Biopharmaceutical manufacturing is undergoing a significant transformation with the advancement of genome editing technologies, particularly through the innovative use of transposases. Surya Karunakaran, PhD, and Ferenc Boldog, PhD, both from ATUM, highlight the potential of these technologies to enhance the production of complex biologics, which are increasingly necessary in today’s medical landscape.
Since their introduction in the 1980s, Chinese Hamster Ovary (CHO) cells have served as the primary mammalian host for biopharmaceutical production. The first CHO-derived product received FDA approval in 1987. The cells are favored for their efficient growth in high-density cultures and their ability to perform human-compatible glycosylation. As the demand for more complex biologics rises, including bi- and trispecific antibodies and antibody-drug conjugates (ADCs), the need for advanced genome engineering technologies becomes critical.
Traditional gene editing techniques have limitations. Early strategies relied heavily on homologous recombination, which is a rare event in mammalian cells, occurring at a frequency of approximately 1 in 10^6 to 10^7 cells. CHO cells are particularly resistant to this method, making it impractical for genome engineering. Alternative methods such as recombinase-mediated cassette exchange (RMCE) can be labor-intensive and require extensive screening for effective integration.
Targeted nucleases like CRISPR-Cas9, TALENs, and ZFNs were developed to induce site-specific double-strand breaks in DNA. While effective for gene knockouts, these methods often result in unwanted insertions or deletions due to non-homologous end joining, the dominant DNA repair pathway in mammalian cells. Furthermore, the efficiency of homology-directed repair, necessary for precise gene insertion, remains low, particularly in CHO cells.
An emerging solution is the combination of the DNA-binding capabilities of targeted nucleases with transposase enzymes. Researchers have successfully fused catalytically inactive Cas9 with piggyBac transposase to create a “chimeric transposase.” This innovative approach allows for highly efficient site-directed transposition, streamlining the modification process in CHO cells.
Key Advantages of Transposase Systems
Transposon systems, such as Leap-In Transposase®, offer significant advantages over traditional gene editing methods. They facilitate a semi-targeted “cut-and-paste” integration of transgenes into the host genome, achieving multi-copy integrations and high transposition efficiency. This leads to the generation of stable cell lines with a consistent genetic makeup, which is crucial for scaling production.
Transposases require only a short target sequence, enabling between 2 and 50 integration copies per CHO genome. This capacity for high-copy integration ensures that most clones exhibit high productivity and maintain the quality attributes of the original cell pool. The efficiency of this method was particularly evident during the COVID-19 pandemic, when several organizations utilized transposase-derived cell pools for early-stage manufacturing, potentially accelerating the time to market for critical therapeutics.
The integration of synthetic biology principles into transposase technology has further enhanced its utility. The Design-Build-Test-Learn (DBTL) cycle allows for rapid prototyping and testing of hyperactive transposase variants, leading to significant improvements in transposition efficiency. By using modular expression vector architectures, researchers can create libraries of genetic elements that optimize the expression of complex biologics.
Future Directions in CHO Cell Engineering
The future of CHO cell engineering lies in the serial application of orthogonal transposase enzymes. These enzymes, derived from various species, can recognize distinct inverted terminal repeats without interfering with previously inserted expressions. This capability opens avenues for multiplexing genome engineering, allowing simultaneous modifications to the genome.
This method has practical applications in enhancing the production of therapeutic proteins. For example, modifying glycosylation patterns can be achieved by overexpressing specific glycosyltransferases or knocking down endogenous enzymes. The precision of transposase technology also allows for the engineering of entire metabolic pathways, enabling tailored production processes.
In conclusion, transposase technology marks a significant advancement in the field of biopharmaceutical development. By providing a scalable and efficient genetic engineering tool, it allows for the rapid testing and refinement of complex biologics in the early stages of research. The ongoing integration of synthetic biology principles promises to further enhance the capabilities of this powerful technology, paving the way for faster and more effective biopharmaceutical production.
