Abstract
Conditionally replicative adenovirus (CRAd) is a cervical cancer gene therapy strategy which seeks to achieve selective viral replication in tumor cells. Oncolysis of infected cells creates a |[ldquo]|bystander effect|[rdquo]|, leading to infection of neighboring cancer cells with progeny virus. However, cervical cancer cells are relatively resistant to infection by adenovirus (Ad), thus limiting the capability of CRAd agents to achieve effective lateralization within the tumor. The objective of this proposal was to establish an efficient and selective infectivity-enhanced CRAd-based therapeutic modality for cervical cancer. We have developed methods to dramatically improve the ability of Ad to infect cervical cancer cells by replacing the knob region of Ad serotype 5 fiber with Ad serotype 3 knob (Ad5/3 chimera) and by putting an Arg-Gly-Asp (RGD)-4C motif in the HI loop of the Ad fiber knob region (RGD modification). Human telomerase reverse transcriptase (hTERT) promoter, which is active in many cancers but mostly inactive in normal organs including the liver, maintains this useful profile in our CRAd construct for cervical cancer. We employed recombinant Ad vectors containing luciferase reporter genes under the control of cytomegalovirus (CMV) promoter with or without fiber mutation (Ad5/3LucRGD and Ad5Luc) to evaluate transductional activity in cervical cancer cell lines in vitro. Then, we evaluated the oncolytic effect of hTERT-CRAd (Ad5/3hTERT E1-RGD and AdhTERT E1) in cervical cancer cell lines by both an MTS assay and crystal violet assay. hTERT-CRAd showed selective oncolysis in hTERT-positive cervical cancer cells but not in the hTERT negative, normal cells. Ad5/3Luc-RGD Ad showed increased reporter gene expression in cervical cancer cell line, HeLa (12.7 times more compare to Ad5Luc in a luciferase assay). Moreover, oncolysis with hTERT-CRAd in cervical cancer cells was much enhanced in Ad5/3RGD retaining the specificity both in a MTS assay and crystal violet assay. These results suggest that infectivity enhancement with mutant fiber is especially effective for enhancing oncolysis. This infectivity-enhanced hTERT-CRAd may thus be useful in the gene therapy of cervical cancer.
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