June 1, 2016 (Vol. 36, No. 11)

Jamie Freeman Product Manager Horizon Discovery

Gene Editing Has the Potential to Have a Significant Impact on Product Quality

The first recombinant protein licensed for use by the U.S. FDA was human insulin in 1982. This was closely followed by tissue plasminogen activator (tPA), the first complex glycosylated protein generated in mammalian cells to be licensed for therapeutic use in 1987. Since then, the discovery, development, and regulatory approval of biological therapeutics (biologics) has grown rapidly, with an average of 15 new entities being approved by the U.S. FDA every year between 2006 and 2011.1

Protein therapeutic products are expressed by the mammalian cell system in which they are generated, and then secreted into the surrounding media, which can expose them to extracellular proteases. These can impact product stability and aggregation2 as well as leading directly to product degradation.3

These by-products can also be co-purified with the product during the downstream processing (DSP) steps used to purify the protein product from the cell culture media. These DSP steps can also lead to the co-purification of host cell proteins (HCPs) that can co-elute due to binding directly to the recovery column4,5 or interactions with the product via protein-protein interactions in a product specific manner.6–8

HCPs have been shown to have adverse clinical effects9, and must be minimized in the final product to levels under 1-100 ppm.10 Due to both the direct impact on the product as well as the cost of removing HCPs as part of the downstream process, there is considerable interest in mechanisms that reduce the presence of undesirable product-specific or nonspecific proteins that contribute significantly to these contaminants. Genome engineering approaches can be used to stop the expression of specific proteins.

In this tutorial we describe using a gene engineering approach to remove a single isoform of a protein from a high-producing CHO line intended for commercial manufacturing. Removal of this protein through DSP methods was impractical, so an engineering solution was sought.

The specific isoform comprised a single base pair mutation in a high copy number gene, which had a negative effect on the expressed product. This engineering method can be applied to any number of similar scenarios where knockout of a specific gene may have a profound impact on the quality of the final product.

The engineering technology used was based on the precise editing capabilities of recombinant Adeno-Associated Virus (rAAV), comprising single-stranded DNA homologous to the target region. The use of rAAV vectors in gene targeting has been refined to enable single base pair resolution editing without sequence error, critical to this application.

Initially, the target cell line was characterized and the presence of a single nucleotide polymorphism (SNP) at a defined location among a large number of gene copies was confirmed. The gene and flanking genomic region were sequenced and droplet digital PCR (ddPCR) performed to identify the presence of the SNP (Figure 1). There were 30 copies of the gene in the genome, of which ddPCR demonstrated that only one was mutated.

Figure 1. ddPCR copy number assay. ddPCR SNP assay indicating the absence of the SNP in the target cell line. The SNP was detected to be present in 1 out of 30 copies of the gene in the parent population. It was no longer detectable in the clones after engineering. Blue bars represent the copy number of the mutant gene. Orange bars represent the copy number of the wild type gene.

After characterization, a targeting strategy was developed that consisted of designing a vector to directly remove the mutated copy of the gene through deletion of a 3.6 Kb section and directly replacing it with the targeting vector containing an antibiotic selection marker (Figure 2A).

As well as targeting only the mutated locus, it was essential to target the correct allele. At this locus the copy number was two, so a two-stage screening strategy was employed to ensure that the correct allele was targeted, and the recombination did not occur on the incorrect, wild type allele (Figure 2B).

After introducing the designed vector the cells underwent a two-week selection period under antibiotic pressure. Surviving colonies were screened using PCR. The first round of PCR screening revealed which colonies contained the selection cassette at the correct locus, irrespective of which allele had been targeted. Those clones successfully targeted by rAAV were then re-screened using PCR specific to the mutated copy (Figure 3A).

Figure 2. Engineering strategy. (A) A schematic representation of the targeting construct and subsequent homologous recombination event. (B) A schematic representation of possible targeting events. The selection vector can integrate in one of two locations, the wild type allele or the transgene-containing allele. Option two is the intended integration.

In this instance, the lack of a PCR product indicated the mutated gene had been successfully removed. This was confirmed by a further round of PCR specific to the wild type allele (Figure 3B). The presence of a wild type band together with the absence of a mutant band indicated successful recombination to remove the mutated copy from the correct allele.

All clones identified through these rounds of PCR were sequenced and validated for the presence of the transgene using the SNP ddPCR (Figure 1). Further analysis using PCR and copy number ddPCR confirmed there were no off target integrations of the rAAV selection cassette.

This project demonstrates that it is possible to use gene-editing technologies to specifically and precisely remove a single copy of a high copy number gene from a production CHO cell line. As well as the application described, this approach can be used to remove a variety of unwanted co-purifying protein contaminants. This could lead to the removal of product-specific, potentially clinically harmful HCPs. This process can also be used to improve a cell-line platform that may be expressing proteins that have deleterious effects on the stability of a number of different products.

It can therefore be concluded that using gene editing in this manner has the potential to have a significantly beneficial impact on the product quality of biotherapeutics.

Figure 3. Identification of successful recombination. (A) A wild type specific PCR. This image demonstrates the band detected by amplifying the mutated gene at the defined locus. Clones C and D lack the PCR product amplified in the parent control population, showing that the transgene is no longer present. (B) A wild type allele PCR. The PCR will amplify the wild type allele where no integrations have been detected. Clones C and D still retain a wild type allele.

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Jamie Freeman (jamie.freeman@horizondiscovery.com) is sr. product manager at Horizon Discovery.