Generation of high-titer retroviral vector-producing macrophages as vehicles for in vivo gene transfer

The goal of this project was to develop a novel gene transfer system based on macrophages (M phi) as shuffles of recombinant retroviral vectors carryi ng therapeutic or marker genes. The murine M phi cell line WGL5 was used as a source of M phi for this study. We generated retrovirus-producing M phi by transducing the WGL5 cells with a replication-defective retroviral vecto r carrying the enhanced green fluorescent protein (EGFP) reporter gene and the Moloney murine leukemia virus (MoMLV) as helper virus. We demonstrated stable integration of the recombinant retrovirus in the M phi genome, effic ient recombinant retrovirus production, and EGFP gene delivery to different cell lines in vitro. To evaluate M phi -mediated EGFP gene transfer in viv o, allogeneic mice were injected s.c. with the retrovirus-producing WGL5 M phi, that gave rise to solid tumor masses at the injection site, highly inf iltrated with host leukocytes. We observed EGFP fluorescence in tumor-infil trating CD4(+) and CD8(+) host T lymphocytes, providing direct evidence of the ability of engineered M phi to mediate EGFP gene delivery to host cells in vivo. Moreover, we showed that retrovirus-producing M phi could home to different organs in vivo following i.v. injection into mice. These data de monstrate that M phi can be engineered as cellular vehicles for recombinant retroviruses carrying heterologous genes and suggest potential application s of this novel vector system for gene therapy.