Site-Directed Engineering of Defined Chromosomal Sites for Recombinant Protein and Virus Expression - Site-directed engineering of defined chromosomal sites. - BioPharm International

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Site-Directed Engineering of Defined Chromosomal Sites for Recombinant Protein and Virus Expression
Site-directed engineering of defined chromosomal sites.


BioPharm International
Volume 22, Issue 7


Figure 3C. Predictable and homogeneous product expression after targeting a tagged locus C. Based on a chromosomal site tagged with a retroviral vector, a packaging cell line was established.3 The tagging vector was replaced by various viral vectors. The productivity of subclones obtained by the targeting of one of those vectors reveals that the expression levels are strictly homogenous among all analyzed isogenic subclones.
With these modular packaging cells, infectious particles can be efficiently produced with high reproducibility, achieving titers up to 2 ×107 IU/106 cells in 24 hours (Figure 3C). Importantly, this technology can be used to produce clinically relevant viral vectors. Indeed, high-titer producer cells for a therapeutic vector that encodes the 8.9-kb collagen VII cDNA in a marker-free cassette were obtained within three weeks without screening.3 Because the master cell line is fully characterized with respect to retroviral vector production conditions, the establishment of a producer cell line is reduced to the replacement step to introduce the vector of interest.


Figure 3D. Predictable and homogeneous product expression after targeting a tagged locus D. To prove the flexibility of the RMCE system, an antibody expression cassette was targeted into the tagged chromosomal loci of clones of different origin, including Chinese hamster ovary cells (CHO), and human embryonic kidney cells (HEK293). The expression of the targeted subclonal producer was measured by ELISA and, although different between the species, is homogenous amongst each other.
Finally, the high capacity of this strategy is demonstrated by its use in the predictable expression of antibodies. For this purpose, we used single-step vectors that encode both the heavy- and the light-chain genes. Upon targeting into the prescreened integration sites, antibody expression clones are generated. Importantly, they show homogeneous antibody expression levels which are expected from the defined genetic modification (Figure 3D).

CONCLUSION

The illustrated data presented here give evidence that this technology is a powerful method for achieving defined and reproducible genetic modification of cells, thereby making it possible to establish producer cells in a short period of time. After a potent integration site is tagged and identified on screening, characterized with respect to safety, and optimized for production, it constitutes a platform for integrating other expression cassettes of interest. Thus, it makes it possible to establish expression clones with predictable properties in a minimal time. Apart from this, it makes it possible to adjust expression cassettes to the requirements of the individual chromosomal sites.11,17 In this way, it contributes to a predictable modification of the mammalian genome and its exploitation for the production of biopharmaceutical molecules.

Dagmar Wirth, PhD, is head of the research group Model Systems for Infection and Immunity (MSYS), +49 531 6181 5040,
This group is associated with the division of Molecular Biotechnology at the Helmholtz Centre for Infection Research, Braunschweig, Germany. Leonor Gama-Norton is a research fellow in MSYS, and Kristina Nehlsen, PhD, and Roland Schucht, PhD, are scientists in MSYS and this division, respectively. Leonor Gama-Norton is also a member of the Instituto de Biologia Experimental e Tecnológica, Universidade Nova de Lisboa (IBET/ITQB/UNL), Oeiras, Portugal.


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