Genome editing has played a prominent role in the development of Chinese Hamster Ovary (CHO) cells for biopharmaceutical processes.
DUKXB11 cell line was created in 1980 by introducing mutations in the dihydrofolate reductase (DHFR) locus. Although the
engineered cells were not intended for stable recombinant protein production, the DHFR modification provided a potent metabolic
selection marker, and the cell line was quickly used to create stably transfected pools.
As knowledge of the CHO genome has increased, many more potential genomic targets have been identified. Genome editing is
now routinely used as a tool to aid biopharmaceutical production. When the DUKXB11 cell line was developed in 1980, the only
available genomic modification techniques were exposure to chemical mutagens or radiation. Massive screening and selection
methods were therefore required to identify cells with the desired genotype. Mutations in the DUKXB11 cell line were introduced
by exposing CHO cells to ethyl methanesulfonate, or gamma radiation. The cells went through many rounds of selection using
[3 H] deoxyuridine to isolate clones that contained the desired genotype. These mutagenesis techniques could introduce undesired
random mutations throughout the genome and require massive selection strategies to identify clones with the desired genotype.
Today, there are several technologies that enable the user to edit the genome more precisely. One of these technologies is
the use of zinc finger nucleases (ZFNs).
ZINC FINGER NUCLEASES
A zinc finger motif is a naturally occurring small protein made up of approximately 30 amino acids, stabilized by at least
one zinc ion. Each zinc finger motif binds to a specific set of three nucleotide bases. When several of these zinc finger
motifs are connected, they target a precise genomic sequence. A ZFN is formed when a FokI endonuclease is fused to these
zinc finger motifs.
ZFNs are designed in pairs that bind to adjacent sequences. When the pair of ZFNs binds to the adjacent sequences, their
FokI endonucleases heterodimerize, cutting the DNA at that location. In other words, ZFNs target a specific sequence of DNA
and create a double-stranded break (DSB) at that precise location.
Once the DSB has been created, the user can then create specific deletions or insertions at that location, using the natural
repair mechanisms of the cell. The precision and accuracy of ZFNs reduce the screening and selection processes needed to identify
cells with the desired genotype, hence, reducing timelines.
Other technologies, such as meganucleases or TALENS (i.e., transcription activator-like effector nucleases), can also create
targeted changes in a genome. Mega-nucleases (from Precision Biosciences and Cellectis) are restriction enzymes found in
single-celled organisms that recognize a large (>20bp) DNA sequence. The disadvantage of this technology is that the protein-engineering
process takes several months and cutting efficiencies can be low. TALENS consist of a TALE DNA binding domain that gives sequence-specific
recognition, fused to the catalytic domain of an endonuclease. Much like ZFNs, TALENS bind to a specific sequence of DNA and
create a DSB. There is, however, a lack of precedence for using them clinically and no clear path to commercial use. In contrast,
ZFNs have been used in gene therapy trials. Sigma-Aldrich holds an exclusive license for the ZFN technology through Sangamo
The ZFN technology enables scientists to explore many potential gene modifications that improve cell lines for biopharmaceutical
production. The modified cell lines can have characteristics such as improved metabolic selection mechanisms, increased r-protein
yield, improved post-translational modifications, and reduced risk profiles.
Improved metabolic selection mechanisms
Two widely used selection systems are the DHFR and glutamine synthetase (GS) systems. The ZFN technology can be used to
create cell lines with improved selection capabilities by knocking out the endogenous DHFR and GS genes. By improving the
selection process, the productivity of the final production clones can be increased.
The DHFR-based selection system requires the elimination of DHFR, an enzyme responsible for purine synthesis. This elimination
can be achieved through the addition of methotrexate (MTX), a DHFR inhibitor, or by mutation of the DHFR gene. As previously
mentioned, existing DHFR knock-out cell lines were created using mutagens such as ethyl methanesulfonate or gamma radiation.
These techniques may have introduced undesired mutations throughout the genome with unknown effects on the cell's performance.
ZFNs allow the user to create a precise knock-out of the DHFR gene without the risk of non-specific mutations.
The GS selection system requires the elimination of the activity of glutamine synthetase, an enzyme responsible for the production
of L-glutamine. The activity of GS can be reduced by the addition of methionine sulfoximine (MSX). This approach, however,
raises regulatory concerns as well as raw material cost. Targeted ZFN-mediated knock-out of the GS gene eliminates the need
for MSX and makes the selection process more stringent.