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.