Inactivation of GDP-fucose transporter gene (Slc35c1) in CHO cells by ZFNs, TALENs and CRISPR-Cas9 for production of fucose-free antibodies.

Kah Fai Chan, Wahyu Shahreel, Corrine Wan, Gavin Teo, Noor Hayati, Shi Jie Tay, Wen Han Tong, Yuansheng Yang, Pauline M. Rudd, Peiqing Zhang, Zhiwei Song
Source: Biotechnology Journal
Publication Date: (2015)
Issue: -: -
Research Area:
Gene Expression
Cells used in publication:
Species: hamster
Tissue Origin: ovarian
4D-Nucleofector® X-Unit
Anti-Her2 (trastuzumab) antibody-producing CHO DG44 cells (CHO-HER) were transfected with ZFNs or TALENs targeting GDP-fucose transporter using 4D Nucleofector (Lonza, Cologne, Germany) system according to manufacturer’s protocol. Briefly, 1.5 million cells were harvested from exponentially growing suspension culture and washed with Dulbecco’s phosphate buffered saline (DPBS) (Life Technologies). The cells were suspended in 100 µl of SG cell line Nucleofector solution containing 5 µg of plasmid DNA (2.5 µg for each ZFN or TALEN) in a Nucleocuvette. Electroporation was then carried out using a Lonza 4D-Nucleofector™ X unit. Following transfection, cells were incubated in suspension in a 3 ml CD CHO : HyClone PF CHO medium in a 6-well plate overnight before they were transferred into a 25 ml fresh medium in a 125-ml shake flask for further culture. Cells were then harvested for AAL-staining and FACS at 7-10 days post-transfection. For CRISPR-Cas9 modification, 5 million suspension CHO-K1 cells were electroporated using 4D-Nucleofector system with 5 µg of CRISPR-Cas9 plasmids in 100 µl of SG cell line Nucleofector solution. Following transfection, cells were recovered in growth medium in a suspension 6-well plate overnight before being scaled up to shake flask culture. Cells were then harvested for AAL-staining and FACS at 7-10 days post-transfection.
Removal of core fucose from N-glycans attached to human IgG1 significantly enhances its affinity for the receptor Fc?RIII and thereby dramatically improves its antibody-dependent cellular cytotoxicity (ADCC) activity. While previous works have shown that inactivation of fucosyltransferase 8 (FUT8) results in mutants capable of producing fucose-free antibodies, we report here the use of genome editing techniques, namely ZFNs, TALENs and the CRISPR-Cas9, to inactivate the GDP-fucose transporter (SLC35C1) in CHO cells. A FACS approach coupled with a fucose-specific lectin was developed to rapidly isolate SLC35C1-deficient cells. Mass spectrometry analysis showed that both EPO-Fc produced in mutants arising from CHO-K1 and anti-Her2 antibody produced in mutants arising from a pre-existing antibody-producing CHO-HER line lacked core fucose. Lack of functional SLC35C1 in these cells does not affect cell growth or antibody productivity. Our data demonstrate that inactivating Slc35c1 gene represents an alternative approach to generate CHO cells for production of fucose-free antibodies