ZFN Technology Advances Biopharmaceutical Manufacturing

Genome editing has played a prominent role in the development of Chinese Hamster Ovary (CHO) cells for biopharmaceutical processes. The 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 [3H] 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 natural lyoccurring 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, 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 Biosciences.

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 r-antibodies.

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.

Improved downstream processing. ZFNs can be used to improve downstream processing by knocking out genes that encode interfering host-cell proteins. If the CHO cell line contains an endogenous protein that copurifies with the r-protein during chromatographic purification, additional and costly steps may be required to remove the endogenous protein. ZFNs can be used to knock out the gene that encodes this endogenous protein. Another potential target may be a protein within the CHO cell that binds the therapeutic r-protein. By knocking out the gene that encodes such a protein, growth and productivity can be improved. The CHO host cell may also produce proteolytic enzymes that could degrade the product before purification. Diminishing protease expression can minimize this effect.

Risk mitigation. The risk of prion or viral infection can be mitigated through genome editing. Retroviral titer in a cell could be reduced by targeting and removing retroviral elements. Additionally, viral uptake pathways can be targeted, conferring resistance to viral attack. Similarly, genes for prion proteins can be targeted and removed.

Combining ZFN modifications. Another benefit of ZFNs is that multiple modifications can be performed in the same clone. Desirable ZFN modifications can be trait stacked into the same cell line, enabling the potential development of a "super" CHO line precisely engineered to efficiently produce safe and effective therapeutic proteins.

Genomic changes improve productivity

Genome editing has vastly improved since the creation of the DUKXB11 cell line. Since 2009, SAFC has applied the ZFN technology to the development of robust CHO cell lines by introducing genomic changes that improve the productivity and processing characteristics of biopharmaceutical manufacturing cell lines. More than 30 specific modifications are available to the biopharmaceutical industry. Through microarray experiments, several key genes that impact cell growth and productivity have been identified and explored.

SAFC has several R&D scientists who identify and validate new genetic alterations that are relevant to the biopharmaceutical industry. They have created the CHOZN GS (GS-/-) and CHOZN DHFR (DHFR -/-) knock-out cell lines. Other available cell lines include knock-outs of GGTA (-/-) and CMAH (-/-), which result in cell lines that produce r-proteins without alpha-gal or Neu5Gc moieties, respectively.

Kate Achtien is an R&D scientist at SAFC, kate.achtien@sial.com