Achieving therapeutically efficacious levels of gene transfer in tumors has been a major obstacle for cancer gene therapy using replication-defective virus vectors. Recently, replicating viruses have emerged as attractive tools for cancer therapy, but generally achieve only transitory tumor regression. In contrast to other replicating virus systems, transduction by replication-competent retrovirus (RCR) vectors is efficient, tumor-selective, and persistent. Correlating with its efficient replicative spread, RCR vector expressing the yeast cytosine deaminase suicide gene exhibited remarkably enhanced cytotoxicity in vitro after administration of the prodrug 5-fluorocytosine. In vivo, RCR vectors replicated throughout preestablished primary gliomas without spread to adjacent normal brain, resulting in profound tumor inhibition after a single injection of virus and single cycle of prodrug administration. Furthermore, stable integration of the replicating vector resulted in persistent infection that achieved complete transduction of ectopic glioma foci that had migrated away from the primary tumor site. Thus, efficient and stable integration of suicide genes represents a unique property of the RCR vector that achieved multiple cycles of synchronous cell killing upon repeated prodrug administration, resulting in chronic suppression of tumor growth and prolonged survival.

The first large randomized phase III trial in gene therapy demonstrated no improvement in the survival of patients injected with packaging cells that produced conventional replication-defective retroviral vectors carrying the herpes simplex virus thymidine kinase gene, a disappointing result that was attributed to extremely poor levels of transduction efficiency. To circumvent this problem, we have developed a modified replication-competent retrovirus (RCR) that is capable of transducing human glioma cell lines A-172, U-87, T-98G, U-373, and U-138 and rat glioma cell lines C6 and 9L, over multiple infection cycles in vitro, resulting in a tremendous enhancement in transduction efficiency over conventional replication-defective retroviral vectors at the same dose. Whereas the transduction efficiency of conventional retroviral vectors injected into preestablished subcutaneous U-87 tumors at a dose of 1.0 x 10(5) transducing units (TU) was only 0.2% at 6 weeks postinjection, the same dose of RCR vector resulted in up to 97.2% transduction. When RCR vectors at a dose of 1.0 x 10(4) TU were injected into preestablished intracranial U-87 tumors, transduction efficiency at 2 and 3 weeks was 74 and 98.1%, respectively. Notably, however, intracranial injection of RCR vectors did not result in detectable infection of normal brain cells. Furthermore, using a sensitive polymerase chain reaction assay, no detectable RCR signal could be observed in any extracerebral tissues, including lung, liver, kidney, upper gastrointestinal tract (esophagus and stomach), lower gastrointestinal tract (colon and small intestine), skin, spleen, and bone marrow. Treatment of U-87 intracranial gliomas with RCR vectors carrying the yeast cytosine deaminase suicide gene followed by 5-fluorocytosine prodrug administration resulted in 100% survival over a 60-day follow-up period, compared with 0% survival of control groups receiving vector alone or prodrug alone. Our results demonstrate that RCR vectors can achieve therapeutically significant levels of transduction in malignant human gliomas, and that RCR vector spread after intratumoral injection is restricted to the tumor itself.

Retroviruses containing inserts of exogenous sequences frequently eliminate the inserted sequences upon spread in susceptible cells. We have constructed replication-competent murine leukemia virus (MLV) vectors containing internal ribosome entry site (IRES)-transgene cassettes at the env-3 untranslated region boundary in order to examine the effects of insert sequence and size on the loss of inserts during viral replication. A virus containing an insertion of 1.6 kb replicated with greatly attenuated kinetics relative to wild-type virus and lost the inserted sequences in a single infection cycle. In contrast, MLVs containing inserts of 1.15 to 1.30 kb replicated with kinetics only slightly attenuated compared to wild-type MLV and exhibited much greater stability, maintaining their genomic integrity over multiple serial infection cycles. Eventually, multiple species of deletion mutants were detected simultaneously in later infection cycles; once detected, these variants rapidly dominated the population and thereafter appeared to be maintained at a relative equilibrium. Sequence analysis of these variants identified preferred sites of recombination in the parental viruses, including both short direct repeats and inverted repeats. One instance of insert deletion through recombination with an endogenous retrovirus was also observed. When specific sequences involved in these recombination events were eliminated, deletion variants still arose with the same kinetics upon virus passage and by apparently similar mechanisms, although at different locations in the vectors. Our results suggest that while lengthened, insertcontaining genomes can be maintained over multiple replication cycles, preferential deletions resulting in loss of the inserted sequences confer a strong selective advantage.

A major obstacle in cancer gene therapy is the limited efficiency of in vivo gene transfer by replication-defective retrovirus vectors in current use. One strategy for circumventing this difficulty would be to use vectors capable of replication within tumor tissues. We have developed a replication-competent retrovirus (RCR) vector derived from murine leukemia virus (MuLV). This vector utilizes a unique design strategy in which an internal ribosome entry site-transgene cassette is positioned between the env gene and the 3' long terminal repeat (LTR). The ability of this vector to replicate and transmit a transgene was examined in culture and in a solid tumor model in vivo. The RCR vector exhibited replication kinetics similar to those of wildtype MuLV and mediated efficient delivery of the transgene throughout an entire population of cells in culture after an initial inoculation with 1 plaque-forming unit (PFU) of vector per 2000 cells. After injection of 6 x 10(3) PFU of vector into established subcutaneous tumors, highly efficient spread of the transgene was observed over a period of 7 weeks, in some cases resulting in spread of the transgene throughout the entire tumor. MuLV-based RCR vectors show significant advantages over standard replication-defective vectors in efficiency of gene delivery both in culture and in vivo. This represents the first example of the use of an RCR vector in an adult mammalian host, and their first application to transduction of solid tumors.

Resistance to TGF-b is frequently observed in ovarian cancer, and disrupted TGF-b/SMAD4 signaling results in the aberrant expression of downstream target genes in the disease. Our previous study showed that ADAM19, a SMAD4 target gene, is downregulated through epigenetic mechanisms in ovarian cancer with aberrant TGF-b/SMAD4 signaling. In this study, we investigated the mechanism of downregulation of FBXO32, another SMAD4 target gene, and the clinical significance of the loss of FBXO32 expression in ovarian cancer. Expression of FBXO32 was observed in the normal ovarian surface epithelium, but not in ovarian cancer cell lines. FBXO32 methylation was observed in ovarian cancer cell lines displaying constitutive TGF-b/SMAD4 signaling, and epigenetic drug treatment restored FBXO32 expression in ovarian cancer cell lines regardless of FBXO32 methylation status, suggesting that epigenetic regulation of this gene in ovarian cancer may be a common event. In advanced-stage ovarian tumors, a significant (29.3%; Po0.05) methylation frequency of FBXO32 was observed and the association between FBXO32 methylation and shorter progression-free survival was significant, as determined by both Kaplan-Meier analysis (Po0.05) and multivariate Cox regression analysis (hazard ratio: 1.003, Po0.05). Reexpression of FBXO32 markedly reduced proliferation of a platinum-resistant ovarian cancer cell line both in vitro and in vivo, due to increased apoptosis of the cells, and resensitized ovarian cancer cells to cisplatin. In conclusion, the novel tumor suppressor FBXO32 is epigenetically silenced in ovarian cancer cell lines with disrupted TGFb/SMAD4 signaling, and FBXO32 methylation status predicts survival in patients with ovarian cancer. Ovarian cancer is the fifth leading cause of death in women and the most deadly of gynecological malignancies. 1 The lifetime risk of ovarian cancer in women is B1.5%. 2 As ovarian cancer has few symptoms early in its course, the majority of patients are diagnosed with advanced-stage disease. Despite advances in chemotherapy, the poor prognosis for patients with ovarian cancer is reflected in the o20% 5-year survival rate after initial diagnosis for patients with stage III and IV disease, whereas survival of patients with stage I or II disease is 480% for the same period. 3 Current