A New-Generation Stable Inducible Packaging Cell Line for Lentiviral Vectors

Abstract

We have successfully generated and characterized a stable packaging cell line for HIV-1-based vectors. To allow safe production of vector, a minimal packaging 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 virus protein G (VSV-G) envelope gene. When the cells were grown in the presence of tetracycline the expression of both HIV-1-derived and VSV-derived packaging 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 produced by transient transfection both for dividing and growth-arrested cells. The vector could be effectively concentrated to titers reaching 10(9) transducing units/ml and allowed for efficient delivery and stable expression of a GFP transgene in the mouse brain. The packaging cell line and all vector 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 and the assays developed will advance lentiviral vectors toward the stringent requirements of clinical applications.

Lentiviral vectors pseudotyped with baculovirus gp64 efficiently transduce mouse cells in vivo and show tropism restriction against hematopoietic cell types in vitro

“…3 However, the high fusogenicity of VSV-G causes rapid syncytia formation and cell death, making it difficult to generate stable cell lines expressing the protein. 4 A number of groups have generated stable packaging and producer cell lines using VSV-G, but in all cases a repressible or inducible promoter regulates the expression of VSV-G. [4][5][6] While regulated expression avoids the problem of VSV-G cytotoxicity, it makes stable production of viral vectors more complicated and cell line generation more timeconsuming.…”

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.…”

“…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

“…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. Simian lentivirus system was successfully used for human DC transduction.…”