To generate T cell-specific retroviral vectors an scFv phage display library derived from immunized mice was selected for binding to the human T cell line Molt-4/8. The scFv cDNAs recovered from the selected phages were transiently expressed as an N-terminal fusion of the spleen necrosis virus (SNV) transmembrane protein (TM) subunit of the viral envelope protein (Env) in the cell line DSH-cxl, which packages the beta-galactosidase gene into SNV particles. Screening of supernatants from about 150 transfections resulted in the identification of 5 scFvs that mediated efficient transduction of Molt-4/8 cells. Using stable packaging cell lines vector preparations with titers greater than 10(4) EFU/ml on human T cells were obtained. The scFv 7A5 in particular was able to mediate selective transduction of human T cells with high efficiency. Titers of up to 106 EFU/ml were reached on Molt-4/8, Jurkat, and A301 cells, while titers on HeLa cells, TE671 cells, 293T cells, and HT1080 cells were below 102 EFU/ml. Transduction of stimulated primary human peripheral blood cells, which consisted mainly of T cells, was about fivefold more efficient than transduction of B cells. Western blot analysis of supernatant from the 7A5 packaging cells demonstrated incorporation of 7A5-TM into vector particles and indicated proteolytic processing of the coexpressed unmodified TM during particle formation. Binding of bacterially expressed 7A5-scFv to a panel of cell lines correlated well with the transduction results. These data provide the first proof of concept that a general approach can be taken to obtain scFvs able to mediate selective gene transfer into target cells.
Retroviral vectors derived from amphotropic murine leukemia viruses (MLV) mediate gene transfer into almost all human cells and are thus not suitable for in vivo applications in gene therapy in which cell-specific gene delivery is required. We and others recently reported the generation of MLV-derived vectors pseudotyped by variants of the envelope glycoproteins (Env) of human immunodeficiency virus type 1 (HIV-1), thus displaying the CD4-dependent tropism of the parental lentivirus (Mammano et al., 1997, J. Virol. 71, 3341-3345; Schnierle et al., 1997, Proc. Natl. Acad. Sci. USA 76, 8640-8645). However, because of their HIV-1-derived envelopes these vectors are neutralized by HIV-specific antibodies present in some infected patients. To circumvent this problem, we pseudotyped MLV capsid particles with variants of Env proteins derived from the apathogenic simian immunodeficiency virus (SIVagm) of African green monkeys (AGM; Chlorocebus pygerythrus). Truncation of the C-terminal domain of the transmembrane protein was found to be necessary to allow formation of infectious pseudotype vectors. These [MLV(SIVagm)] vectors efficiently transduced various human CD4-expressing cell lines using the coreceptors CCR5 and Bonzo to enter target cells. Moreover, they were resistant to neutralization by antibodies directed against HIV-1. Therefore, [MLV(SIVagm)] vectors will be useful to study the mechanisms of SIVagm cell entry and for the selective gene transfer into CD4+ T-cells of AIDS patients.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.