BAK and BAX deletion using zinc‐finger nucleases yields apoptosis‐resistant CHO cells

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.

Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases

“…Alternately, the small deletion of one base may have happened by coincidence or by a biased DNA repair process at this locus. The type of mutations recovered are similar to previously observed ZFN-created alleles and include deletions and insertions (9,10,(25)(26)(27)(28). These small mutations (−7 bp to +4 bp) (Fig.…”

Zinc Finger Nuclease Knock-out of NADPH:Cytochrome P450 Oxidoreductase (POR) in Human Tumor Cell Lines Demonstrates That Hypoxia-activated Prodrugs Differ in POR Dependence

“…This specificity and magnitude of effect is difficult to achieve using RNAi or inhibitor approaches. In common with other studies with ZFNs (33,37,45,56), most (9/12) of the sequenced mutations at the POR target site are frameshifting indels (Table 3) that are predicted to encode truncated proteins lacking the FAD/NADPH domains critical for catalytic activity (46,47). As also noted in earlier studies (35,45,57), there is evidence for reduction to homozygosity in some clones (e.g.…”

“…When repaired by error-prone non-homologous end joining, this generates base substitutions and indels at high frequency, many of which will result in targeted gene knock-out. The successful disruption of target genes has been demonstrated in human cell lines (33)(34)(35)(36)(37) and a number of model organisms, including rat (38), zebrafish (39,40), and Drosophila (41,42), although these reagents have rarely been used in tumor cell lines (43)(44)(45). The custom-designed ZFNs utilized in this study target a 33-bp sequence in the 8th exon of POR (Fig.…”

Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome

“…To improve bioprocessing efficiencies, many researchers have sought to improve cell-line longevity by suppressing apoptosis in CHO cells. These efforts involve modulating protein activity by overexpressing antiapoptotic pathways 24 and blocking pro-apoptotic pathways with chemicals 25 , short interfering RNA (siRNA) 26 and gene deletions 27 . However, the complex nature of apoptosis has made it nontrivial to optimize in CHO cells.…”