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.
Increased r-protein production.
There are several other ways to boost the r-protein yield besides improving the selection process. Genes related to apoptosis
can be targeted and knocked out, resulting in longer culture life. Genes that correlate with growth and productivity can be
manipulated by changing existing elements that control gene expression.
Another potential method for boosting r-protein yield is a targeted integration approach. Traditionally, r-protein DNA integrates
randomly into the genome. Several clones must be screened to isolate a stable, high-producing clone. If a desirable integration
region is identified, ZFNs can be used to precisely integrate the transgene at that location, which can lead to higher-producing
and consistently stable clones.
Managing post-translational modifications.
Because of genetic differences between CHO and human cells, r-proteins that are manufactured in CHO cells may have different
glycosylation patterns compared with proteins manufactured by human cells. These differences can cause an immunogenic response
when the drug is administered to the patient. Two examples of glycosylation differences include Neu5Gc moieties and alpha
1, 3-galactose (alpha-gal) moieties. The genes responsible for these glycosylation patterns are functional in CHO cells, but
not in humans. A r-protein produced in CHO cells may therefore contain Neu5Gc or alpha-gal moieties that could cause an immunogenic
response when administered. Knocking out the genes responsible for these glyco-proteins can eliminate this risk.
Molecule efficacy can also be increased by engineering glycosylation patterns that increase the residence time of the drug
in the bloodstream or by increasing the binding of the Fc region of the antibody to the Fc receptor. The circulating half-life
of therapeutic recombinant glycoproteins can be improved by increasing the sialic acid concentration. Targeting genes that
increase sialic acid concentrations can increase the residence time of the drug. Increased antibody-dependent cellular cytotocicity
(ADCC) can be achieved by creating antibodies that have greater binding affinity to Fc receptors. Non-fucosylated glyco-proteins
have greater binding affinity to Fc receptors, and knocking out genes responsible for fucosylation can result in more efficacious
Management of post-translational modifications is also important in biosimilar manufacturing, when the glyco-profile of the
original product must be matched. In these cases, ZFNs can be used to target genes that impact the glyco-profile to engineer
a cell line that can produce a r-protein that matches the innovator material.