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A method to rapidly generate lentiviral vector producer cell lines

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Transient transfection of multiple plasmids into adherent HEK293T cells is currently the standard approach to manufacture clinical-grade lentiviral vectors.1-2 However, this strategy is (1) uneconomical, (2) has lengthy schedules to source plasmid DNA and (3) presents scalability challenges. Therefore, scientists at GSK sought for alternatives that would allow them to increase yield. To this end, a suspension-adapted stable producer cell line presented itself as an attractive option.

Past attempts could only achieve stability through low-toxicity envelope glycoproteins rather than the commonly adopted vesicular stomatitis virus envelope glycoprotein (VSVg) or with adherent cell lines.3-5 Moreover, previous approaches mostly involved stable transfection or transduction of DNA encoding each vector component into host cells at separate loci, thereby subjecting those cell line development campaigns to genetic or transcriptional instability (in at least one locus), fragilizing in turn productivity.

In this study, scientists at GSK described a novel approach that consists in introducing a single large (BAC) DNA construct - encoding all vector components - into 293T cells via stable transfection, toward the generation of lentiviral vector producer cell lines.6

 

Design, cloning & testing of BAC constructs

GSK designed their BAC constructs such that the CMV promoter-driven transcription units would be head-to-tail in the order transfer-gagpol-VSVg-rev with 2 copies of the 1.2-kb chicken HS4 insulator between transcription units in order to avert promoter interference (Figure 1).

CGT_GSK (2020)_Fig 1A-C_screenshot

Figure 1. (A) Schematic representation of GSK’s approach to manufacture lentiviral vectors (B) Design and cloning of BAC constructs
(C) Genome-wide coverage plot (via TLA-based assay) revealing a single integration site (of BAC DNA) – at chromosome 3 - in the 293Tsa EGFP clone 2 cell line.6

Lentiviral vector supply via transient transfection or stable producer cell lines?

To supply clinical grade lentiviral vectors by transient transfection, cell banking of E.coli containing the plasmids and high-quality or GMP-grade plasmid production is commonly done by a CMO. This approach is attractive early in a drug development project, but complexity typically ensues in the supply chain (i.e. plasmid and transfection reagent supply must be maintained throughout the drug’s lifetime). Moreover, this strategy also has restrictions with regards to possible upstream conditions.6

On the other hand, producing lentiviral vectors through stable producer cell lines is met with complexity at the start (i.e. need to perform a cell line development campaign and setting up GMP mammalian cell banks) but simplifies supply chain by enabling more a manageable upstream process (i.e. less constraints on optimization of upstream conditions). As a result, this strategy could be beneficial for maximizing cell growth, vector titer as well as infectivity.6

The balance between the pros and cons could possibly be specific to each drug development project. Furthermore, the authors suggest that the equilibrium might shift toward stable producer cell lines “if cell line development for lentiviral vector production becomes a standard service offered by contract manufacturing organizations, as is currently available for monoclonal antibodies.”6

A widely applicable method for the industrialization of lentiviral vector manufacture

The strategy described in this paper can be done with standard plasmid subcloning, conventional mammalian tissue culture techniques, and does not necessitate automation for high-throughput clone screening. As such, the reported method is accessible and suitable for both academic setting as well as industrial environment.

Although the resulting volumetric titers is as high as with transient transfection, the flexibility of this platform allows to further optimize yield (e.g. can be scaled up in single-use stirred-tank bioreactors) and the suspension cell lines are genetically and functionally stable in extended cell culture.6 Therefore, the authors are convinced that transitioning to an inherently scalable suspension cell culture format will enable significantly higher batch yields than permitted with current manufacturing processes. Ultimately, this method could enable better patient access to medicines - based on lentiviral vectors - in the future.6

Genetic QC in cell line development: a requirement by regulatory authorities

To ensure the consistency and safety of manufactured products, the FDA and EMA have issued well-defined guidelines that need to be adhered to.  Among them, we count: extensive genetic characterization of producer cell lines to scrutinize identity, genetic stability and purity. Our TLA-based solutions are ideally suited for such endeavors, thanks to its unmatched ability to yield all essential genetic characteristics in a single experiment (see comparison table). Furthermore, our comprehensive proprietary solutions will be able to support you and your team in a cost-attractive and time-efficient manner. Of note, we offer (upon request) detailed reporting suitable for use in the regulatory application for pharmaceuticals for human use, according to FDA and EMA expectations (CTD Quality module, section 3.2.S.2.3.).

With close to 10 years of experience, our dedicated team of PhD-level scientists have built in-depth experience analyzing genetic alterations in various forms, as our TLA-based method is routinely applied by leading biotech and pharmaceutical companies globally as part of their cell line development process. In fact, our data have been featured in over 50 peer-reviewed publications and have been used as part of filings. As such, TLA-based is widely regarded as the gold-standard for comprehensive genetic QC of pharmaceutical cell lines to guarantee correct genetic engineering.

In fact, FDA & NIH researched also availed themselves of our TLA-based assays. To mitigate the risk of activating oncogenes, Reiser et al. used CRISPR-Cas9 to remove the SV40 T antigen-encoding sequences from HEK293T cells and leveraged our technology to QC their T-antigen-negative cell clones, towards the manufacturing of safer vector-producing HEK293T cell lines. Click here to read a summary of this work and to learn about our contribution there: 

FDA & NIH apply TLA-based solutions

 

For more information on TLA-based solutions for cell line development, please visit the page below.

Cell Line Development

 

 

References

[1] Segura, M.M., Mangion, M., Gaillet, B., and Garnier, A. (2013). New developments in lentiviral vector design, production and purification. Expert Opin. Biol. Ther. 13, 987–1011.

[2] McCarron, A., Donnelley, M., McIntyre, C., and Parsons, D. (2016). Challenges of up-scaling lentivirus production and processing. J. Biotechnol. 240, 23–30.

[3] Merten, O.W., Hebben, M., and Bovolenta, C. (2016). Production of lentiviral vectors. Mol. Ther. Methods Clin. Dev. 3, 16017.

[4] Ikeda, Y., Takeuchi, Y., Martin, F., Cosset, F.L., Mitrophanous, K., and Collins, M. (2003). Continuous high-titer HIV-1 vector production. Nat. Biotechnol. 21, 569–572.

[5] Throm, R.E., Ouma, A.A., Zhou, S., Chandrasekaran, A., Lockey, T., Greene, M., De Ravin, S.S., Moayeri, M., Malech, H.L., Sorrentino, B.P., and Gray, J.T. (2009). Efficient construction of producer cell lines for a SIN lentiviral vector for

[6] Chen YH, Pallant C, Sampson CJ, Boiti A, Johnson S, Brazauskas P, Hardwicke P, Marongiu M, Marinova VM, Carmo M, Sweeney NP, Richard A, Shillings A, Archibald P, Puschmann E, Mouzon B, Grose D, Mendez-Tavio M, Chen MX, Warr SRC, Senussi T, Carter PS, Baker S, Jung C, Brugman MH, Howe SJ, Vink CA. Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct. Mol Ther Methods Clin Dev. 2020 Aug 14;19:47-57. doi: 10.1016/j.omtm.2020.08.011. PMID: 32995359; PMCID: PMC7501408.

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