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Updated: Jun 20, 2026
Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)
Published on: June 14, 2012
BAK and BAX deletion using zinc-finger nucleases yields apoptosis-resistant CHO cells
Gregory J Cost1, Yevgeniy Freyvert, Annamaria Vafiadis
1Sangamo BioSciences, Inc., Richmond, California 94804, USA.
Abstract:
Anoxic and metabolic stresses in large-scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro-apoptotic proteins and over-expression of anti-apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro-apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis. We demonstrate here the complete and permanent elimination of both the Bak and Bax proteins in combination in Chinese hamster ovary (CHO) cells using zinc-finger nuclease-mediated gene disruption. Zinc-finger nuclease cleavage of BAX and BAK followed by inaccurate DNA repair resulted in knockout of both genes. Cells lacking Bax and Bak grow normally but fail to activate caspases in response to apoptotic stimuli. When grown using scale-down systems under conditions that mimic growth in large-scale bioreactors they are significantly more resistant to apoptosis induced by starvation, staurosporine, and sodium butyrate. When grown under starvation conditions, BAX- and BAK-deleted cells produce two- to fivefold more IgG than wild-type CHO cells. Under normal growth conditions in suspension culture in shake flasks, double-knockout cultures achieve equal or higher cell densities than unmodified wild-type cultures and reach viable cell densities relevant for large-scale industrial protein production.
Insights
Eliminating pro-apoptotic Bak and Bax proteins in Chinese hamster ovary (CHO) cells prevents apoptosis. These engineered cells show enhanced IgG production and robust growth, improving biopharmaceutical manufacturing.
Area of Science:
- Biotechnology
- Cell Biology
- Molecular Biology
Background:
- Anoxic and metabolic stresses in large-scale cell culture can trigger apoptosis, impacting biopharmaceutical production.
- Current strategies to reduce apoptosis involve mRNA knockdown or protein over-expression.
- Apoptosis in cell culture is mediated by pro-apoptotic proteins Bak and Bax, where either protein's activity is sufficient to induce cell death.
Purpose of the Study:
- To investigate the complete and permanent elimination of both Bak and Bax proteins in Chinese hamster ovary (CHO) cells.
- To assess the impact of Bax and Bak gene knockout on cellular resistance to apoptosis and IgG production.
Main Methods:
- Utilized zinc-finger nuclease-mediated gene disruption to create a double knockout of BAX and BAK genes in CHO cells.
- Evaluated apoptosis resistance under various stress conditions (starvation, staurosporine, sodium butyrate) using scale-down bioreactor systems.
- Quantified IgG production in BAX- and BAK-deleted cells compared to wild-type CHO cells under starvation conditions.
Main Results:
- Successfully achieved complete and permanent elimination of both Bak and Bax proteins in CHO cells via gene disruption.
- BAX- and BAK-deleted CHO cells exhibited significantly enhanced resistance to apoptosis induced by multiple stimuli.
- These knockout cells demonstrated a two- to fivefold increase in IgG production under starvation conditions and comparable or higher cell densities under normal growth conditions.
Conclusions:
- Complete elimination of Bak and Bax proteins confers robust apoptosis resistance to CHO cells.
- BAX- and BAK-deleted CHO cells offer significant advantages for biopharmaceutical production, including increased IgG yield and improved cell viability.
- This gene-editing strategy presents a promising approach for optimizing cell culture processes in biopharmaceutical manufacturing.
