Discussion
The current focus in the biopharmaceutical industry is to identify cells that exhibit predictable expression levels with the expected amount of protein (Wang et al., 2018). However, traditional procedures for cell line development are based on random transgene integration into the genome (Lai et al., 2013). Thus, the crosstalk between cis- and trans-acting factors in the neighboring chromosome influences whether transgenes are silent or active (West & Fraser, 2005). Even after establishing single cell clones, silencer sequences present on adjacent chromosomes contribute to the spread of condensed chromatin to the transgene, leading to transgene silencing (Wang et al., 2018). To circumvent possible positional effect, the use of SSI has been proposed (Lee, Kallehauge, Pedersen, & Kildegaard, 2015). Targeted integration of transgenes into predefined chromosomal locations leads to more predictable and stable transgene expression (Lee et al., 2015). Recently, SSI has been further improved using RMCE, a technology that exchanges existing genetic cassettes established by SSI with similar cassettes carrying GOI (Turan et al., 2011). RMCE allows re-establishment of new clones carrying new GOI from preexisting clones carrying different GOI (Turan et al., 2013). Clones that established via RMCE behave similarly to the preexisting clones in terms of productivity, cell growth and metabolism (Turan et al., 2013) (Fig. 6). In this study, we established Cre/Lox-based RMCE for efficient GOI exchange in CHO cells. CRISPR/Cas9-mediated SSI integrated LP into a genomic hotspot region (Fer1L4 ). Cre/Lox-mediated RMCE replaced LP with TV, as evidenced by DNA sequencing results showing the recombinant region inFer1L4 . Furthermore, optimal conditions for RMCE, which significantly improved RMCE efficiency, were established. To our knowledge, this study provides the first demonstration that Cre/Lox-based RMCE can reestablish a derivative clone from an existing one previously established by SSI. As the cellular characteristics and productivity of clones regenerated by RMCE were as expected and the variation between clones was minimal (Turan et al., 2013), our results can be actively used in establishing new clones with predictable productivity from preexisting clones previously established by SSI (Fig. 6).
Genomic hotspots refers to genomic loci that confer exceptional stability and enhanced transcriptional activity (Nathaniel K. Hamaker & Kelvin H. Lee, 2018). Thus, targeting GOI in such genomic hotspots is prerequisite process for SSI-based cell line development (Nathaniel K. Hamaker & Kelvin H. Lee, 2018). The importance of genomic hotspots in SSI-based cell line development is supported by the fact that it enables antibody production regardless of antibody format (Crawford et al., 2013; Zhang et al., 2015). Genomic hotspots can guarantee higher protein productivity, but are limited to include up to two copies of LPs (Gaidukov et al., 2018). Therefore, a strategy of integrating multiple copies of LPs would increase the protein productivity in clones established by SSI-based RMCE. Thus, we propose the application of a multi-cistronic gene vector using the 2A system to produce two or more independent proteins at the stoichiometric level (Donnelly, Hughes, et al., 2001; Donnelly, Luke, et al., 2001). Up to 9 proteins linked to 2A sequence have been co-translated at equivalent levels (Geier, Fauland, Vogl, & Glieder, 2015). Thus, multi-cistronic (GOI-2A)8-GFP cassettes can be used to simultaneously express GFP as a sorting marker, and GOI for protein production (Fig. 7). The exchanged clones expressing GFP in this study were concentrated to 80% through FACS sorting, so that the application of multi-cistronic gene cassettes would help establish new clones via RMCE. Furthermore, multiple GOI linked to 2A can generate GOI at the stoichiometric level. Hence, the application of a multi-cistronic gene cassette can solve the shortcomings of SSI-based RMCEs that incorporate up to two copies of GOI (Fig. 7). Taken together, the possible disadvantages of SSI-based RMCEs can be solved by using multi-cistronic gene cassettes on TVs, as new clones can be easily obtained through RMCE and show an improvement in GOI production.
In summary, we performed SSI to target LP incorporation into a genomic hotspot, and established a Cre/Lox-based RMCE system to exchange LP with TV. This system efficiently re-establish new clones from existing ones by only exchanging a preexisting gene cassette for an analogous cassette carrying GOI. RMCE efficiency was indirectly improved by up to 80% when FACS sorting was performed after RMCE. Therefore, our results provide evidence that Cre/Lox-based RMCE system will be a useful strategy for iteratively establishing clones that confer predictable productivity and properties.