A stable system for the high-titer production of multiply attenuated lentiviral vectors

Lentiviral vectors open exciting perspectives for the genetic treatment of a wide array of inherited and acquired diseases, owing to their ability to govern the efficient delivery, integration, and long-term expression of tra nsgenes into nondividing cells both in vitro and in vivo. The genomic compl exity of HIV, where a whole set of genes encode virulence factors essential for pathogenesis but not required for gene transfer, allowed a major step toward clinical acceptability through the creation of multiply attenuated p ackaging systems. Until now, however, vector particles could only be produc ed by transient transfection because no high-output, stable packaging cell line was available that produced the latest generation of HIV-based vectors . Here we describe such a line, based on the doxycycline-repressible expres sion of HIV-1 Rev/Gag/Pol and of the vesicular stomatitis virus G envelope (VSV G) in 293 human embryonic kidney cells. Upon induction, the LVG clones can produce 1 to 20 HeLa-transducing units per cell per day for about a we ek, a yield that compares favorably with that of transiently transfected 29 3T cells. These virions exhibit functional properties similar to those of v iruses produced transiently, in particular the ability to transduce nonmito tic targets. This system will facilitate the further development of lentivi ral vectors for gene therapy.