A new-generation stable inducible packaging cell line for lentiviral vectors

D. Farson, R. Witt, R. McGuinness, T. Dull, M. Kelly, J. Song, R. Radeke, A. Bukovsky, A. Consiglio, L. Naldini

Research output: Contribution to journalArticle

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 Teto minimal promoter. 293G cells express the chimeric TetR/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 109 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.

Original languageEnglish
Pages (from-to)981-997
Number of pages17
JournalHuman Gene Therapy
Volume12
Issue number8
DOIs
Publication statusPublished - 2001

ASJC Scopus subject areas

  • Genetics

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    Farson, D., Witt, R., McGuinness, R., Dull, T., Kelly, M., Song, J., Radeke, R., Bukovsky, A., Consiglio, A., & Naldini, L. (2001). A new-generation stable inducible packaging cell line for lentiviral vectors. Human Gene Therapy, 12(8), 981-997. https://doi.org/10.1089/104303401750195935