Developing adenoviral vectors encoding therapeutic genes toxic to host cells: comparing binary and single-inducible vectors expressing truncated E2F-1

Abstract

Adenoviral vectors are highly efficient at transferring genes into cells and are broadly used in cancer gene therapy. However, many therapeutic genes are toxic to vector host cells and thus inhibit vector production. The truncated form of E2F-1 (E2Ftr), which lacks the transactivation domain, can significantly induce cancer cell apoptosis, but is also toxic to HEK-293 cells and inhibits adenovirus replication. To overcome this, we have developed binary- and single-vector systems with a modified tetracycline-off inducible promoter to control E2Ftr expression. We compared several vectors and found that the structure of expression cassettes in vectors significantly affects E2Ftr expression. One construct expresses high levels of inducible E2Ftr and efficiently causes apoptotic cancer cell death by activation of caspase-3. The approach developed in this study may be applied in other viral vectors for encoding therapeutic genes that are toxic to their host cells and/or inhibit vector propagation.

Figure 1. Schematic diagram of binary- and single Adv systems

(A) CMV promoter drives the expression of the tTA protein. EGFP or E2Ftr is under the control of a synthetic minimal promoter composed of Tet-responsive element (TRE) and CMV mini-promoter (phcmv), which is silent unless activated by tTA. In the absence of Tet or Dox, tTA binds to phcmv and triggers the expression of EGFP or E2Ftr. When Tet is added to the medium, tTA is bound by Tet and unable to bind to phcmv and activate the expression of EGFP or E2Ftr. SK-MEL-2 cells in the absence or presence of Dox (1μg/ml) were infected with AdTet-EGFP at a MOI of 50. After 24 hours, cells were observed for EGFP expression under fluorescence microscopy and photographs were taken with Kodak MDS 290 software at × 20 magnification. (B) Schema of three single bicistronic Ads encoding E2Ftr, the left and right inverted terminal repeat sequences (LITR or RITR, respectively); encapsidation signal (ES) and E1/E3 deleted genes are shown in the Ad structure.

Figure 2. Regulation of E2Ftr expression from binary- and single Adv systems

(A) SK-MEL-2 cells in the absence or presence of Dox (1μg/ml) were infected with Ad-E2Ftr at a MOI of 50 or co-infected with Adhv at a MOI of 5. After 48 hours, cells were harvested and protein extracts were used for a western blot assay. (B) SK-MEL-2 cells in the absence or presence of Dox (1μg/ml) were infected with AdTet-E2Ftr1, AdTet-E2Ftr2, and AdTet-E2Ftr3 at a MOI of 50. After 48 hours, cells were harvested and protein extracts were used for a western blot assay. (C) SK-MEL-2 cells in the absence or presence of Dox (1μg/ml) were co-infected with AdTet-E2Ftr1, AdTet-E2Ftr2, and AdTet-E2Ftr3 at a MOI of 50 and Adhv at a MOI of 5. After 48 hours, cells were harvested, protein extracts were used for a western blot assay, and a mAb mouse-antihuman E2F-1 was used to detect E2Ftr. α-actin was used to demonstrate equal loading for each lane. Similar results were obtained in two additional experiments.

Figure 3. Effect of E2Ftr expresssion on Ad yield and lytic plaque formation

HEK-293 cells in either the absence or presence of Dox (1μg/ml) were infected with AdTet-EGFP, AdTet-E2Ftr1, AdTet-E2Ftr2, or AdTet-E2Ftr3 at a MOI of 5. After 72 hours, cells were harvested and divided in two samples; one sample was used to conduct Western Blot analysis and the second sample was used to determine virus titers. (A) For Western blot, a mAb mouse-antihuman E2F-1 was used to detect E2Ftr. α-Actin was used to demonstrate equal loading for each lane. (B) For virus titering, cultures were collected at 72 hours after infection and went through three cycles of freezing and thawing to release viruses from the cells. Lysates were serially diluted to determine the titers in HEK-293 cells. The dashed line shows the level of viruses added in the cultures. Each point represents the mean of three independent experiments ± standard deviation (SD; bars). C) HEK-293 cells were cultured in presence or absence of Dox (1 μg/ml) and infected with AdTet-EGFP or AdTet-E2Ftr3 at a MOI of 5; a standard plaque assay was performed to assess formation of lytic plaques. A representative experiment is shown from three performed.

Figure 4. Comparison between Dox and Tet in HEK-293 survival E2Ftr regulation, and Ad yield

(A) Toxicity of Dox vs Tet at 0, 1, 5, and 10 μg/ml of concentration were compared in HEK-293 cells by a MTT assay at 72 hours after drug treatments. Each point represents the mean of three independent experiments ± (SD; bars). (B) HEK-293 cells in the presence of Dox or Tet at 0, 1, 5, and 10 μg/ml were infected with AdTet-E2Ftr3 at a MOI of 5. At 24 hours after infection, cell extracts were used for western blot assay and a mAb mouse-antihuman E2F-1 was used to detect E2Ftr. α-Actin was used to demonstrate equal loading for each lane. C) HEK-293 cells were infected with AdTet-E2Ftr3 at a MOI of 5. Ad yield was determined in the presence of Dox or Tet at 0, 1, 5, and 10 μg/ml of concentration. The cultures were collected three days after infection and went through three cycles of freezing and thawing to release viruses from the cells. Lysates were serially diluted to determine the titers in HEK-293 cells. The dashed line shows the level of viruses added in the cultures. Each point represents the mean of three independent experiments ± (SD; bars).

Figure 5. Effect of E2F-1 or E2Ftr expressions on melanoma cell morphology

SKMEL-28 cells were uninfected (mock) or infected at a MOI of 100 with Ad-LacZ, Ad-E2F-1, AdTet-E2Ftr3, or Ad-E2Ftr alone or co-infected with Adhv at a MOI of 5. After 24 hours, cell morphology was analyzed by light microscopy. Apoptotic cells are indicated by arrowhead; vesiculated cells are indicated by arrow. Pictures were taken with bright-field optics at × 40 magnification.

Figure 6. Evaluation of apoptosis induced by three different Adv expressing E2F-1 or E2Ftr

SK-MEL-28 cells were uninfected (mock) or infected at a MOI of 100 with Ad-LacZ, Ad-E2F-1, AdTet-E2Ftr3, or Ad-E2Ftr alone or co-infection with Adhv at a MOI of 5. After 72 hours, cells were analyzed for activation of the caspases pathway and apoptosis. (A) Western blot was used to detect activation of the caspases pathway, a mAb mouse-antihuman E2F-1 was used to detect E2Ftr, a mAb mouse-antihuman PARP was used to detect PARP, and a mAb mouse-antihuman caspase-3/CPP3 was used to detect caspase-3. α-actin was used to demonstrate equal loading for each lane. (B) Hoechst staining was performed by adding Hoechst 33258 dye to a final concentration of 10μM to each well in the 24-well plate. Condensation and fragmentation of nuclei (examples indicated by white arrows) were observed under a fluorescence microscope (Olympus Microsystems) at × 20 magnification. (C) For annexin V staining, cells were stained with annexin V-PE and 7-Amino-actinomycin D (7-AAD); positive cells for annexin V-PE and 7-AAD staining were analyzed by FACScan flow cytometer with FlowJo software. Similar results were obtained in three independent experiments. A representative experiment is shown.

Figure 7. Assessment of melanoma cell killing activity from Adv encoding E2F-1 or E2Ftr

SK-MEL-28 cells were infected with (A) Ad-E2Ftr alone (at a MOI of 100) or co-infected with Adhv at a MOI of 5 or (B) uninfected (mock) or infected at a MOI of 100 with Ad-LacZ, Ad-E2F-1, AdTet-E2Ftr3. At 72 hours after infection, cell viability was analyzed by MTT assay; each point represents the mean of three independent experiments ± (SD; bars).