White Paper –
Probing the CHO Genome and Gene Function Using ZFNs

Introduction

Zinc finger nucleases (ZFNs) have proven to be a powerful tool for modifying genes. In comparison to traditional techniques, such as adding chemical or radiation based mutagens that cause various random mutations, ZFNs can create very accurate, targeted changes to the genome in a much more rapid fashion. For quite some time, SAFC has been applying ZFNs to the development of CHO (Chinese hamster ovary) cells to improve their productivity and processing characteristics for biopharmaceutical manufacturing.

SAFC’s CHOZN® cell lines have been designed to make creating production clones for biopharmaceuticals faster and simpler. Utilizing ZFN technology, customers have reported a time savings of up to eight weeks in their cell line development processes.

Targeted Knockouts

From a biopharmaceutical cell line perspective, the ability to modulate the activity of two genes in particular has been seen to be important. The first is the modification of the dihydrofolate reductase (DHFR) gene and subsequent elimination of this enzyme’s activity. This modification is commonly used as a selection process for production cell line development. Traditional CHO DHFR null mutants were created using a combination of chemical and radiation based mutagenesis. Often, these cells lines were found to grow poorly in chemically defined media and have additional liabilities that make them less than ideal as a platform cell line. Using ZFNs has allowed accurate and precise gene deletion within the DHFR gene leading to cell lines with improved growth or production characteristics.

Another important modification for production cell line selection processes is one that targets the glutamine synthetase (GS) gene. Current methods use addition of L-methionine sulfoximine (MSX) to the cell culture to inhibit the endogenous GS activity. ZFN modification of the GS gene and subsequent ablation of enzymatic activity eliminates the need for adding MSX for the selection of recombinant producing clones. However, this is not the only way that ZFNs are being used to improve CHO cell lines. The technology allows elimination of endogenous proteins and their respective activities that may cause antigenic response of biopharmaceuticals which improves therapeutic efficacy or risk profiles.

Further Applications

Most biopharma drugs are manufactured as injectables instead of oral delivery because as r-proteins, they would be destroyed if they were to pass through the digestive tract. These drugs, that may be recognized as foreign to the immune system, need to be produced as human-like as possible to avoid any type of antigenic response. This does not mean they have to be made in human cells; rather, that they should be produced to bear human-like post translational modifications and scaffolding.

The cellular machinery in CHO cells adds many of the same post-translational modifications to r-proteins as would human cells. Some of these modifications are important, particularly glycosylation, for the protein’s function and activity, and from a drug perspective, increases stability and circulating half-life when injected into a patient. But some of the modifications added onto r-proteins by wild type CHO cells can cause an antigenic response and might make the drug less efficacious, or more dangerously, it could cause an anaphylactic-like response in the patient.

Therefore, it is important to engineer the CHO cell to be more human-like and eliminate its ability to make any potentially harmful modifications to the protein it produces. Two genes within the CHO cell – CMAH and GGTA1 – have been identified to cause modifications to the carbohydrate chain that can become antigenic in human patients. ZFN technology was used to create deletions within these genes and eliminate their activity within the CHO cell. This removed the enzyme activities that catalyse these modifications to the carbohydrate chains of the glycoproteins, thus ensuring that therapeutic proteins produced within such modified cell lines will have lower antigenic risk potential.

The Future of ZFN Technology

ZFN technology now makes it possible to make precise genetic alterations to complex eukaryotic cells in a way that has been possible in bacteria for many years – by easily editing the genomes to make cell lines with improved characteristics. The recent completion and publication of the complete genome for the CHO-K1 cell line enables the possibility of additional modifications to improve production characteristics or r-protein product quality and/or safety.

SAFC researchers are currently investigating some 50 potential genes and subsequent modification using ZFN techniques. Such modified cell lines could lead to cell lines with improved characteristics such as post translation modification, productivity or longevity, or even eliminate the production of host cell proteins to simplify the downstream purification process. At this point, it is possible to imagine the CHOZN® Platform cell line as being continually enhanced, leading to Super-CHO cell lines in the future that are extremely efficient and make safer, more effective therapeutic proteins.

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