Applications of transposase systems to CHO cells

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Application of transposase expression systems to CHO cells: Lonza’s GS piggyBac® system 

The goal of cell-line development for biopharmaceutical manufacturing is to generate high-performing cells that afford high yields of the desired therapeutic molecule in high quality with few impurities. Generation of high concentrations of high-purity recombinant proteins, monoclonal antibodies (mAbs), and multispecies antibodies means less downstream purification is required, starting materials are efficiently used, and overall costs are reduced. 

One way to increase the expression of recombinant proteins is to modify the Chinese hamster ovary (CHO) host-cell genome using transposons with high integration activity and the ability to insert the gene of interest (GOI) into areas of the host-cell genome where gene expression levels are high.  

That is just what Lonza’s GS piggyBac® transposon system does. Part of the GS Xceed® Gene Expression System, the technology comprises an engineered, hyperactive transposase that recognizes GS piggyBac® transposons and inserts them, including their GOI cargos, into sites of open chromatin within the host-cell genome. GS piggyBac® transposons contain GOIs flanked by unique inverted terminal repeat (ITR) sequences. The transposase enzyme recognizes these unique ITR sequences and cleaves the DNA. The expression cargo (which can contain up to four different GOIs) is then pasted into the genome of the host-cell. 

Genome integrations catalyzed by the GS piggyBac® transposase enzyme are typically characterized by TTAA tetranucleotide repeat sequences at both the 5' and 3' vector-genome junctions and are biased towards regions of open chromatin. Consequently, GS piggyBac® technology therefore enables insertion of GOIs into highly transcriptionally active sites that are associated with long-term, stable, high expression. 

In the GS piggyBac® system, the GS piggyBac® transposase can be delivered with the transposon-containing vector using messenger RNA (mRNA) or plasmid DNA (pDNA). The mRNA is preferable for the creation of stable pools or clones because it has a short half-life, and thus the transposase enzymes are not around long enough to start moving the newly integrated vectors. When using pDNA, however, it is possible for the transposases to remain active for long enough to mobilize the newly integrated vector cargoes. If the goal is transient gene expression, either approach is suitable as the host-cell culture is not maintained after harvesting of the recombinant protein. 

There are several advantages to Lonza’s GS piggyBac® transposon system. First, it can accommodate large DNA cargos, which makes it highly suited for cell-line engineering for complex proteins including multispecific antibodies. Second, this cut-and-paste DNA insertion technology targets stable regions of the CHO genome associated with highly expressed genes. Third, the GS piggyBac® transposase enzyme is engineered to be much more efficient than its wild type counterpart. Fourth, GS piggyBac® expression vectors can be assembled efficiently using a one-pot method whether the transposon will include one to four product genes.  

Fifth, Lonza’s GSquad® suite of vectors are fully compatible with GS piggyBac® technology. The cloning workflow (a two-step protocol) is identical regardless of the number of product genes, and incorporation of sequencing primer binding sites also enables easy sequencing of final vectors. GSquad® vectors exist for cloning of both full-length product genes and antibody variable domains and the same GSquad® vectors can be used to support transient, stable pool, and clone expression workflows. 

Finally, Lonza’s GS Xceed® and GS Effex® cell lines within the GS® toolbox are fully compatible with GS piggyBac® transposon technology. The CHOK1SV GS-KO® GS Xceed® cell line has been widely used for the expression of biotherapeutics and has a long track record of regulatory approval (over 80 commercial products have been made with the GS System®). GS Effex®, which is derived from GS Xceed®, lacks the enzyme responsible for fucosylation, affording afucosylated recombinant mAbs with increased potency. 

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