Enhanced baculovirus-mediated transduction of human cancer cells by tumor-homing peptides

Abstract

Tumor cells and vasculature offer specific targets for the selective delivery of therapeutic genes. To achieve tumor-specific gene transfer, baculovirus tropism was manipulated by viral envelope modification using baculovirus display technology. LyP-1, F3, and CGKRK tumor-homing peptides, originally identified by in vivo screening of phage display libraries, were fused to the transmembrane anchor of vesicular stomatitis virus G protein and displayed on the baculoviral surface. The fusion proteins were successfully incorporated into budded virions, which showed two- to fivefold-improved binding to human breast carcinoma (MDA-MB-435) and hepatocarcinoma (HepG2) cells. The LyP-1 peptide inhibited viral binding to MDA-MB-435 cells with a greater magnitude and specificity than the CGKRK and F3 peptides. Maximal 7- and 24-fold increases in transduction, determined by transgene expression level, were achieved for the MDA-MB-435 and HepG2 cells, respectively. The internalization of each virus was inhibited by ammonium chloride treatment, suggesting the use of a similar endocytic entry route. The LyP-1 and F3 peptides showed an apparent inhibitory effect in transduction of HepG2 cells with the corresponding display viruses. Together, these results imply that the efficiency of baculovirus-mediated gene delivery can be significantly enhanced in vitro when tumor-targeting ligands are used and therefore highlight the potential of baculovirus vectors in cancer gene therapy.

FIG. 1.

(A) Schematic representation of the recombinant baculovirus constructs. (B) A schematic illustration of a recombinant baculovirus displaying the LyP-1-VSVG, F3-VSVG, or CGKRK-VSVG fusion proteins on the viral envelope. Abbreviations: gp64ss, the signal sequence of AcMNPV gp64; LyP-1, F3, and CGKRK, targeting peptides; polyAla, a linker sequence encoding 20 alanine residues; VSVG TM, transmembrane/cytoplasmic domains of vesicular stomatitis virus G protein; p10, polh, and SV40, promoters of the p10, polyhedrin, and simian virus 40 genes, respectively.

FIG. 2.

(A) Expression of the LyP-1-VSVG, F3-VSVG, and CGKRK-VSVG fusion proteins on the surface of infected Sf9 cells at 38 h p.i. Fusion proteins were detected with rabbit anti-VSVG-tag antibody and analyzed by confocal microscopy. Images are single confocal optical midsections of cells, approximately 0.8 μm in thickness. Differential interference contrast and fluorescence images are merged. Scale bar, 10 μm. (B) Bioluminescence of luciferase-expressing infected Sf9 cells in microtiter plate wells at 38 h p.i. The images were captured with a digital camera (Nikon Coolpix 4100). (C) Immunoblot analysis of the recombinant baculoviruses (10⁸ PFU) displaying LyP-1-VSVG, F3-VSVG, and CGKRK-VSVG fusion proteins on the viral envelope. Ac-luc served as a negative control. Viral proteins were probed under reducing conditions with rabbit anti-VSVG-tag antibody. (D) Determination of the ratio of total particle number versus the count of infectious virus (2.5 × 10⁷ PFU) by immunoblot analysis with AcMNPV anti-vp39 MAb.

FIG. 3.

Flow cytometric analysis of the binding of AcLyP-1-luc, AcF3-luc, AcCGKRK-luc, and the control virus, Ac-luc (100 PFU/cell), to the surface of MDA-MB-435 and HepG2 cells. Viral binding was detected with mouse anti-gp64 MAb. Normalized mean fluorescence values (Ac-luc = 1) for each virus ± standard deviations of triplicate samples are indicated. The data were compared using the unpaired Student t test with a two-tailed P value, and statistical significance was determined. ***, P < 0.001; **, P < 0.002.

FIG. 4.

(A) Viral transduction efficiency of MDA-MB-435 and HepG2 cells measured by luciferase activity. The cells were transduced with Ac-luc (control), Ac-LyP-1-luc, AcF3-luc, and AcCGKRK-luc viruses, followed by monitoring the luciferase activity at 24 and/or 48 h p.t. Mock-infected cells were used as the background control. The PFU counts used are indicated. The results are shown as means of relative luciferase activity (Ac-luc = 1) ± standard deviations of three individual experiments, each performed in triplicate per variable. The data were compared using the unpaired Student t test with a two-tailed P value, and statistical significance was determined; **, P < 0.05. (B) Expression profile of Ac-luc, Ac-LyP-1-luc, AcF3-luc, and AcCGKRK-luc viruses (100 PFU/cell) in HepG2 cells at 0 to 36 h p.t. The luciferase activity (relative light units [RLU]) was monitored every 2 h.

FIG. 5.

Effect of ammonium chloride on gene transduction in HepG2 cells at 24 h p.t. Transduction efficiency of the Ac-LyP-1-luc, AcF3-luc, and AcCGKRK-luc display viruses and the control virus, Ac-luc, was monitored by measuring luciferase activity. Influence of ammonium chloride per se on the luminescence signal was determined by addition of 10 mM ammonium chloride to transduced control cells 1 h prior to harvesting (single values on the right).

FIG. 6.

Cross-inhibitory effect of the synthetic LyP-1 (A), F3 (B), and CGKRK (C) peptides on the binding of recombinant viruses AcLyp-1-luc (A), AcF3-luc (B), and AcCGKRK-luc (C) and control virus Ac-luc (A to C) to MDA-MB-435 cells. Cells were incubated with the corresponding soluble peptides (indicated in the figure) prior to virus attachment at 4°C followed by detection of the bound viruses by flow cytometry using anti-gp64 MAb. The data represent the means ± standard deviations of three independent experiments, each performed in duplicate.

FIG. 7.

Inhibitory effect of the synthetic LyP-1 (A), F3 (B), and CGKRK (C) peptides on the transduction efficiency of AcLyP-1-luc, AcF3-luc, and AcCGKRK-luc, respectively. HepG2 cells were preincubated with the corresponding peptides at 4°C prior to addition of the virus (100 PFU/cell). An irrelevant RKK peptide was used as a control (not shown). The luciferase activity was monitored at 24 h p.t. The peptide concentrations and viral constructs are indicated.