Gene therapy holds great promise for the treatment of debilitating and incurable diseases. On this note, lentiviral vectors (LVs) have become a popular choice as a therapeutic carrier.1 Their safety, ability to infect and capacity to sustain long-term expression in both proliferating and nonproliferating cells1, have rendered LVs as a tool of choice for the stable genetic modification of various cell types, for both in vivo and ex vivo applications2.
Removing SV40 T antigen-encoding sequences from HEK293T cells for safety
Research has shown that human embryonic kidney 293 T (HEK293T) cells yield higher vector titers than their parental HEK293 cells3,4. Therefore, HEK293T are commonly used in LV manufacturing. However, the presence of SV40 T antigen-encoding sequences poses safety concerns since their bearing can promote tumor formation as well as hamper key cellular genes following random integration into the host genome4. Furthermore, HEK293 and HEK293T cell lines carry between five and six copies of Ad5 DNA5, which give rise to impurities during the production process of viral vector lots. As such, FDA guidance - for human gene therapy IND applications - underscores the need to limit the presence of such residual substrate DNA, to ensure the production of safe therapeutic products.6
To mitigate the risk of activating oncogenes, Reiser et al. used CRISPR-Cas9 to ablate the SV40 T antigen-encoding sequences from HEK293T cells.7
TLA picks up integration sites and reveals large structural variations
Here, scientists from FDA and NIH availed themselves of our proprietary TLA technology for the complete genomic characterization of their T antigen-negative cell clones. Furthermore, TLA was used to assess integration events of the pRTAK SV40 T antigen plasmid, originally used to obtain the derived HEK293T cell line.7,8,9,10
Our generated TLA data suggested a single plasmid integration on chromosome 3 and revealed multiple plasmid copies within a large 550-kb deleted endogenous DNA sequence on this chromosomal position.7,8,9
Championing the design of safer and more efficacious lentiviral vectors in the future
This work revealed that the engineered HEK293T cells did not affect the overall capacity to produce vectors. However, lentiviral vector titers derived from those knock-out clones were reduced compared to HEK293T that still harbored intact T antigen-encoding sequences, thereby casting doubt upon their usefulness for large-scale viral vector manufacturing.7
Nevertheless, this paper will unquestionably inspire scientists to continue designing safer and more efficacious lentiviral vectors in the future. We are honored that Reiser and his colleagues recognize the high-quality standard of our TLA technology and are proud to have contributed to this consequential work.
TLA-based solutions for the improved analytical QC of cell and gene therapy products
Unlike conventional approaches, TLA technology allows the:
- Complete targeted next generation sequencing of any (trans)gene of interest
- Detection of all sequence variants, be it SNVs and (larger) structural variants in integrated vector sequences and integration sites, unlike PCR-based approaches which are mostly hypothesis-driven.
Given the unique capabilities of our proprietary technology, Cergentis has witnessed an increasing adoption of its TLA-based solutions for the improved analytical QC of cell and gene therapy products.
For more detailed information, we invite you to check out our 2 most recent application notes, describing the relevance of our TLA-based solutions in both the upstream and downstream processes of cell and gene therapy manufacturing:
Targeted complete NGS and QC of transgenes and integration sites in upstream cell and gene therapy manufacturing
Targeted complete NGS and QC of transgenes, integration sites and gene editing in heterogeneous cell and gene therapy products
 Milone, M. C., & O'Doherty, U. (2018). Clinical use of lentiviral vectors. Leukemia, 32(7), 1529–1541. https://doi.org/10.1038/s41375-018-0106-0
 Perry C, Rayat ACME. Lentiviral Vector Bioprocessing. Viruses. 2021 Feb 9;13(2):268. doi: 10.3390/v13020268. PMID: 33572347; PMCID: PMC7916122.
 Merten, O.W., Charrier, S., Laroudie, N., Fauchille, S., Dugué, C., Jenny, C., Audit, M., Zanta-Boussif, M.A., Chautard, H., Radrizzani, M., et al. (2011). Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application. Hum. Gene Ther. 22, 343–356.
 Merten, O.W., Hebben, M., and Bovolenta, C. (2016). Production of lentiviral vectors. Mol. Ther. Methods Clin. Dev. 3, 16017.
 Lin, Y.C., Boone, M., Meuris, L., Lemmens, I., Van Roy, N., Soete, A., Reumers, J., Moisse, M., Plaisance, S., Drmanac, R., et al. (2014). Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations. Nat. Commun. 5, 4767.
 U.S. Food and Drug Administration. (2020, January). Chemistry, manufacturing, and control (CMC) information for human gene therapy investigational new drug applications (INDs). FDA. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/chemistry-manufacturing-and-control-cmc-information-human-gene-therapy-investigational-new-drug
 Bae DH, Marino M, Iaffaldano B, Fenstermaker S, Afione S, Argaw T, McCright J, Kwilas A, Chiorini JA, Timmons AE, Reiser J. Design and Testing of Vector-Producing HEK293T Cells Bearing a Genomic Deletion of the SV40 T Antigen Coding Region. Mol Ther Methods Clin Dev. 2020 Jul 9;18:631-638. doi: 10.1016/j.omtm.2020.07.006. PMID: 32775497; PMCID: PMC7397404.
 de Vree, P.J., de Wit, E., Yilmaz, M., van de Heijning, M., Klous, P., Verstegen, M.J., Wan, Y., Teunissen, H., Krijger, P.H., Geeven, G., et al. (2014). Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nat. Biotechnol. 32, 1019–1025.
 Hottentot, Q.P., van Min, M., Splinter, E., and White, S.J. (2017). Targeted locus amplification and next-generation sequencing. Methods Mol. Biol. 1492, 185–196.
 Heinzel, S.S., Krysan, P.J., Calos, M.P., and DuBridge, R.B. (1988). Use of simian virus 40 replication to amplify Epstein-Barr virus shuttle vectors in human cells. J. Virol. 62, 3738–3746.