The present work shows the use of the regulatory sequences of the H19 gene for the development of DNA-based therapy for human ovarian cancer related ascites. The successful development of anti-tumor gene therapy depends on the use of a combinatorial approach aimed at targeted delivery and specific expression of effective anti-tumor agents. Various gene therapy strategies for the treatment of ovarian cancer are currently under development and aim towards maximal treatment efficacy and minimal adverse effects. In this study, a tumor-selective promoter was used in conjunction with a cytotoxic gene to achieve targeted tumor cell destruction. Trials in animal models showed that tumor specific promoters exhibit a clear advantage compared to strong viral promoters such as CMV promoter currently used in clinical trials . While most tumor-specific promoters are relatively weak, resulting in insufficient transgene expression levels, the H19 promoter is known to be highly activated in various tumor types and to show no or only marginal activity in the surrounding normal tissue [26, 28].
The goal of the present study was to evaluate the therapeutic potential of expression vectors carrying the "A" fragment of the diphtheria toxin (DT-A) gene under the control of the H19 regulatory sequences in an ovarian carcinoma animal model. We have previously shown that these constructs are able to selectively kill tumor cell lines and inhibit tumor growth in vitro and in vivo [[28, 24, 23] and ]. The choice of the DT-A as a toxin gene ensured not only high killing activity but its use has a great advantage in avoiding unintended toxicity to normal cells, since the DT-A protein released from the lysed cells is not able to enter neighboring cells in the absence of the DT-B fragment .
In order to determine the feasibility of this approach for the therapy of ovarian cancer in a human patient, both RT-PCR and ISH analyses were applied on cells isolated from OCAF to determine the level of H19 gene expression. High levels of H19 transcript were detected in the ascites malignant cells (Figure 1A+B+C). The high level of H19 RNA found in the OCAF is in accordance with previous results obtained from our study on the expression profile of H19 in epithelial ovarian cancer .
The therapeutic potential of the toxin vector was evaluated in vitro using different human ovarian cancer cell lines and in cells isolated from OCAF (Figures 3A and 1B). The H19 regulatory sequences were able to drive DTA expression in the ovarian cancer cell lines that led to cell death. Therefore, we further investigated the therapeutic potential of the toxin vector in vivo using the ES-2 cell line which has high tumorogenic properties. Although ES-2 cells (carrying a p53 mutation) showed no endogenous expression of the H19 gene when tested in culture (Figure 2, lane 6), H19 RNA were detected at high levels in all the tumors developed following injection of these cells into the animal (Figure 4), supporting the possible role of H19 in tumor growth which is upregulated under hypoxic stress. In addition, it was previously shown that in certain bladder carcinoma cell lines H19 RNA is either not or weakly expressed in normal culture conditions, but strongly expressed when tumors are grown by injecting these cell lines into nude mice [30, 31].
H19 expression was also detected in ascites developed after intraperitoneal injection of these cells into the peritoneum of nude mice (data not shown). Furthermore, the apparent non-correlation between transgene expression under the regulation of the H19 promoter and endogenous H19 expression might be explained by the absence of negative regulatory sequences in the DTA-H19 construct. The promoter activity of endogenous H19 gene is determined by the naked chromatin structure which differs from that of the constructs transfected into the cells. Thus, transcription factors may be able to induce transcription from the plasmid, but not from the endogenous gene.
We have shown the existence of a tight association between the p53 status and H19 induction under hypoxic stress (manuscript sent for publication). In this case, it is possible that the enhanced H19 expression observed in these tumors is related to selection and clonal expansion of H19 expressing cells, under the severe and harsh conditions (for example: low oxygen levels) of a rapidly growing tumor in vivo, which is the real situation in the target tumors to be treated.
The heterotopic model for ovarian cancer used in this research has the advantage of rapidly developing tumors, allowing short turn-around times for the experiments (three weeks). In addition, the developed tumors are easily manageable because of relatively large size and accessibility. The DTA-H19/PEI complex was able to highly inhibit the growth rate of the subcutaneous tumors induced in mice by subcutaneous injection with the ES-2 cell line (Figure 5A). At least 40% inhibition of tumor growth by DTA-H19/PEI was obtained compared to tumors treated with the control plasmid Luc-H19/PEI (P < 0.05) (Figure 5B). Moreover, it is very important to note that no signs of unwanted toxicity were detected in normal mice treated subcutaneously by DTA-H19/PEI.
We used the cationic polymer PEI (JetPEI™), a linear polyethylenimine derivative as a transfection promoter agent in the heterotopic animal model described in Figure 5. The JetPEI™ compacts the DNA into positively charged particles capable of interacting with anionic proteoglycans at the cell surface, thereby facilitating the entering of the DNA by endocytocis . No toxic effect was detected in the treated animals participating in these experiments.
Although this is a preliminary study, our working hypothesis is that intraperitoneal administration of DTA-H19 has the potential to reach ascites tumor cells, deliver its intracellular toxin without targeting normal tissues, and thus may help reduce tumor burden, fluid accumulation; improve the quality of life of the patient; and prolong their life span. This suggested approach was further demonstrated in a compassionate patient treatment in which the DTA-H19 plasmid was intraperitoneally injected into the peritoneum of a woman with advanced and recurrent ovarian carcinoma. Following several infusions, a complete resolution of ascites was shown (Case report in preparation).