Director, Product Management
ATUM
Chinese Hamster Ovary (CHO) cells have been the industrial workhorse for production of biopharmaceuticals since the 1980s, with the first CHO-derived product approved by the FDA in 1987. Nearly five decades later, CHO cells are the primary mammalian host for biopharmaceutical manufacturing due to their ease of manipulation, robust growth in high-density suspension cultures and post-translational modification machinery that yields human-compatible glycosylation profiles. The increasing complexity of next-generation biologics such as bi- and trispecific antibodies, antibody-drug conjugates (ADCs), and vaccines demands novel, enabling technologies for CHO genome engineering.
Director Emeritus, Cell Line Development,
While tools like CRISPR-Cas9, Transcription Activator-Like Effector Nucleases (TALENs), and Zinc-Finger nucleases (ZFNs) have demonstrated great utility, primarily for genetic knock-out applications, none have been adopted as widely for biopharmaceutical manufacturing as transposases. Since their discovery by Nobel laureate Barbara McClintock, transposase/transposon pairs (jumping genes) have profoundly changed how we perceive genomic fluidity and structural integrity. Recent efforts in the development of hyperactive transposase systems coupled with synthetic biology have created a single, efficient tool for genetic knock-ins, knock-downs, and for complex bioengineering needs. Transposases are fundamentally altering the paradigm of CHO-cell engineering.
Limits of traditional gene editing
Early strategies for site-specific integration relied on homologous recombination driven by extensive homology between the recombinant sequence and its genomic target.1 Homologous recombination is a rare event in mammalian cells, occurring with a frequency of approximately 1 in 106-107 cells per generation. Moreover, CHO cells are particularly recalcitrant to homology dependent repair (HDR), which makes this approach unviable for CHO engineering.
Another approach involves the engineering of “landing pads” with recombinase recognition sequences at genomic hotspots. Upon expression of the recombinase enzymes (Cre, FLIP or FC31), donor plasmids containing FRT, Lox, or attP/attB sequences can be site-specifically integrated via recombinase-mediated cassette exchange (RMCE). While conceptually elegant, this is a labor-intensive process. It requires the creation of the cell line with the landing pad, followed by recombinase mediated cassette exchange to generate the recombinant cell line. This approach also requires extensive screening for transcriptional hot spots, which is not trivial. The levels of productivity afforded by a single transgene copy were also rarely viable for biomanufacturing.
Targeted nucleases like CRISPR-Cas9, TALENs, and ZFNs sought to address these limitations by inducing site-specific double-strand breaks (DSBs) at defined genomic loci. However, non-homologous end joining (NHEJ), which is the dominant repair pathway in mammalian cells, quickly ligates broken DNA ends, resulting in insertions or deletions. While well-suited for gene knockout, this is detrimental for precise gene insertion.2 Homology-directed repair (HDR) pathway, required for template-guided, precise gene knock-in, is significantly less active and restricted primarily to the S and G2 phases of the cell cycle. This leads to low insertion efficiencies in industrially relevant CHO cells (estimated to be around 1 in 100,000), requiring the need for selection mechanisms or high-throughput screening methods to find the appropriate clones. This efficiency disparity has largely limited the utility of the targeted nucleases for applications that require integration of expression cassettes or other genetic elements.
An attractive solution to overcome the inefficiency of the HDR mechanism is to associate the DNA-binding domains of targeted nucleases with transposase enzymes. For instance, researchers have fused catalytically dead Cas9 (dCas9) with piggyBac transposase to accomplish site-directed transposition. This “chimeric transposase” approach combines targeting capabilities with high-efficiency transposition and has high potential to streamline site-specific modifications in CHO cells.
Key features of transposase systems
Transposon systems, such as the widely employed Leap-In Transposase® and piggyBac transposase, overcome the limitations of NHEJ-dependent repair by facilitating enzyme-catalyzed, semi-targeted “cut-and-paste” integration of a transgene cassette into the host genome. In contrast to RMCE and targeted nucleases, transposases offer two critical advantages: multi-copy integrations across the CHO genome and high transposition efficiency, resulting in highly productive homogeneous cell populations.
The transposase enzyme typically requires only a short target sequence (e.g., TTAA) and an open chromatin region, resulting in anywhere between 2 and 50 integration copies per CHO genome.3 The integrity of the transposed DNA is stably maintained at every integration site, resulting in high-titer stable cell lines with exceptional genetic stability.3 Furthermore, transposases promote highly efficient transposition even for large cargo sizes approaching 10–20 kb.
This mechanism generates stable, homogenous cell pools where most clones have high productivity and product quality attributes that closely resemble the original pool. These stable pools have been used to accelerate the transition from discovery to early stage manufacturing. During the COVID-19 pandemic, several organizations utilized transposase-derived pools for IND-enabling studies and for early manufacturing slots, suggesting a future where CHO-derived recombinant biopharmaceuticals could be brought to market a year faster than is possible today, saving lives and reducing drug development costs.
Synthetic biology, modular vectors
The rapid adoption of transposases is in part due to the application of synthetic biology (Synbio) principles, specifically the Design-Build-Test-Learn (DBTL) cycle, to engineer hyperactive transposase variants that exhibit significantly enhanced transposition efficiency. Leading technology platforms have paired these advances with the development of modular expression vector architectures and cloning tools to rapidly test and build a library of genetic elements (promoters, untranslated regions, polyadenylation signals, selection marker cassettes) to optimize the expression of any given biologic architecture.
This marriage of synthetic biology and protein engineering empowers the commercialization of complex biologics by providing a toolbox to refine transposon design in early research while creating a robust workflow for stable cell line generation. Recent publications4,5 report the generation of high-titer stable cell lines for expression of complex 4-chain bispecifics, protein-nanoparticle vaccines, and trispecific T cell engagers, all of which required an early screen of transposon vector design to optimize chain ratios, expression, and critical quality attributes (CQAs).
Advanced CHO engineering
The most sophisticated application of transposon technology lies in the serial engineering of the CHO host using orthogonal transposase enzymes. These enzymes, derived from different species, recognize distinct inverted terminal repeats (ITRs) and do not cross-mobilize or interfere with previously inserted expression cassettes.6 Orthogonal transposase-transposon pairs open the door to multiplexing genome engineering, where simultaneous sequence knock-in and/or functional knock-down is performed either serially or in parallel. The intrinsic stability of transposon-integrated elements enables consistent 1-to-1 transfer of information from the experimental design to the genome of the CHO cell, the so-called “what you see is what you get” of biotechnology. Transposon-mediated genome engineering is almost entirely devoid of gene concatemers, truncations, duplications, and other non-designed genome recombination, making genome engineering a more predictable science.
Orthogonal transposases are already used to rapidly establish bespoke host cell lines for product-specific applications. The original CHO host can be engineered to overexpress key glycosyltransferases or to knock down endogenous CHO enzymes (e.g. FUT8) to pre-program glycosylation characteristics. Subsequently introducing the target protein using a second orthogonal transposase will not interfere with the already integrated modifications. Alternatively, an existing antibody-expressing cell line can be retrofitted with glycan modifying enzymes on a second orthogonal transposon without impacting existing integration sites.
Modular vectors and orthogonal transposases also enable the engineering or enhancement of entire metabolic pathways. For example, improving sialylation of therapeutic proteins requires the coordinated overexpression of multiple enzymes, such as the sialic-acid substrate-generating enzyme GNE, along with B4GALT1 (galactosyltransferase) and ST6GAL1 (sialyltransferase). The expression level of each enzyme in this cascade can be fine-tuned in the transposon vector, and complex pathways can be engineered with the help of orthogonal transposase enzymes.
In summary, transposase technology has matured over the last 10 years from a simple integration method into a scalable, widely applicable, genetic engineering tool used by companies developing biopharmaceuticals. By enabling stable, multi-copy integrations through hyperactive enzymes and leveraging synthetic biology principles in transposon vector design, it allows for quick testing and refinements for new complex biologic formats early in preclinical stages and for the establishment of robust, scalable processes for stable cell line generation. Both features have encouraged wide adoption and application of the transposase technology in the field of biopharmaceutical development.
Surya Karunakaran, PhD, is the director of product management at ATUM. Ferenc Boldog, PhD, is director emeritus, cell line development at ATUM.
References
1. Folger, KR, Wong, EA, Wahl, G, et al. Patterns of integration of DNA microinjected into cultured mammalian cells: Evidence for homologous recombination between injected plasmid DNA molecules. Mol Cell Biol 1982; 2(12), 1372–1387.doi: 10.1128/mcb.2.11.1372-1387.1982
2. Lieber, MR, The mechanism of human nonhomologous DNA end joining. J Biol Chem 2010; 285(28), 18871–18875. doi: 10.1074/jbc.R700039200.
3. Balasubramanian S, Rajendra Y, Baldi L, et al. Comparison of three transposons for the generation of highly productive recombinant CHO cell pools and cell lines. Biotechnol and Bioengi 2016; 113(6), 1234–1243. doi: 10.1002/bit.25888.
4. Weidenbacher, P. AB, Sanyal, M, Friedland, N, et al. A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates. Nat Commun 2023, 14(1), 2149. doi:10.1038/s41467-023-37417-9
5. Carretero-Iglesia, L, Hall, OJ, Berret, J, et al. (2024). ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells. Nat Cancer 2024, 5(10), 1494–1514. doi:10.1038/s43018-024-00821-1
6. ATUM. (2025). Leveraging orthogonal transposase/transposon pairs as an alternative genetic engineering tool in CHO cells. [Poster presented at Cell Culture Engineering XIX].
