3. Targeted Genome Editing in CHO Using Engineered Zinc Finger Nucleases (ZFN’s) x Wild type cells ZFNs introduces a double stranded DNA break in the gene (transient exposure). Break repaired imperfectly by non-homologous end-joining (NHEJ). Gene ORF disrupted. Single cell clone x
4.
5. Cell Line Engineering Using ZFNs Purpose: Create a dhfr- genotype in CHO K1 parental cell line DG44 CHO K1 CHO K1/ DHFR- Superior transfection efficiency Shorter doubling time Higher cell densities Does not clump in suspension DHFR selection and gene amplification Easy to adapt to CD formulations Cells mutagenized
6. CHO Dihydrofolate Reductase (dhfr-) Clones - 83% - 73% - - 5% Not tested 7% 1% 33% Transfection Efficiency. Compared to CHOK1 Clone Peak VCD Days in culture (> 60% viable) Doubling time (hours) Glucose depletion (< 1.0 mmol/L) Glutamine depletion (< 0.5 mmol/L) Max lactate production (g/L) Max NH4+ production (mmol/L) DE7 3.0E+06 18 23.95 D13 Not depleted 1.8 7.71 8E7 2.6E+06 18 27.70 D13 Not depleted 1.67 6.35 GE6 2.8E+06 18 26.64 Not depleted Not depleted 1.96 6.67 FN18 2.1E+06 11 31.03 Not depleted Not depleted 1.9 7.03 6G7 2.5E+06 15 27.25 Not depleted Not depleted 2.81 7.97 CHO K1 6.8E+06 9 20.11 D6 D4 1.57 5.39 Chasin DG44 2.4E+06 18+ 25.23 D6 D4 1.76 5.61 Commercially purchased DG44 2.5E+06 7 26.55 Not depleted Not depleted 2.35 9.78
8. Cell Line Engineering Using ZFNs: Glutamine Synthetase (GS-) Cells Purpose: c reate a GS genotype in CHO K1 parental cell line Benefit: Better growth characteristics than CHOK1SV in your parental cell line Arrow = withdrawal of glutamine
hard-to-transfect cells, including primary, non-dividing, and differentiated cell lines
hard-to-transfect cells, including primary, non-dividing, and differentiated cell lines
These are examples of CHO knockouts that have been developed by CCES using ZFN technology. We will discuss this further in the next slides.
Ch in ese hamster ovary cells are an attractive mammalian expression host that have been extensively employed for the production of recombinant prote in therapeutics. These cells of fer the advantages that they are easily genetically manipulated, can be adapted for large-scale suspension culture, and most importantly, can give rise to proteins with glycans that are similar, although not identical, to those found on human glyco proteins . Certain features of CHO K1 cells make it a more attractive host than the widely used DG44 dhfr deficient CHO line CHO-K1 yields higher transfection efficiencies, shorter doubling times, higher cell densities, is easy to adapt to CD formulations and does not clump in suspension but it is lacking in the ability to provide gene amplification and methotrexate selection like DG44. The DG44 dhfr- knockout was created using chemical mutagenesis and gamma irradiation but the methods of mutation created other deleterious effects on CHO cell line. Is it possible to combine the certain qualities of CHOK1 such as higher transfection efficiencies with the amplification ability provided by a dhfr- line (such as DG44) ?
The answer is yes. This data shows several dhfr knockouts that were generated with ZFN targeted deletion of the dhfr gene in CHOK1 cells compared to the original CHOK1 cell line and two versions of the DG44 cell line. Notice the transfection efficiency in DG44 is much lower in comparison than KI or our dhfr- CHOK1
The data shown here is from the knockout clone, DE7, shown in the previous slide. The cells were passaged in media with and without hypoxanthine and thymidine (HT) supplement. Cells without the dhfr gene require HT in the media to survive. When the HT is removed, the DE7 clone does not grow and decreases in viability, showing by phenotypic assay that the clone has no functional dhfr gene.
In addition to dhfr (-/-) cell lines, another cell line in the industry are GS (-/-) cells from Lonza. These cells are deficient in Glutamine Synthase and therefore have a requirement for glutamine in the media until the GS gene is co-expressed with the gene of interest. Therefore this is another metabolic selection system, and it can be amplified with MSX. However, several groups have reported unfavorable growth characteristics with the CHOK1SV cell line (Lonza’s version). The data shows the growth of several GS KO clones generated from ZFN targeted deletion. While the cells are passaged in media containing glutamine, they grow normally and have high viability. Once passaged into media without glutamine (shown by the arrow), the cells die.
Fut8 and Neu3 knockouts would be of interest to biopharma, because as mentioned earlier, glycosylation patterns of biological drugs are extremely important. There is a Fut8 cell line in the industry, but again, some people find it a difficult cell line to work with. Biologics are produced in large scale culture which is expensive to set up and run. Cells such as Bax/Bak knockouts have increased longevity in the bioreactor, giving the cells more time to produce recombinant proteins, increasing yield and decreasing overall cost to produce.
Conclusion: Three successful rounds of knockout are shown resulting in a triple knockout. The first Western blot (left panel) shows no expression for GS in knockout (GS-/-). Middle figure top pane shows that DHFR is not expressed in clones 1F1.6 and 2B12.8 or the DHFR knockout control DG44, but is expressed in GS knockout control cells (GS knockout from first panel) and WT cells. Second panel show none of the clones express GS except the DG44 control cells which should express GS (control cells). Right panel, FUT8 KO cells don’t bind the fluorescent lectin (LCA) while WT cells do (see note below). Demonstrating cells are triple knockouts. Have many examples of single gene knockout…. But how far can we push this technology….? Treat cells with successive sets of ZFNs – first glutamine synthetase (GS), then DHFR, then FUT8. KO clones isolated at each stage. FUT8 KO kills 1-6 fucosylation of a core glycan, which is required for fluorescent lectin A binding. FUT8 KO cells thus don’t bind the fluorescent lectin (LCA) Glutamine synthetase (GS) has an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. In other words, you need GS to make glutamine. Cells need glutamine for growth.
