Oncoretrovirus and lentivirus vectors pseudotyped with lymphocytic choriomeningitis virus glycoprotein: generation, concentration, and broad host range

Abstract

Lymphocytic choriomeningitis virus (LCMV) is a noncytopathic arenavirus shown to infect a broad range of different cell types. Here, we combined the beneficial characteristics of the LCMV glycoprotein (LCMV-GP) and those of retroviral vectors to generate a new, safe, and efficient gene transfer system. These LCMV-GP pseudotypes were systematically compared with vectors containing the widely used amphotropic murine leukemia virus envelope (A-MLVenv) or the vesicular stomatitis virus G protein (VSV-G). Production of LCMV-GP-pseudotyped oncoretroviral and lentiviral vectors by transient transfection resulted in vector titers similar to those with A-MLVenv or VSV-G. In contrast to A-MLVenv particles, LCMV-GP pseudotypes could be efficiently concentrated by ultracentrifugation without loss of vector titer. Unlike the cell-toxic VSV-G, a stable retroviral packaging cell line constitutively expressing LCMV-GP could be established. Vectors pseudotyped with LCMV-GP efficiently transduced many cell lines from different species and tissues relevant for gene therapy. Transduction of human glioma cells was studied in detail. These cells are a major target for cancer gene therapy and were transduced more efficiently with LCMV-GP-pseudotyped vectors than with the generally used A-MLVenv particles. The high stability, low toxicity, and broad host range make LCMV-GP-pseudotyped vectors attractive for gene transfer applications. The recombinant LCMV-GP-pseudotyped vectors will also allow functional characterization of naturally occurring and recombinant LCMV-GP variants.

FIG. 1.

Stable retroviral packaging cell lines that constitutively produce LCMV-GP-pseudotyped retroviruses. (A) The envelope protein-negative packaging cell line TELCeB was transfected with LCMV-GP and a zeocin resistance expression plasmid. After selection with zeocin, the pseudotype titers of clone TELCeB-GP #2-3 were analyzed for several weeks of cell culture by end-point dilutions on TE671, the parental cell line of TELCeB. Results are shown as TU of the lacZ marker gene per milliliter, which was carried by the retroviral vector. A cell clone from mock-transfected cells (TELCeB) as well as parental TELCeB cells, which were cocultivated with clone #2-3 supernatants (TELCeB+ #2-3 supernatants), were analyzed in parallel. (B) Fluorescence-activated cell sorter analysis of TELCeB-GP #2-3. Mock-transfected cells (shaded lines) or TELCeB-GP #2-3 cells (open lines) were analyzed by flow cytometry with the LCMV-GP-directed monoclonal antibody KL25.

FIG. 2.

LCMV-GP-pseudotyped vectors are superior to A-MLVenv particles for transduction of human glioma cell lines. Target cells were transduced with serial dilutions of LCMV-GP-, VSV-G-, or A-MLVenv-pseudotyped oncoretroviral vectors encoding eGFP. Transduced, eGFP-positive cells were quantified by flow cytometry. Results are the mean vector titers and standard deviations from three end-point dilutions.

FIG. 3.

LCMV-GP-pseudotyped vectors transduce primary human glioma cells. Cells from a primary human glioblastoma were resuspended and directly plated on coverslips. The cells we transduced 4 days after plating with LCMV-GP-pseudotyped lentiviral vectors encoding eGFP. Three days after transduction, the cells were analyzed by immunostaining with monoclonal anti-eGFP antibody (A) and polyclonal anti-GFAP antibody (B). Shown are the phase contrast (C) and the eGFP/GFAP overlay (D). Bar, 100 μm.