Generation of retroviral vector for clinical studies using transient transfection

Transient transfection of 293T cells was utilized to produce high-titer mur ine recombinant retroviral vectors for clinical studies. This system was in itially optimized by gene transfer using different retroviral envelope prot eins into activated human CD4(+) T lymphocytes in vitro, Higher titer and i nfectivity were obtained than with stable murine producer lines; titers of 0.3-1 x 10(7) infectious units per milliliter for vectors encoding the gree n fluorescent protein (GFP) were achieved. Virions pseudotyped with envelop e proteins from gibbon ape leukemia virus or amphotropic murine leukemia vi rus resulted in gene transfer of greater than or equal to 50% in CD4(+) hum an T lymphocytes with this marker. Gene transfer of Rev M10 with this vecto r conferred resistance to HIV infection compared with negative controls in the absence of drug selection, Thus, the efficiency of transduction achieve d under these conditions obviated the need to include selection to detect b iologic 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 10(7) IU/ml, These packaging cells can be used to generate high-t iter virus in sufficient quantities for clinical studies and will facilitat e the rapid, cost-effective generation of improved retroviral, lentiviral, or other viral vectors for human gene therapy.