell construction process reduce the cost of research and development.
  • the first object of the present invention is to provide a stable protein expression site in the CHO cell genome, and the stable protein expression site is located within the 101274-101574th base of the CHO cell gene NW_003613756.1.
  • nucleotide sequence of bases 101274-101574 of the CHO cell gene NW_003613756.1 is shown in SEQ ID NO.1.
  • the site for stably expressing the protein can be recognized by the 5'NNNNNNNNNNNNNNNNNNNGG 3' sequence of the CRISPR/Cas9 technology.
  • this site is located at the intron of the cep170 gene on the CHO cell genome NW_003613756.1.
  • the 5'NNNNNNNNNNNNNNNNNNNNNNNNNNNNNGG 3' sequence in the embodiment of the present invention selects the following 9 sequences: 5'-TTCTTTTATGACATTCCCACTGG-3', 5'-ATATGTTCAAAGTTCACCAGTGG-3', 5'-ACTTTGAACATATTTCAGTGGGG-3', 5'-GAGATTTAAAAGTCATATGCAGG-3', 5'-GTTGGAAAAAAAATAAAATTTGG-3', 5'-CAACTTTTTTTATTTGAATTAGG-3', 5'-TTTTTATTTGAATTAGGAACAGG-3', 5'-ATGGGTAGGAGAAGGGAAATGGG-3', 5'-GCAGGTAGGGGAGGATGGGTAGG- 3'.
  • the above nine groups of sequences are distributed within the 101274-101574th base of CHO cell gene NW_003613756.1, covering most of the upstream and downstream sequences within 300 bases of the present invention, indicating that the 300 bases of the present invention Both can be used as sites for stably expressing proteins.
  • the present invention does not limit the above nine groups of sequences.
  • the above nine groups of sequences are only used as the preferred technical means to introduce the coding gene of the target protein into the stable expression site.
  • the purpose of stably expressing the protein of the present invention can be achieved.
  • the protein is a protein with a molecular weight less than 160KDa.
  • the protein is one of polypeptides, functional proteins, antibodies, and fusion proteins.
  • the second object of the present invention is to provide the application of the stable protein expression sites in the CHO cell genome in CHO cells to stably express foreign proteins.
  • the application specifically involves constructing a gene encoding an exogenous protein or polypeptide at the site of stably expressing the protein in the CHO cell gene NW_003613756.1.
  • the third object of the present invention is to provide an expression vector for expressing protein in CHO cells, the coding gene of the protein is located between the 5' homology arm and the 3' homology arm of the expression vector region, the 5' homology arm and the 3' homology arm are sequences with a length of 600 bp upstream and downstream of the stable protein expression site respectively.
  • the expression vector is a vector suitable for expression in CHO cells.
  • the expression vector also includes a promoter sequence located upstream of the gene encoding the protein, and the promoter controls the expression of the protein.
  • the promoters are: CMV (strong mammalian expression promoter derived from human cytomegalovirus), EF-1a (strong mammalian expression promoter derived from human elongation factor 1 ⁇ ), SV40 (simian vacuolar virus 40 derived mammalian expression promoter), PGK1 (mammalian promoter derived from phosphoglycerate kinase gene), UBC (mammalian promoter derived from human ubiquitin C gene), human beta actin ( ⁇ -actin Gene-derived mammalian promoter), CAG (strong hybrid mammalian promoter) and the like.
  • an expression vector for expressing a protein in CHO cells comprising the following steps: inserting the gene encoding the protein into the region between the 5'arm and the 3'arm of the plasmid, making the protein encoding The gene is located downstream of the promoter and is regulated by the promoter to obtain the expression vector for expressing the protein in CHO cells.
  • the fourth object of the present invention is to provide a CHO recombinant cell with site-specific integration and stable expression of protein.
  • the CHO recombinant cell is obtained by using the expression vector for expressing protein in CHO cells and the sgRNA corresponding to the target sequence. Plasmids and Cas9 plasmids were transformed into CHO cells.
  • the target sequence is preferably 5'-TTCTTTTATGACATTCCCACTGG-3', 5'-ATATGTTCAAAGTTCACCAGTGG-3', 5'-ACTTTGAACATATTTCAGTGGGG-3', 5'-GAGATTTAAAAGTCATATGCAGG-3', 5'-GTTGGAAAAAAAATAAAATTTGG-3', 5'-CAACTTTTTTTATTTGAATTAGG-3', 5'-TTTTTTATTTGAATTAGGAACAGG-3', 5'-ATGGGTAGGAGAAGGGAAATGGG-3' or 5'-GCAGGTAGGGGAGGATGGGTAGG-3'.
  • a method for constructing CHO recombinant cells comprising the steps of:
  • the plasmids are respectively the expression vector for expressing protein in CHO cells, the sgRNA plasmid and the Cas9 plasmid corresponding to the target sequence;
  • the stable expression site obtained by the present invention is located within 150 bp upstream and downstream of the 101424th base of the CHO cell gene NW_003613756.1, that is, the 101274-101574th base, which can integrate foreign protein genes and perform stable expression.
  • the present invention integrates the target gene into the stable expression region through fixed-point integration, which solves the problem of unclear integration sites caused by random integration; the present invention uses the 101424th base of the stable expression site NW_003613756.1 in the CHO genome
  • the site-specific integration of exogenous genes within the range of 101274-101574 bases upstream and downstream of the base overcomes the expression instability caused by the position effect and the repeated and tedious cell line screening process, reducing the original screening time of 6-12 months to 1-3 months, effectively shortening the R&D time for constructing stable expression cell lines and reducing costs.
  • Fig. 1 is a schematic diagram of fixed-point integration of the present invention
  • Figure 2 shows the expression of EGFP at different passages in cells constructed with different target sequences.
  • Figure 3 shows the expression of IL2-HSA at different passages by cells constructed with different target sequences.
  • the method for measuring the average fluorescence intensity of cells culture the cells until the confluence reaches about 90%, digest the cells with 0.25% trypsin, and terminate the digestion with a complete medium equal to the amount of trypsin, and collect the cells in a sterile centrifuge tube at 1000rpm/ Centrifuge for 5 min, discard the supernatant and resuspend with PBS, pass through a cell strainer and collect in a flow loading tube, and use a blank CHO-K1 cell as a control to analyze the fluorescence intensity of the cells with a flow cytometer.
  • the CHO-K1-1b7 cells expressing the Zsgreen1 reporter gene screened by high-throughput flow cytometry were cultured in an adherent culture until they were in good condition, and the CHO cells at this time were regarded as the 0th generation, and then cultured continuously for 20 days.
  • the expression of Zsgreen1 protein in cells at passages 0, 10, and 20 was observed under an inverted fluorescence microscope, and the average fluorescence intensity of cells at passages 0, 10, and 20 was detected by BD flow cytometry.
  • CHO-K1-1b7 cells can still express 100% Zsgreen1 protein in the adherent culture state, and Zsgreen1 protein between different passages
  • the expression levels are basically the same, and there is a strong green fluorescent signal.
  • the CHO-K1-1b7 cells that passed the verification of anchorage stability were subjected to suspension acclimation, and the CHO-K1-1b7 cells that were successfully acclimatized in suspension were continuously passaged for 60 passages, and the success of acclimation in suspension was regarded as the 0th passage.
  • the expression of Zsgreen1 protein in the cells at passages 0, 10, 20, 30, 40, 50, and 60 was observed under an inverted fluorescence microscope, and at the same time, the cells at passages 0, 10, 20, 30, 40, and 50 were detected by flow cytometry. Mean fluorescence intensity of cells at passage 60.
  • CHO-K1-1b7 cells could still express 100% Zsgreen1 protein in suspension culture state, and Zsgreen1 protein expression between different passages The levels are basically the same, and there is a strong green fluorescent signal. It shows that CHO-K1-1b7 cells can stably express the Zsgreen1 reporter gene, and at the same time, it shows that the integrated site of the lentivirus carrying the Zsgreen1 reporter gene is a stable expression site.
  • Collect CHO-K1-1b7 cells use the DNA extraction kit to extract the genome, use DraI, SspI, and HpaI three restriction endonucleases to digest the genome at 37°C for 16-18h, and the digestion system is as follows:
  • the digested product was purified and recovered using a PCR purification kit, and the chromosome walking adapter GenomeWalker Adaptor was connected to both ends of the purified digested fragment.
  • the connection system was as follows:
  • a round of PCR reaction system is as follows:
  • the second round of PCR reaction system is as follows:
  • the second round of PCR products were subjected to agarose gel electrophoresis and gel recovery sequencing, and the sequencing was performed according to the kit Lenti-X Integration Site Analysis Kit (Clontech: 631263).
  • the lentivirus integration site information can be obtained by comparing the sequencing results with the CHO cell genome on NCBI.
  • the lentivirus integration site in CHO-K1-1b7 cells is located at base 101424 of the CHO cell genome NW_003613756.1.
  • Sequences in which the predicted editing efficiency was higher than 0.56 were selected as target sequences.
  • Target sequence 5'-TTCTTTTATGACATTCCCACTGG-3'(SEQ ID NO.2), score 0.63
  • Target sequence 5'-ATATGTTCAAAGTTCACCAGTGG-3'(SEQ ID NO.3), score 0.79
  • Target sequence 5'-ACTTTGAACATATTTCAGTGGGG-3'(SEQ ID NO.4), score 0.75
  • Target sequence 5'-GAGATTTAAAAGTCATATGCAGG-3'(SEQ ID NO.5), score 0.57
  • Target sequence 5'-CAACTTTTTTTATTTGAATTAGG-3'(SEQ ID NO.7), score 0.69
  • Target sequence 5'-TTTTTATTTGAATTAGGAACAGG-3'(SEQ ID NO.8), score 0.56
  • Target sequence 5'-GCAGGTAGGGGAGGATGGGTAGG-3'(SEQ ID NO.10), score 0.62
  • CRISPR/Cas9-mediated genome editing technology Using CRISPR/Cas9-mediated genome editing technology and homologous recombination, the green fluorescent protein gene (EGFP, 26.7KDa) will be integrated at the target site.
  • CRISPR/Cas9-mediated homologous recombination technology needs to construct sgRNA plasmid and Donor Plasmid, the construction process is as follows:
  • the PSK-u6-gRNA plasmid is digested with BBsI enzyme, and the vector after digestion is gel recovered;
  • Donor plasma The information of Donor plasma is shown in Figure 1, which was obtained by transforming the existing plasmid vector expressing EGFP. 5'arm and 3'arm are the upstream and downstream homology arms of the target sites recognized by each pair of sgRNAs, with a length of 600bp, and GOI is the integrated target gene.
  • the EGFP sequence of the target gene is carried by the original plasmid.
  • interferon-beta-human serum albumin fusion protein IL2-HSA, 82KDa
  • CRISPR/Cas9-mediated homologous recombination technology needs to construct sgRNA plasmid and Donor Plasmid, the construction process is as follows:
  • sgRNA-R1 5'TAAAACGTGGGAATGTCATAAAAGAA 3'
  • sgRNA-R6 5'TAAAACAATTCAAATAAAAAAAAGTTG 3'
  • the PSK-u6-gRNA plasmid is digested with BBsI enzyme, and the vector after digestion is gel recovered;
  • Donor plasma information is shown in Figure 1. 5'arm and 3'arm are the upstream and downstream homology arms of the target site, respectively, with a length of 600bp, and GOI is the integrated target gene.
  • Detection method the cell lines obtained in Example 4 were continuously passaged for 60 passages, and the cells were collected for 15 passages per passage, and the cell fluorescence was detected with a flow cytometer and the intensity was detected, as shown in Figure 2.
  • the fluctuation range of green fluorescence intensity between the 0th passage and the 60th passage was not more than 30%.
  • Urinary Microalbumin Assay Kit Detects the Expression of IL2-HSA in the Cell Line Constructed in Example 5
  • Detection method The cell lines obtained in Example 5 were continuously passaged for 60 generations under serum-free culture conditions, and the cell fermentation supernatant of this generation was collected every 15 generations, and the urinary microalbumin assay kit was used to detect HSA content n mg/L, according to The expression level of IL2-HSA was converted, and the analysis of the detection results showed that the cells constructed according to different target sequences in Example 5 had a stable ability to express IL2-HSA at different passages, as shown in FIG. 3 .
  • the genome fixed-point editing technology mediated by CRISPR/Cas9 is mainly used for fixed-point integration. Therefore, the target sequence is mainly designed for the upstream and downstream sequences within 300 bp, and the target sequence obtained by screening in Example 3 of the present invention
  • the 9 sets of target sequences cover most of the upstream and downstream sequences within 300 bp of the present invention, and the 101274-101574 base range in the CHO cell gene NW_003613756.1 of the present invention can successfully construct a site-specific integrated stable expression cell line, And all can stably express the target protein.

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Abstract

L'invention concerne un site capable d'exprimer de manière stable une protéine dans un gène de cellule CHO NW_003613756,1 et son utilisation. Le site d'expression stable obtenu se trouve sur des bases situées à moins de 150 pb en amont et en aval de la base 101424 du gène NW_003613756.1 des cellules CHO, c'est-à-dire les bases 101274 à 101574. Un gène de protéine exogène peut être intégré et exprimé de manière stable. Un gène de destination est intégré à ladite zone d'expression stable au moyen d'une intégration spécifique au site, et le problème d'un site d'intégration peu clair causé par une intégration aléatoire est résolu. L'intégration spécifique d'un gène exogène dans la plage des bases 101274 à 101574 en amont et en aval de la base 101424 d'un site d'expression stable NW_003613756.1 dans un génome CHO permet de surmonter l'expression instable causée par l'effet de position et d'éviter le processus répété et fastidieux de criblage des souches cellulaires.
PCT/CN2022/099460 2021-11-23 2022-06-17 Site capable d'exprimer de manière stable une protéine dans le gène de cellule cho nw_003613756,1 et son utilisation Ceased WO2023093005A1 (fr)

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CN113969283B (zh) * 2021-11-23 2022-07-12 江南大学 一种cho细胞基因nw_003613756.1内稳定表达蛋白质的位点及其应用

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