A conditionally replicative adenovirus, CRAd-S-pK7, can target endometriosis with a cell-killing effect

Abstract

Background: Novel therapeutic approaches for endometriosis based on molecular strategies may prove to be useful. Conditionally replicative adenoviruses (CRAds) are designed to exploit key differences between target and normal cells. The wild-type adenovirus (Adwt) promoter can be replaced by tissue-specific promoters, allowing viral replication only in target cells. Viral infectivity can be enhanced by altering Ad tropism via fiber modification. We investigated whether CRAds can be used to target endometriosis and determined the most efficient transcriptional- and transductional-targeting strategy.

Methods: An in vitro study was carried out using human endometriotic cell lines, 11Z (epithelial) and 22B (stromal), normal human ovarian surface epithelial cell line (NOSE006) and primary human endometriosis cells. A total of 9 promoters and 12 Ad tropism modifications were screened by means of a luciferase reporter assay. From this screening data, three CRAds (CRAd-S-pK7, CRAd-S-RGD, CRAd-S-F5/3sigma1, all incorporating the survivin promoter but with different fiber modifications) were selected to perform experiments using Adwt and a replication-deficient virus as controls. CRAds were constructed using a plasmid recombination system. Viral-binding capacity, rates of entry and DNA replication were evaluated by quantitative real-time PCR of viral genome copy. Cell-killing effects were determined by crystal violet staining and a cell viability assay for different concentrations of viral particles per cell.

Results: Comparison of promoters demonstrated that the survivin promoter exhibited the highest induction in both endometriotic cell lines. Among the fiber-modified viruses, the polylysine modification (pK7) showed the best infection enhancement. CRAd-S-pK7 was validated as the optimal CRAd to target endometriosis in terms of binding ability, entry kinetics, DNA replication and cell-killing effect. CRAd-S-pK7 also exhibited a high level of DNA replication in primary endometriosis cells.

Conclusions: CRAd-S-pK7 has the best infection and cell-killing effect in the context of endometriosis. It could prove to be a useful novel method to target refractory cases of endometriosis.

Figure 1

Transcriptional activity in endometriotic cell lines. Luciferase activity by transcriptionally targeted Ads at an MOI of 100 vp/cell, expressed as a percent of Ad5luc activity in (A) 11Z and (B) 22B. Luciferase activities of the candidate promoters are expressed as a percentage of the CMV promoter activity. Each bar represents the mean of three experiments ± SD. *P < 0.05 versus other viruses.

Figure 2

Transductional activity in endometriotic cell lines. Luciferase activity by transductionally targeted Ads at an MOI of 100 vp/cell, expressed as a percent of control virus activity in (A) 11Z and (B) 22B. Each bar represents the mean of three experiments ± SD. *P < 0.05 versus other viruses.

Figure 3

Viral-binding ability in endometriotic cell lines. Viral binding at an MOI of 1000 vp/cell, expressed as E4 gene copy numbers normalized to β-actin in (A) 11Z and (B) 22B. Each bar represents the mean of three experiments ± SD. *P < 0.05 versus other viruses.

Figure 4

Entry kinetics in endometriotic cell lines. Viral entry rates at 1 and 2 h after infection at an MOI of 1000 vp/cell, expressed as E4 gene copy numbers normalized to β-actin in (A) 11Z and (B) 22B.

Figure 5

Viral replication in endometriotic cell lines. Cells were infected with CRAd-S-pK7, CRAd-S-RGD, CRAd-S-F5/3σ1, Adwt and Ad5luc at an MOI of 100 vp/cell. Cells and growth medium were harvested on Days 1, 3 and 6 after infection and then subjected to qPCR. E4 gene copy numbers were normalized to β-actin in (A) 11Z and (B) 22B. Each bar represents the mean of three experiments ± SD. *P < 0.05 versus other viruses.

Figure 6

Viral cytopathic assay in endometriotic cell lines and control cell line (NOSE006). Cells were infected at different vp/cell (1, 10, 100 and 1000). Cells were stained with crystal violet after 6 days of incubation. (A) 11Z, (B) 22B and (C) NOSE006.

Figure 7

MTS assay in endometriotic cell lines, (A) 11Z and (B) 22B. OD is the difference between the reading obtained at 490 nm and the background reading at 630 nm. Each bar represents the mean of three experiments ± SD. *P < 0.05 versus other CRAds.

Figure 8

CRAds replicate in primary endometriosis cells. CRAd-S-pK7 has the best DNA replication profile. Each bar represents the mean of three experiments ± SD. *P < 0.05 versus other viruses.