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Construction method and application of fucose transferase 8 (FUT8) function-deleted cell strain

A technology of fucosyl transferase and function loss, which is applied in the field of molecular biology and can solve problems such as reducing the production and quality of antibody drugs and growth pressure

Active Publication Date: 2021-07-02
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the common problem of these methods is: introducing a large amount of exogenous proteins (such as overexpressing GnTIII) or reducing the expression of endogenous proteins (such as knocking out FUT8), which may bring unnecessary damage to the mature engineered cells. Growth pressure, thereby reducing the yield and quality of antibody drugs produced by it

Method used

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  • Construction method and application of fucose transferase 8 (FUT8) function-deleted cell strain
  • Construction method and application of fucose transferase 8 (FUT8) function-deleted cell strain
  • Construction method and application of fucose transferase 8 (FUT8) function-deleted cell strain

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Experimental program
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Effect test

Embodiment 1

[0074] The selection of embodiment 1 mutation site

[0075] In the present invention, the inventors found that H363, R365, D368, K369, E373, Y382, D409, D453 and S469 are all active sites of FUT8, but not all mutations at all sites are operable.

[0076] The requirements for screening suitable active sites in the present invention are based on the following conditions:

[0077] 1. Since CRISPR technology relies on the positioning of PAM (protospacer adjacent motif), this requires that the designed gRNA (guide RNA) must target the sequence near PAM;

[0078] 2. The base editing tool can only effectively realize the conversion of specific bases in the active window of the gRNA-targeted sequence at a fixed distance from the PAM sequence;

[0079] 3. Due to the degeneracy of codons, nonsense mutations may occur.

[0080] Based on the screening of the above three conditions, the inventors found three operable sites R365, D368 and D453.

[0081] The FUT8 protein sequence is as fo...

Embodiment 2

[0096] Embodiment 2 constructs plasmid

[0097] 1. Synthesize a targeting sequence DNA fragment with a restriction endonuclease BbsI site as follows:

[0098] For gRNA1:

[0099] Forward: 5'-CACCGCTTTGTCAGTGCGTCTGACA-3' (SEQ ID NO.11)

[0100] Reverse: 5'-AAACTGTCAGACGCACTGACAAAGC-3' (SEQ ID NO.12)

[0101] For gRNA2:

[0102] Forward: 5'-CACCGTCAGACGCACTGACAAAGT-3' (SEQ ID NO.13)

[0103] Reverse: 5'-AAACACTTTGTCAGTGCGTCTGAC-3' (SEQ ID NO.14)

[0104] For gRNA3:

[0105] Forward: 5'-CACCTGTCAGACGCACTGACAAAG-3' (SEQ ID NO.15)

[0106] Reverse: 5'-AAACCTTTGTCAGTGCGTCTGACA-3' (SEQ ID NO.16)

[0107] For gRNA4:

[0108] Forward: 5'-CACCGGATATACACTTTTCTCTCCC-3' (SEQ ID NO.17)

[0109] Reverse: 5'-AAACGGGAGAGAAAGTGTATATCC-3' (SEQ ID NO.18)

[0110] The oligonucleotide chains of the above sequences were synthesized, and the forward and reverse strands of each group were mixed to make the final concentration 50 μM. Denatured at 94°C for 5 minutes, annealed at 50°C to obtai...

Embodiment 3

[0130] Example 3 transfection of CHO engineered cells

[0131] Co-transfect the HP180-dCas9 plasmid encoding gRNA (HP180-dCas9-gRNA1, 2, 3, 4) and the base editor plasmid (ABEmax or BE3) into CHO engineered cells, the process is as follows:

[0132] 1. Preparing Cells

[0133] at 37°C, 5% CO 2 CHO cells were cultured with CHO Fusion medium (containing 2% Glutamax and 1% penicillin and streptomycin (P / S) double antibody) in a cell culture box with a certain concentration, and after they grew to the logarithmic growth phase, count the cells Density, take the number of cells as 2×10 6 The cell solution was placed in a 15ml centrifuge tube, centrifuged at 100g for 5min, the supernatant was discarded, and then the cells were resuspended with 1.8ml Opti MEM medium (containing 2% GlutaMax and 1% P / S double antibody).

[0134] 2. Incubate the Transfection Reagent

[0135] After vortexing the FectoPRO reagent (purchased from Polyplus Transfection Company) for 5 seconds, take 2 μL i...

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Abstract

The invention discloses a construction method and application of a fucose transferase 8 (FUT8) function-deleted cell strain. According to the invention, active sites of FUT8 are screened and selected, a base editing technology is adopted, and under the conditions that foreign proteins are not introduced and the overall structure of endogenous proteins is not destroyed, the purpose that constructed mature engineering cells lose the function of fucose transferase (FUT8) is achieved by virtue of the minimal change of a gene level, so a host for normally producing an antibody drug containing fucose is converted into a host for producing an antibody drug without fucose; and thus, the yield and the quality of a produced antibody drug are improved.

Description

technical field [0001] The invention relates to the field of molecular biology, and specifically relates to a method for constructing a fucosyltransferase 8 (FUT8) function-deficient cell line and its application. Background technique [0002] Therapeutic antibody drugs are a rapidly growing field of drug development in recent years. In addition to neutralizing the effect of antigens, a considerable number of therapeutic antibody drugs (such as anti-tumor antibody drugs), after binding to target cells, through antibody-dependent cell-mediated cytotoxicity (antibody-dependent cell-mediated cytotoxicity) , ADCC) to kill target cells. Therefore, improving the ability of therapeutic antibody drugs to produce ADCC is crucial to improving the efficacy of anti-tumor antibody drugs. [0003] Studies have shown that compared with therapeutic antibodies containing α1,6-linked core fucose, ADCC of therapeutic antibodies with fucose-deleted glycosylation modifications is enhanced by t...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N5/10C12N15/85C12N15/11C12Q1/6869C12Q1/6888C12Q1/04
CPCC12N15/1137C12N15/85C12N9/1051C12N5/0682C12Q1/6869C12Q1/6888C12Y204/01068C12N2310/20C12N2510/00C12Q2535/122
Inventor 袁燕秋毛洋王圣钧王军舰何羽骐钟雨伦
Owner SUN YAT SEN UNIV
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