As part of one of the world’s top industries, pharmaceutical companies bring in billions of dollars and save millions of lives. Time and money goes into the process of research and development, drug discovery, and bioprocessing – the necessary parts of getting drugs with life-saving potential to market. Eliminating potential pitfalls and saving time where possible while not decreasing quality is essential for biotherapeutics. Many product candidates will not be selected for commercial development but still need to be manufactured appropriately for preclinical and clinical testing.
In terms of bioprocessing, scientists have found that modifying current mammalian expression platforms could be the answer. Of these platforms, Chinese hamster ovary (CHO) cells are most commonly used.
Figure 1. Glutamine synthetase in mammals.
(A) GS is crucial in metabolizing nitrogen; it catalyzes glutamate and ammonia to condense glutamine; (B) Gene structure of CHO GS locus. (Fan et al.)
CHO Expression Systems In Biotherapeutics
According to a review on how CHO cells are used in bioprocessing by Fan et al., CHO cells are used in more than half of approved therapeutic proteins due to their robust growth that fairs nicely in producing large-scale pharmaceuticals.1 Possessing the ability for human-like post-translational modifications is an added benefit as this is typically necessary in biotherapeutic development.
One drawback to using CHO cells is the clonal selection process. This is the rate-limiting step in bioprocessing since it takes a lot of time and money to ensure pools or single clones with high and stable expression of an intended biologic are selected out of all the transfected cells.
Conversely, targeting GS with gene editing technology to obtain a full phenotypic knockout (zero GS protein expression) is far more effective compared to the typical GS MSX inhibition system. GS knockout cells allow for faster and more complete cell line selection and overall reduce the cell line development timeline.1
Role Of Novel Gene Editing To Enhance CHO Expression Systems
Novel gene editing proteins – such as Cas-CLOVER – target specific genes in a deliberate manner. This is compared to the older, labor-intensive processes of random insertion, sequential homologous recombination, or causing a frameshift in many genomic sites with the hopes that a mutation will occur in the target sequence.
Combining newer gene editing tools that have specific targeting capabilities with robust CHO expression systems has significantly impacted bulk cell culture productivity. As demonstrated in the figure below from Fan et al., GS KO cells (edited with relatively low efficiency with zinc-finger nuclease), yield an increased rate of high-producing cells resulting in improved efficiency.1
Figure 2. Comparing selected bulk cultures between CHOK1SV and GS-KO cells.
(A) HPLC titers of monoclonal antibody x bulk cultures. KO: GS-KO cells with 25 μM MSX, K1: CHOK1SV cells with 50 μM MSX. (B) One-way analysis of day 14 ACES titer data against parental cells used in the experiment. Data was analyzed by JMP 8.0 software. The significance of the mean difference was tested by Tukey–Kramer test. Histograms indicated the titer distribution of individual clones. CHO: Chinese hamster ovary cells; CHOK1: CHOK1SV parental cells; HPLC: High-performance liquid chromatography; KO: CHOK1SV-derived GS-knockout cells. (Fan et al.)
Cas-CLOVER GS knockout CHO Expression Systems
Demeetra’s proprietary Cas-CLOVER utilizes guide RNA targeting with an ultraspecific dimeric nuclease Clo051. In our system, dCas serves as a fusion protein only. It is mutated and unable to cut DNA. As opposed to the use of a single guide RNA (gRNA) like that in CRISPR, the Cas-CLOVER system utilizes two gRNAs in addition to the Clo051 nuclease activity that requires dimerization of subunits associated with each guide RNA. Using two gRNA makes the Cas-CLOVER gene editing system highly targeted and only functional when the paired gRNAs dimerize at the correct target site.
Figure 3. Our Cas-CLOVER gene editing technology
It helps eliminate off-target mutations, has been validated in plants, mammalian cells and yeast and is available through simple licenses.
Cas-CLOVER requires two gRNA and “PAM” (NGG) target sites. But fortunately, the number of nucleotides allowable for Clo051 function between the two gRNAs – the “spacer region” – is relatively large and flexible. Leveraging Cas-CLOVER’s flexible design, high efficiency and precision we set out to establish genome edited suspension CHO cell products.
Multiplexing by co-transfection of two gRNA pairs with Cas-CLOVER resulted in 196 base-pair deletion, eliminating nearly an entire exon of the GS gene. These large deletions resulted in single-cell clones that had a full GS knockout phenotype as shown by complete loss of viability without glutamate media supplementation.
Figure 4: GS-/- Suspension CHO Cells Demonstrate Loss of Viability Without Glutamine. WT CHO-K1 cells demonstrate viability with or without glutamine supplementation (blue,+or–Glu). GS-/- cells on the other hand lose viability without glutamine supplementation red.
Furthermore, Cas-CLOVER mediated GS knockout CHO cell viability was recovered when the GS gene was stably transformed. Cultures in the figure were kept under +/- Glutamine conditions for 19 days.
Figure 5: GS -/- Cells Transformed With GS Recover Viability
Suspension CHO GS -/- cells transformed with or without GS constructs. Cells not transformed with GS gene (left, GS-) lose viability without Glutamine supplementation in the media. Cells stably transformed with the GS gene (right, GS+) recover viability even without Glutamine supplementation.
References
Fan, L., Frye, C. C., & Racher, A. J. (2013). The use of glutamine synthetase as a selection marker: recent advances in Chinese hamster ovary cell line generation processes. Pharmaceutical Bioprocessing, 1 (5), 487–502. https://www.openaccessjournals.com/articles/the-use-of-glutamine-synthetase-as-a-selection-marker-recent-advances-in-chinese-hamster-ovary-cell-line-generation-proc.pdf