Safe two-plasmid production for the first clinical lentivirus vector that achieves >99% transduction in primary cells using a one-step protocol.

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.