A Stable System for the High-Titer Production of Multiply Attenuated Lentiviral Vectors

Abstract

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 transgenes into nondividing cells both in vitro and in vivo. The genomic complexity 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 packaging systems. Until now, however, vector particles could only be produced 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 expression 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 week, a yield that compares favorably with that of transiently transfected 293T cells. These virions exhibit functional properties similar to those of viruses produced transiently, in particular the ability to transduce nonmitotic targets. This system will facilitate the further development of lentiviral vectors for gene therapy.

Development of inducible EIAV-based lentiviral vector packaging and producer cell lines

“…The level of regulation provided by coTetR, and the rate that vector is produced post-induction seems to be superior to other inducible systems, particularly the Tet-Off system, in which leaky expression was seen in the off-state. 16,21 Furthermore, the Tet-Off system relies on withdrawal of dox/tetracycline to relieve repression which takes a minimum of 4 days to induce vector production, 16,[18][19][20]25 and up to 13 days to reach maximal levels. 20 In this study, vector production is observed within 24 h of induction.…”

“…16,21 Furthermore, the Tet-Off system relies on withdrawal of dox/tetracycline to relieve repression which takes a minimum of 4 days to induce vector production, 16,[18][19][20]25 and up to 13 days to reach maximal levels. 20 In this study, vector production is observed within 24 h of induction. The timing of dox addition and harvest is being further investigated with a view to optimising vector yields and collection of multiple harvests.…”

“…Stable packaging and producer cell lines have been developed for the production of human immunodeficiency virus 1-based LVs [14][15][16][17][18][19][20][21][22][23][24][25] but, to date, none have been described for EIAV-based LVs. The glycoprotein of the vesicular stomatitis virus (VSV-G) is often used for pseudotyping LVs because of its high stability, broad tropism and generation of high-titre viral stocks.…”

“…However, a complicating factor is that VSV-G is cytotoxic 26,29 and therefore its expression must be regulated. For many of the human immunodeficiency virus 1 based packaging cell lines VSV-G expression is controlled using tetracycline-inducible systems, 15,16,18,20,21,24 enabling VSV-G expression to be switched on at the time of vector production. In these systems, gene expression is controlled by expression of the tetracycline transactivator fusion protein comprising the tetracycline repressor (TetR) and the transcription activation domain of VP16.…”

Vectors for the treatment of autoimmune disease

“…This method provides stable cell lines, capable of controlled production of high-titre stocks of lentiviral particles. 117 A similar method, but with all the viral genes expressed from regulated promoters was also recently described. 118 Lentiviral vectors based on viruses other than HIV-1 have been made including HIV-2, 119 bovine, 120 feline, 121 and simian 122,123 lentiviruses.…”