Establishment of a Parvovirus B19 NS1-Expressing Recombinant Adenoviral Vector for Killing Megakaryocytic Leukemia Cells

Abstract

Adenoviral viral vectors have been widely used for gene-based therapeutics, but commonly used serotype 5 shows poor transduction efficiency into hematopoietic cells. In this study, we aimed to generate a recombinant adenovirus serotype 5 (rAd5) vector that has a high efficiency in gene transfer to megakaryocytic leukemic cells with anticancer potential. We first modified the rAd5 backbone vector with a chimeric fiber gene of Ad5 and Ad11p (rAd5F11p) to increase the gene delivery efficiency. Then, the nonstructural protein NS1 of human parvovirus B19 (B19V), which induces cell cycle arrest at the G2/M phase and apoptosis, was cloned into the adenoviral shuttle vector. As the expression of parvoviral NS1 protein inhibited Ad replication and production, we engineered the cytomegalovirus (CMV) promoter, which governs NS1 expression, with two tetracycline operator elements (TetO₂). Transfection of the rAd5F11p proviral vectors in Tet repressor-expressing T-REx-293 cells produced rAd in a large quantity. We further evaluated this chimeric rAd5F11p vector in gene delivery in human leukemic cells, UT7/Epo-S1. Strikingly, the novel rAd5F11p-B19NS1-GFP vector, exhibited a transduction efficiency much higher than the original vector, rAd5-B19NS1-GFP, in UT7/Epo-S1 cells, in particular, when they were transduced at a relatively low multiplicity of infection (100 viral genome copies/cell). After the transduction of rAd5F11p-B19NS1-GFP, over 90% of the UT7/Epo-S1 cells were arrested at the G2/M phase, and approximately 40%-50% of the cells were undergoing apoptosis, suggesting the novel rAd5F11P-B19NS1-GFP vector holds a promise in therapeutic potentials of megakaryocytic leukemia.

Keywords: adenoviral vector; anti-cancer; apoptosis; cell cycle arrest; parvovirus B19.

Figure 1

Modifications of the adenoviral backbone vector pacAd5 9.2-100. The proviral gene of units 9.2-100 of the Ad5 and chimeric fiber Ad are diagramed. The chimeric Ad5 and Ad11P fiber gene is indicated.

Figure 2

Modifications of the adenoviral shuttle vectors. (A,B) Modification of the CMV promoter. The transfer plasmid pacAd5 CMV-B19NS1-EGFP is diagrammed. Two tetracycline operator elements were inserted into the CMV promoter, which govern B19V NS1 expression. (C) Validation of the CMVTetO₂ promoter activity. HEK293 cells and T-REx-293 cells were transfected with pacAd5 CMV-B19NS1-GFP and pacAd5 CMVTetO₂-B19NS1-GFP, respectively. At 48 h post-transfection, cells were collected for Western blotting using anti-strep antibody to check B19V NS1 expression. Lane 1: HEK293 cells control; Lane 2: HEK293 cells transfected with pacAd5 CMV-B19NS1-GFP; Lane 3: HEK293 cells transfected with pacAd5 CMVTetO₂-B19NS1-GFP; Lane 4: T-REx-293 cells transfected with pacAd5 CMV-B19NS1-GFP; Lane 5: T-REx-293 cells transfected with pacAd5 CMVTetO₂-B19NS1-GFP; Lane 6: protein ladder.

Figure 3

Production of recombinant adenovirus expressing GFP and B19NS1-GFP. (A) The schematic flowchart of recombinant Ad production. Both the Ad shuttle and proviral backbone plasmids are transfected into cells. After homologous recombination, a rAd proviral genome is generated, which further replicates to produce rAd virions as diagramed. (B) Recombinant adenovirus production. T-REx-293 cells were co-transfected using linearized pacAd5 CMVTetO₂-GFP or pacAd5 CMVTetO₂-B19NS1-GFP with linearized pacAd5F11P 9.2-100. After one week, GFP expression was monitored under a Nikon Eclipse Ti-S inverted microscope at 10× magnification.

Figure 4

rAd5F11p-B19NS1-GFP is more effective than the parent rAd5 in the transduction of leukemia cells UT7/Epo-S1 at a relatively low MOI. UT7/Epo-S1 cells were transduced with rAd5-B19NS1-GFP or rAd5F11p-B19NS1-GFP at three different MOIs (100, 500, and 1000 vgc/cell), respectively. At 48 h post-transduction, GFP expression was monitored under a Nikon Eclipse Ti-S inverted microscope at 10× magnification.

Figure 5

rAd5F11p-B19NS1-GFP induces cell cycle arrest at the G2/M phase and apoptosis in UT7/Epo-S1 cells. UT7/Epo-S1 cells were transduced with rAd5F11p-GFP or rAd5F11p-B19NS1-GFP at an MOI of 100 vgc/cell. After 48 h post-transduction, (A) Western blotting. Transduced cells were collected for Western blot analysis using an anti-strep antibody. The same membrane was reprobed by using anti-β-actin antibody. (B,C) Cell cycle analysis and Fluorescent-Labeled Inhibitors of Caspases (FLICA). Transduced cells were fixed and stained using 4′,6-diamidino-2-phenylindole (DAPI) or FLICA caspase-9. Cell cycle and caspase-9 activity were detected by flow cytometry. (D,E) Transduced cells were stained with Annexin-V FITC and Propidium Iodide (PI), followed by flow cytometry analysis. The percentages of both early and late apoptotic cells are presented with averages and standard deviations, which were obtained from at least three independent experiments.