A new-generation stable inducible packaging cell line for lentiviral vectors

We have successfully generated and characterized a stable packaging cell li ne for HIV-1-based vectors. To allow safe production of vector, a minimal p ackaging construct carrying only the coding sequences of the HIV-1 gag-pol, tat, and rev genes was stably introduced into 293G cells under the control of a Tet(o) minimal promoter. 293G cells express the chimeric Tet(R)/VP16 trans-activator and contain a tetracycline-regulated vesicular stomatitis v irus protein G (VSV-G) envelope gene. When the cells were grown in the pres ence of tetracycline the expression of both HIV-1-derived and VSV-derived p ackaging functions was suppressed. On induction, approximately 50 ng/ml/24 hr of Gag p24 equivalent of vector was obtained. After introduction of the transfer vector by serial infection, vector could be collected for several days with a transduction efficiency similar or superior to that of vector p roduced by transient transfection both for dividing and growth-arrested cel ls. The vector could be effectively concentrated to titers reaching 10(9) t ransducing units/ml and allowed for efficient delivery and stable expressio n of a GFP transgene in the mouse brain. The packaging cell line and all ve ctor producer clones described here were shown to be free from replication- competent recombinants, and from recombinants between packaging and vector constructs that transfer the viral gag-pol genes. The packaging cell line a nd the assays developed will advance lentiviral vectors toward the stringen t requirements of clinical applications.