Background A stable packaging cell line facilitates large-scale lentivirus vector manufacture. However, it has been difficult to produce clinical-scale HIV-1-based lentiviral vectors using a packaging cell line, in part due to toxicity of packaging genes, and gene silencing that occurs during the long culture period necessary for sequential addition of packaging constructs.Methods To avoid these problems, we developed a three-level cascade gene regulation system designed to remove tetracycline transactivator (tTA) from cytomegalovirus immediate early promoter (CMV) -controlled expression to reduce cytotoxicity from constitutive expression of tTA and leaky expression of packaging genes. We also performed a one-step integration of the three packaging plasmids to shorten the culture time for clonal selection.Results Although leaky expression of p24 and vector production still occurred despite the three-level regulation system, little cytotoxicity was observed and producer cells could be expanded for large-scale production. Producer cells yielded remarkably stable vector production over a period greater than 11 days with the highest titer 3.5 X 10(7) transducing units (TU)/ml and p24 300 ng/ml, yielding 2.2 x 10(11) TU and 1.8 milligram (mg) p24 from one cell factory. No replication-competent lentivirus (RCL) was detected. Long-term analysis demonstrated that, although the cells are genetically stable, partial gene silencing occurs after 2-3 months in culture; however, the one-step construct integration allowed prolonged vector production before significant gene silencing. Concentrated vector resulted in 90% transduction in CD4(+) lymphocytes at 20 TU per cell. CD34(+) progenitor cells were transduced at 41-46% efficiency, and long-term initiating culture (LTC-IC) was transduced at 45-51%.Conclusions These results demonstrate for the first time HIV-1-based lentiviral vector production on the large scale using a packaging cell line. Copyright (c) 2005 John Wiley & Sons, Ltd.

We report the design of a unique two‐plasmid production system for the first lentiviral vector to be evaluated in humans, VRX496. VRX496 is an optimized VSV‐G pseudotyped vector derived from HIV‐1 that expresses antisense to the HIV envelope gene. We found that a two‐plasmid approach to production resulted in higher vector production titers when compared with a three‐plasmid approach, which is particularly important for vector production at the large scale. Therefore, we carefully designed a single packaging construct, VIRPAC, for safety by reducing its homology with VRX496 and by insertion of functionally validated genetic elements designed to reduce the risk of generation of a replication‐competent lentivirus (RCL). A native cis‐acting ribozyme is used to prevent read through into the envelope gene from the upstream gag‐pol genes in the packaging vector, thus preventing RNAs containing gag‐pol and env together for comparable safety to a three‐plasmid system. We demonstrate that there is no significant in vivo vector mobilization using a primary SCID‐hu mouse transplantation model, which correlates with the presence of an anti‐HIV payload and suggests that inclusion of antisense may be a useful tool to restrict mobilization in other vector constructs. Gene transfer is achieved using a one‐step transduction procedure that is simple and clinically translatable, which reaches stable transduction efficiencies of >99% in CD4 + T lymphocytes within 3 days of culture initiation. Copyright © 2004 John Wiley & Sons, Ltd.