A new-generation stable inducible packaging cell line for lentiviral vectors.
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.
Farson
D
D
Cell Genesys, Foster City, CA 94404, USA.
Witt
R
R
McGuinness
R
R
Dull
T
T
Kelly
M
M
Song
J
J
Radeke
R
R
Bukovsky
A
A
Consiglio
A
A
Naldini
L
L
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015139:Blotting, Southern; D001921:Brain / Q000378:metabolism; D002455:Cell Division; D002460:Cell Line; D015685:Fusion Proteins, gag-pol / Q000235:genetics; D015694:Gene Products, rev / Q000235:genetics; D015696:Gene Products, tat / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins; D051379:Mice; D008810:Mice, Inbred C57BL; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D013752:Tetracycline / Q000494:pharmacology; D013997:Time Factors; D014161:Transduction, Genetic; D014162:Transfection; D019076:Transgenes; D014759:Viral Envelope Proteins / Q000235:genetics; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
D000900:Anti-Bacterial Agents; D015685:Fusion Proteins, gag-pol; C028391:G protein, vesicular stomatitis virus; D015694:Gene Products, rev; D015696:Gene Products, tat; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins; D013752:Tetracycline