Generation of retroviral vector for clinical studies using transient transfection

Shiaolan Yang, Rafael Delgado, Steven R. King, Clive Woffendin, Christopher S. Barker, Zhi Yong Yang, Ling Xu, Garry P. Nolan, Gary J. Nabel

Research output: Contribution to journalArticlepeer-review

Abstract

Transient transfection of 293T cells was utilized to produce high-titer murine recombinant retroviral vectors for clinical studies. This system was initially optimized by gene transfer using different retroviral envelope proteins into activated human CD4+ T lymphocytes in vitro. Higher titer and infectivity were obtained than with stable murine producer lines; titers of 0.3-1 x 107 infectious units per milliliter for vectors encoding the green fluorescent protein (GFP) were achieved. Virions pseudotyped with envelope proteins from gibbon ape leukemia virus or amphotropic murine leukemia virus resulted in gene transfer of ≥ 50% in CD4+ human T lymphocytes with this marker. Gene transfer of Rev M10 with this vector conferred resistance to HIV infection compared with negative controls in the absence of drug selection. Thus, the efficiency of transduction achieved under these conditions obviated the need to include selection to detect biologic effects in T cells. Finally, a protocol for the production of large-scale supernatants using transient transfection was optimized up to titers of 1.9 x 107 IU/ml. These packaging cells can be used to generate high-titer virus in sufficient quantities for clinical studies and will facilitate the rapid, cost-effective generation of improved retroviral, lentiviral, or other viral vectors for human gene therapy.

Original languageEnglish (US)
Pages (from-to)123-132
Number of pages10
JournalHuman Gene Therapy
Volume10
Issue number1
DOIs
StatePublished - Jan 1 1999
Externally publishedYes

ASJC Scopus subject areas

  • Genetics

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