Retroviral Gene Transfer Using Safe and Efficient Packaging Cell Lines

Abstract

One of the requirements for the use of retroviral vectors in human gene therapy is a packaging cell line which is incapable of producing replication-competent virus and which produces high titers of replication-deficient vector virus. Wild-type virus may be produced through recombinational events between the helper virus and a retroviral vector. We have constructed an ecotropic packaging cell line, GP + E-86, and an amphotropic packaging cell line, GP + envAm12, in which the viral gag and pol genes are on one plasmid and the viral env gene is on another plasmid. Both plasmids contain deletions of the packaging sequence and the 3' LTR. The fragmented helper virus genomes, when introduced into 3T3 cells, produce titers of retrovirus which are comparable to the titers produced from packaging cells containing the helper virus genome on a single plasmid. We have found no evidence for the generation of wild-type retrovirus using the GP + E-86 and GP + envAm12 packaging lines, either alone or in combination with the N2 retroviral vector. We also show that these packaging cell lines can be used to transfer the neoR gene of the N2 vector into mouse hematopoietic cells, followed by successful (48-52%), long-term (up to 200 days) transplantation into irradiated recipients. These results indicate that these packaging lines are safe and efficient for use in experiments designed for murine (using GP + E-86) and human (using GP + envAm12) gene therapy.

The treatment of established intracranial tumors by in situ retroviral IFN-γ transfer

“…37 The ecotropic retroviral packaging cell line GP+E-86 6,38 and NIH 3T3 cells (ATCC CCL 163) were also cultured as previously described. 24,39 Packaging cells and retroviral infected NIH 3T3 cells resistant to geneticin G418 (Life Technologies, Melbourne, Australia) were selected and maintained in medium supplemented with this antibiotic at concentrations of 1 and 0.4 mg/ml of medium, respectively.…”

“…Isolation of retrovirus producing GP+E-86 clones Transfection of the GP+E-86 ecotropic retroviral packaging cells (RPCs) 38 with the pZIG(IFN-␥) DNA was performed using Lipofectamine Plus (Life Technologies) reagent according to the manufacturer's protocol. GP+E-86 cells were also transfected with pZIG(SV) DNA (a retrovirus control plasmid that lacks a cytokine gene) by calcium phosphate transfection as previously described.…”

Construction of new retroviral producer cells from adenoviral and retroviral vectors

“…MoMLV-based vectors were either transfected into an intermediate ecotropic packaging cell line GP+E-86 38,39 using calcium phosphate transfection as described above or lipofectamine transfection (Life Technologies, Gibco-BRL, Gaithersburg, MD, USA) and resultant virus used to infect an amphotrophic packaging cell line PA317, 21 or directly transfected into PA317. The three vectors used were as follows: vector pLN which contains neoR, 40 vector pLNPOZ which contains neoR and lacZ 41 and vector pLNCX-␤.…”

Ectopic expression of a mutant form of PKCα originally found in human tumors: aberrant subcellular translocation and effects on growth control

“…This vector contains the viral long terminal repeats from the Moloney murine leukemia virus, a puromycine resistance gene (controlled by the simian SV40 virus promoter) and encapsidation sequences. The pBabe-PKCa constructs were transfected by the calcium phosphate precipitation technique into the amphotropic GPAM 12 cell line (Markowitz et al, 1990). GPAM12 cell lines are NIH3T3 derivatives that contain a helper virus that allowed the generation of defective Moloney murine leukemia virus particles which were then used to infect early passage R6 cells.…”

“…Brie¯y, the full length cDNA encoding hPKCa-wt (Finkenzeller et al, 1990) and hPKCa-mut were cloned directionally into the multiple cloning site of the retroviral expression vector pBabe (Markowitz et al, 1990;Morgenstern and Land, 1990). This vector contains the viral long terminal repeats from the Moloney murine leukemia virus, a puromycine resistance gene (controlled by the simian SV40 virus promoter) and encapsidation sequences.…”