View our CHO GS knock-out application notes - Horizon Discovery

APPLICATION NOTE
CHO Cell Engineering for Bioproduction using rAAV
Joshua Kapp, Jane Elliot, Tom Henley, Christine Schofield, Chris Torrance, Paul Morrill, Mark Stockdale
INTRODUCTION
Chinese hamster ovary (CHO) cells are the most commonly
used mammalian host for industrial production of
recombinant proteins. Significant efforts have resulted in
reducedcycletimetoINDwhileaveragetitrehasrisen
more than five fold in less than ten years. The publication
of the CHO genome has created impetus for new
improvements to the CHO bioproduction platform, through
the use of gene editing technologies such as Horizon’s rAAV
technology.
One of the challenges when working with CHO is achieving
a rapid selection of clones which express the recombinant
gene of interest. In recent times, methionine sulphoxamine
(MSX), an inhibitor of the enzyme glutamine synthetase
(GS), has been used to facilitate recombinant GS selection
of heterologous protein expression cassettes. GS is
required for glutamine production and without a functional
endogenous enzyme, cells require an alternative source of
glutamine to survive. Unfortunately, MSX has cost
implications and increases production timelines due to
inhibition of cell line growth. Furthermore when MSX is
used in a regulatory environment, manufacturers are
required to demonstrate that it is not carried through to
the final product. A GS null CHO line would eliminate the
need for MSX while offering benefits both in terms of cost
and bioprocess timelines.
Horizon’s rAAV technology leads the genome editing
industry in terms of precision and accuracy, making it ideal
for sensitive applications such as engineering of
bioproduction cell lines. Here we demonstrate the
effective use of the rAAV gene editing platform to achieve
the targeted disruption of the endogenous CHO GS gene
METHODS
Parental Cell Line Characterisation
The CHO‐K1 cell line was purchased from the European
Collection of Cell Cultures (ECACC). Prior to targeting, the
cell line was characterised to ascertain its suitability for
modification.
The process of characterisation involves a number of
steps as detailed in Figure 1. Initially the CHO‐K1 line
was single cell diluted and several clones selected based
upon the similarity of growth characteristics compared
to the parent CHO‐K1 cells.
RESULTS & DISCUSSION
The initial rAAV targeting strategy was based on the
study by Pei‐Qi Liu et al, 20101 who demonstrated the
functional knock‐out of CHO GS through the disruption
of the exon six reading frame of the GS gene, which
contains the active site of the enzyme.

AAV incorporating appropriate targeting sequence was
prepared in an HEK 293 packaging cell line and purified.
CHO‐K1 cells were transduced with rAAV and allowed to
incubate 48 hours. Antibiotic selection was then applied for
a two week period after which the culture was single cell
diluted and clones were further expanded for two weeks.
Genotype of individual clones was established using PCR to
identify targeted recombination events.
Sequence analysis performed on three selected clones
confirmed the complete removal of exon six.
The presence of the LoxP sites allows for future excision of
the selection marker if desired.
CONCLUSION
We were able to achieve targeted disruption of the GS
gene by complete removal of the entire exon six. This
project demonstrates the utility of the rAAV platform in
performing precision based modification of the CHO
genome.
HORIZON SUPPORT
Horizon offers access to its GENESIS gene editing platform
on a custom basis for Bioproduction Applications. We are
able to engineer precise genetic changes in CHO and other
mammalian cell hosts for a number of different
applications and purposes including:
GENE DISRUPTION
Our offering for Gene Disruption is unique as it is driven by
homologous recombination. This allows Horizon and the
end user to work together to exactly define the final
sequence that the engineered cell line will have. All
resultant clones will then have the same sequence
outcome. Other technologies give random output and as a
result many of the clones emerging from such processes
have less utility in bioproduction.
Sample applications: Deletion of viral receptors, deletion of
host co‐purifying proteins, generation of auxotrophic lines,
and manipulation of glycosylation pathways
TARGET INTEGRATION
CHO is particularly challenging when it comes to
introduction of new genetic material in a targeted manner.
The CHO genome has a propensity for randomly
incorporating new sequence making off target integration a
significant issue. Horizon’s rAAV gene editing process
eliminates this problem while still maintaining a good level
of efficiency for genome modification projects. This
proposition is strengthened by Horizon’s 100% service
model where Horizon performs all the gene editing and
provides a fully characterized line to the client. We
routinely run over 100 gene editing projects per year and
can confidently run multiple projects in parallel.
Applications: Engineering of high expressing landing pads,
reverse engineering of high producing clones
LANDING PAD DEVELOPMENT
The creation of a pre‐engineered landing pad where
expression cassettes can be quickly targeted into the
genome has clear time saving benefits for isolating high
expressing cells. Horizon’s technology is particularly well
suited to the reverse engineering of known high expressing
regions to remove existing expression cassettes and replace
them with sites suitable for targeted recombination. In
addition Horizon is developing a suite of its own “hot spots”
and can custom tailor its approach to meet the specific
needs of the client.
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