Enhanced gene transfer with fusogenic liposomes containing vesicular stomatitis virus G glycoprotein

Abstract

Exposure of Lipofectin-DNA complexes to the partially purified G glycoprotein of the vesicular stomatitis virus envelope (VSV-G) results in loss of serum-mediated inhibition and in enhanced efficiency of gene transfer. Sucrose density gradient sedimentation analysis indicated that the VSV-G associates physically with the DNA-lipid complex to produce a VSV-G liposome. The ability to incorporate surrogate viral or cellular envelope components such as VSV-G into liposomes may allow more-efficient and possibly targeted gene delivery by lipofection, both in vitro and in vivo.

FIG. 1

SDS-polyacrylamide gel electrophoresis of partially purified VSV-G. The conditioned medium of 293 cells transfected with pCMV-G was centrifuged, and the resulting pellet was examined by electrophoresis (lane 1). The pellet suspension was subjected to velocity sucrose gradient, and VSV-G-containing fractions were pooled, diluted with PBS, and repelleted. The repurified pellet suspension was analyzed as described above (lanes 2 and 3). About 100 ng of protein in each VSV-G preparation was loaded and visualized by silver staining (lanes 1 and 2) and by Western blot analysis with anti-VSV-G monoclonal antibody, P5D4 (lane 3).

FIG. 2

The effect of increasing amounts of the partially purified VSV-G on lipofection efficiency in BHK and 208F cells (6). Increasing amounts of partially purified VSV-G in the form of pelleted conditioned medium from transfected 293 cells were added to the DNA-lipid complex (2.5 μg of Lipofectin and 0.5 μg of pCMV-luc per well) immediately prior to addition to cells. Transfection was performed with 12-well plates in DMEM with 10% FBS. The experiment was performed in triplicate, and the data are means ± standard deviations.

FIG. 3

Effect of the concentration of serum in culture medium on VSV-G-induced lipofection efficiency. Aliquots of partially purified VSV-G (200 ng) were added to the DNA-lipid complex immediately before addition to cells in 12-well plates in DMEM supplemented with various concentrations of FBS. The experiment was performed in triplicate, and the data are means ± standard deviations.

FIG. 4

Buoyant-density analysis by sucrose density gradient centrifugation of uncomplexed VSV-G, Lipofectin-DNA complex, and Lipofectin–DNA–VSV-G complexes. (A) Luciferase activities (represented by the total activity of each fraction) in BHK cells transfected with gradient fractions containing the Lipofectin-DNA complex or the Lipofectin–DNA–VSV-G complex; (B) Western blot analysis of fractions from the gradient containing pelleted VSV-conditioned medium from transfected, VSV-G-producing 293 cells and from a gradient containing the Lipofectin–DNA–VSV-G liposome. VSV-G protein was detected with the anti-VSV-G monoclonal antibody P5D4. Sucrose densities are indicated as the densities in parallel gradients.