A Packaging Cell Line for Lentivirus Vectors

Abstract

Lentivirus vectors can transduce dividing and nondividing cells. Using three-plasmid transient transfections, high-titer (>109 IU/ml) recombinant lentivirus vectors pseudotyped with vesicular stomatitis virus G (VSV-G) protein can be generated (T. Kafri et al., Nat. Genet. 17:314–317, 1997; H. Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319–10323, 1997; L. Naldini et al., Science 272:263–267, 1996). The recombinant lentiviruses can efficiently infect brain, liver, muscle, and retinal tissue in vivo. Furthermore, the transduced tissues demonstrated long-term expression of reporter genes in immunocompetent rodents. We now report the generation of a tetracycline-inducible VSV-G pseudotyped lentivirus packaging cell line which can generate virus particles at titers greater than 106 IU/ml for at least 3 to 4 days. The vector produced by the inducible cell line can be concentrated to titers of 109 IU/ml and can efficiently transduce nondividing cells in vitro and in vivo. The availability of a lentivirus packaging cell line will significantly facilitate the production of high-titer lentivirus vectors for gene therapy and study of human immunodeficiency virus biology.

Generation of a Replication-Competent, Propagation-Deficient Virus Vector Based on the Transmissible Gastroenteritis Coronavirus Genome

“…Although the virus titers generated by complementation do not generally reach the titers provided by wild-type viruses (23,30,50), the virus titers described in this paper are closer to those of the retroviral packaging cell lines, one of the most developed systems, producing virus titers in the range of 10 5 to 10 8 PFU/ml (26,33,34,35,46,51). The system reported in this paper represents highly significant progress in relation to previously reported coronavirus packaging cell systems owing to (i) its higher expression levels (1,000-fold), (ii) the introduction of stably transformed packaging cell lines that provide better safety and realistic application in vaccine development, (iii) quantification of E protein and virus production levels, and (iv) characterization of the replication-competent, propagation-deficient virus in terms of physical and chemical stability and morphogenesis analyzed by electron microscopy.…”

“…Flavivirus (24,30), adenovirus (27,50), lentivirus (23,37), and recently coronavirus (6) have been successfully complemented in packaging cell lines. This type of packaging cell has been developed using noncytopathic Sindbis virus replicon systems (17,24,25,30), Semliki Forest virus-based vectors (39), Venezuelan equine encephalitis virus replicon particles (6), and stably transformed cell lines by placing the genes under noninducible (50) or inducible tetracycline or ecdysone promoters (23,37).In this article, we report the efficient trans-complementation of a recombinant TGEV (rTGEV) deficient in the essential E gene by packaging cell lines expressing the E protein (E ϩ cells). Packaging cell lines were established by using the noncytopathic Sindbis virus replicon or by stable expression of the E gene under the cytomegalovirus (CMV) promoter.…”

“…Complementation of deficient viruses is frequently used to generate safe vectors for vaccination and gene therapy. Flavivirus (24,30), adenovirus (27,50), lentivirus (23,37), and recently coronavirus (6) have been successfully complemented in packaging cell lines. This type of packaging cell has been developed using noncytopathic Sindbis virus replicon systems (17,24,25,30), Semliki Forest virus-based vectors (39), Venezuelan equine encephalitis virus replicon particles (6), and stably transformed cell lines by placing the genes under noninducible (50) or inducible tetracycline or ecdysone promoters (23,37).…”

Hematopoietic Stem cell transplantation and lentiviral vector‐based gene therapy for Krabbe's disease: Present convictions and future prospects

“…The lentiviral vector packaging cassette DNRF and the VSV-G envelope plasmid were described previously (Kafri et al, 1999). Promoter sequences and codon-optimized GALC cDNA's were synthesized by GeneArt (Thermo Fisher).…”

Viruses and Virus-Like Particles in Biotechnology: Fundamentals and Applications

“…The major problem in the production of lentiviruses has been the development of a packaging cell line. Stable expression of lentivirus particles has proven to be more difficult than that of oncoviruses [117,118], partly due to the expression of proteins such as rev and viral proteases which appear to be toxic to cells [119]. Consequently, lentiviral vectors have been produced by transient transfection of high-expressing cell lines such as COS [120] and 293T [121,122] generally using four different plasmids (gag-pol, env, rev, and lv-vector).…”