Peter O'Callaghan

Peter O'Callaghan, Ph.D

In this interview, our expert, Dr. Peter O'Callaghan, addresses your questions on Lonza's GS piggyBac® transposase technology, from how it works, to why you should be using it in your development process for the expression of biologics.

What is GS piggyBac transposase technology and how does it work?

Transposons are mobile genetic elements and their mobility is mediated by transposase enzymes. The transposon, as part of a recombinant protein expression vector, contains the gene(s) of interest (GOI) you want to insert into your host cell, flanked by transposase-recognising inverted terminal repeat (ITR) sequences. Once the vector is transfected into the host cell line along with the GS piggyBac transposase, the transposase recognizes the ITR sequences at each end of the genes you want to integrate into the host cell, at which point the transposase cleaves the DNA. The expression cargo is then pasted into sites of open chromatin within the host cell genome where gene expression is typically high. Genome integrations catalysed by the GS piggyBac transposase enzyme are typically characterised by TTAA tetranucleotide repeat sequences at both the 5' and 3' vector-genome junctions and are biased towards regions of open chromatin.

The GS piggyBac technology therefore allows you to select stable pools and clonal cell lines where vectors have been inserted into highly transcriptionally active sites that are associated with long-term, stable, high expression.

diagram gs piggybac transposase technology
Diagram of GS piggyBac transposase technology

What are the benefits of using GS piggyBac to express my biotherapeutic molecule in stable cell line development?

We, and our customers, have found that when using GS piggyBac, stable CHO cell pools recover faster post transfection, and higher average titers can be achieved from both bulk stable pools and clonal cell lines. Lonza has generated high titre stable pools and clonal cell lines for a range of molecules from hard to express antibodies, next-generation molecular formats, fusion proteins and bispecific antibodies when using GS piggyBac. More product means drug developers can undertake the studies they need and speed towards being first in clinic.

I am performing transient screening of therapeutic candidates, how can GS piggyBac support this work?

At Lonza we have been optimizing the stable pool expression format in recent years, particularly since the introduction of the GS piggyBac hyperactive transpose technology. We therefore sought to apply the learnings from that work to create an optimized transient expression process that merges the best attributes of both traditional transient and stable pool expression to support early discovery work. Our new GS Discovery solution meets this need by offering such a hybrid process powered by GS piggyBac.

It is, to our knowledge, the first example wherein a transposase enzyme has been used to boost titers from a transient transfection process over such a short timeframe. Learn more about the GS Discovery Transient Expression Platform

Do I need to create special vectors to accommodate GS piggyBac?

Lonza have done the hard work for you. Our dedicated team of R&D experts have developed a suite of vectors known as GSquad®. GSquad is fully compatible with GS piggyBac and you can insert up to four genes into a single vector following a simple two-step protocol. The same GSquad vectors can be used to support transient and also stable pool and clone expression workflows.

How is the GS piggyBac transposase delivered alongside the GOI-containing vector?

It can be delivered as either mRNA or plasmid DNA. The mRNA is preferable for the creation of stable pools or clones as it will have a short half-life, and once the transposase activity has completed, the recombinant genes should be stably incorporated into the host cell genome. When using the pDNA form of the transposase, it is possible that the duration of transposase expression will be extended, potentially leading to re-mobilisation of integrated GS vectors in the cell, which would be undesirable from a consistency viewpoint. However, this is not a concern when performing transient gene expression work as the cultures will not be maintained post-harvest.

Is there a limit on the size of the gene that can be inserted with GS piggyBac technology?

Our system can deal with large gene cargos of over 200 kb.

What host cell lines is GS piggyBac compatible with?

Lonza have developed high-performing CHO host cell lines, with GS Xceed and GS Effex being currently available as part of the GS toolbox. Both of these cell lines are fully compatible with the GS piggyBac transposase 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® is derived from GS Xceed and lacks the enzyme responsible for fucosylation. This absence of fucose increases the potency of the mAb product.

Read more about the GS Effex Cell Line

How do I get access to GS piggyBac technology?

With our Lonza in Your Lab® offering, GS piggyBac is available as part of the GS Gene Expression System via a license. The GS Gene Expression System includes host cell lines, GS piggyBac, GS Discovery transient expression platform, GSquad vectors, extensive process know-how and on demand expert technical support throughout your development journey. The GS System also underpins our CDMO services offering.

Do you want to elevate your cell line development process with our GS System and GS piggyBac technology?

Contact us or visit our GS Xceed Gene Expression System page for more information on our offerings.

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