Viral-based modelling and correction of neurodegenerative diseases by RNA interference.

Experimental recapitulation of recessive human genetic neurodegenerative disease in rodents can be classically addressed through genetic disruption of the related gene. Although very informative, this specific gene targeting is restricted to mice and precludes a species scale-up towards non-human primates. Concomitantly, this requirement to silence a specific gene in a broad range of animal models is important in the design of therapeutic approaches to dominantly inherited neurodegenerative diseases. The emergence of RNA interference (RNAi), a highly specific mechanism of post-translational gene silencing, has opened a plethora of biological application ranging from reverse genetic analysis to therapeutic schemes. Recombinant viral vectors, by promoting a long-lasting delivery of genetic instructions in a broad range of cellular types of different species origins, represent potential platforms mandating silencing of specific gene products through RNAi. This review aims at providing an overview of the different viral systems engineered so far for efficient in vitro and in vivo delivery of RNAi instructions. Additionally, the potential of RNAi for functional analysis and therapy for polyglutamine disorders or amyotrophic lateral sclerosis is discussed.

Combined transcriptional and transductional targeting improves the specificity and efficacy of adenoviral gene delivery to ovarian carcinoma.

Adenoviruses are efficient gene delivery vehicles but have broad native tropism. To this end, finding ways to target this virus specifically to carcinomas has become an important focus of cancer gene therapy. Transductional and transcriptional forms of targeting have been used with promising results in ovarian carcinoma. Therefore, we combined both forms of targeting to investigate the effect on the specificity and efficiency of transgene expression in this disease. We used the tissue-specific SLPI promoter and the ovarian cancer associated targeting adaptor protein, sCARfC6.5. This bispecific protein contains the coxsackie-adenovirus receptor ectodomain and a single-chain antibody specific for c-erbB-2. Viruses containing the SLPI or the ubiquitously expressed CMV promoter, with or without sCARfC6.5, were used for infection of ovarian cancer cell lines, primary ovarian tumor cells, and in an orthotopic model of disseminated ovarian carcinoma. This dual-targeting strategy increased the efficiency and specificity of transgene expression in vitro in reporter and cell-killing assays, and in vivo. By using both the SLPI promoter and sCARfC6.5, transgene expression was increased in ovarian tumors and decreased in normal tissues, including the liver. Thus, we show that combining transcriptional and transductional targeting can increase the efficacy and specificity of adenoviral gene therapy for ovarian carcinoma.

Effective single chain antibody (scFv) concentrations in vivo via adenoviral vector mediated expression of secretory scFv.

Single chain antibodies (scFv) represent powerful interventional agents for the achievement of targeted therapeutics. The practical utility of these agents have been limited, however, by difficulties related to production of recombinant scFv and the achievement of effective and sustained levels of scFv in situ. To circumvent these limitations, we have developed an approach to express scFv in vivo. An anti-erbB2 scFv was engineered for secretion by eukaryotic cells. The secreted scFv could bind to its target and specifically suppress cell growth of erbB2-positive cells in vitro. Adenoviral vectors expressing the cDNA for the secretory scFv likewise could induce target cells to produce an anti-tumor anti-erbB2 scFv. In vivo gene transfer via the anti-erbB2 scFv encoding adenovirus also showed anti-tumor effects. Thus, by virtue of engineering a secreted version of the anti-tumor anti-erbB-2 scFv, and in vivo expression via adenoviral vector, effective concentrations of scFv were achieved. In vivo gene transfer clearly represents a powerful means to realize effective scFv-based approaches. This method will likely have applicability for a range of disorders amenable to targeted therapeutic approaches.

Strategies to accomplish targeted expression of transgenes in ovarian cancer for molecular therapeutic applications.

PURPOSE: The purpose of the study was to determine the capability of the midkine (MK) and cycooxygenase-2 (cox-2) gene promoter regions to function as tumor-specific promoters for use in targeted gene therapy of ovarian cancer. EXPERIMENTAL DESIGN: Established and primary ovarian cancer and mesothelial cells were transduced by adenoviral vectors containing a reporter or thymidine kinase gene expressed under the control of the MK, cox-2, or cytomegalovirus (CMV) promoters. SCID or C57BL/6 mice were injected i.p. with these same vectors. In vitro reporter gene expression and cellular cytotoxicity was determined using luciferase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. Acute toxicity in vivo was assessed by histological evaluation of harvested tissues. RESULTS: Consistent activation of the MK and cox-2 promoters was noted in all of the ovarian cancer cell lines in addition to primary ovarian cancer cells. In contrast, reduced reporter activity was reported in mesothelial cells transduced with adenoviruses containing the test promoters, which was especially apparent for the cox-2 promoter. Additionally, the cox-2 promoter exhibited significantly lower reporter gene levels in liver and peritoneum than the control promoter in in vivo experiments. Tumor-cell killing induced by Adcox-2 MTK was comparable to that observed with AdCMVTK. However, a clear differential toxicity pattern was observed in favor of animals treated with Adcox-2 MTK when compared with controls. CONCLUSIONS: These data clearly demonstrate that the transcriptional control afforded by the cox-2 promoter is tumor-specific and is able to mitigate associated toxicity in normal tissue while maintaining therapeutic efficacy in the context of an ovarian cancer molecular chemotherapeutic approach.

An immunomagnetic-based method for the purification of ovarian cancer cells from patient-derived ascites.

OBJECTIVE: Primary ovarian cancer cells obtained from fresh tumor have many advantages over established cell lines. Therefore, a procedure for the specific and efficient purification of such neoplastic cells is critical. We report an effective immunomagnetic method for the isolation of tumor cells from the ascitic fluid of patients diagnosed with ovarian adenocarcinoma. METHODS: This procedure incorporates the use of monoclonal antibody (mAb) CC49, which recognizes the tumor-associated glycoprotein 72 (TAG-72). TAG-72 is highly expressed on ovarian tumor cell surfaces with little or no reactivity with normal tissues. Also used in this protocol are immunomagnetic beads, which bind to the CC49 mAb via a secondary antibody. When ovarian cancer cells adhere to the magnetic beads, a magnetic field is used to separate the tumor cells from all other cellular components. RESULTS: Using ascitic fluid from five patients, we found that preparations before purification contained between 38 and 52% neoplastic cells. Using our method, we produced preparations that were between 63 and 96% pure for cancer cells, thus obtaining an average increase in tumor cell enrichment of 86%. CONCLUSION: We, therefore, believe this method is preferable for producing high yields of pure ovarian neoplastic cells. We are now employing this technique in our laboratory to provide a stringent and pure template for our studies on gene transfer to primary ovarian cancer cells.

Pro-apoptotic treatment with an adenovirus encoding Bax enhances the effect of chemotherapy in ovarian cancer.

BACKGROUND: Tumor cell heterogeneity and resistance to chemotherapy-mediated cell death are major obstacles in cancer therapy. It has been reported that expression of the pro-apoptotic molecule Bax can induce cell death or sensitize tumor cells to chemotherapy in stable cell clones derived from tumor cells. However, these studies are limited in that they cannot represent the heterogeneity of cancer cells observed in vivo. In this study, we have further explored the therapeutic potential of Bax. METHODS: Using an inducible recombinant Bax adenovirus, we screened a panel of ovarian cancer cell lines and primary patient-derived ovarian tumor cells for their sensitivity to Bax-mediated cytotoxicity. Apoptotic cell death was evaluated qualitatively with Hoechst staining and quantitatively with MTS and Annexin V-based assays. Endogenous levels of both Bcl-2 and Bax protein and p53 status were evaluated. The potential of bax to sensitize ovarian cancer lines to chemotherapy was also tested. Dose-response curves were generated to evaluate cell death. RESULTS: Overexpression of Bax directly induced apoptosis in both ovarian cancer cell lines and the patient-derived primary cancer cells. However, the sensitivity of these cells to Bax varied and appeared to be independent of both the status of p53 and the endogenous levels of bcl-2 or Bax, critical molecules in the apoptotic pathway. Importantly, overexpression of Bax significantly enhanced chemotherapy-induced cytotoxicity in both established cell lines and primary ovarian carcinoma cells. CONCLUSIONS: These studies suggest that overexpression of Bax alone or in combination with chemotherapy may provide a means to overcome the problems imposed by the heterogeneous nature of tumors, ultimately augmenting the efficacy of chemotherapy in patients suffering from ovarian cancer.

Phase I trial and pharmacologic trial of sequences of paclitaxel and topotecan in previously treated ovarian epithelial malignancies: a Gynecologic Oncology Group study.

PURPOSE: A phase I and pharmacologic study to evaluate the feasibility of administering paclitaxel (PTX) in combination with topotecan (TPT) without and with granulocyte colony-stimulating factor (G-CSF) in women with recurrent or refractory ovarian cancer. PATIENTS AND METHODS: TPT was administered as a 30-minute infusion daily for 5 days and PTX was given as a 24-hour infusion (PTX-24) either before TPT on day 1 or after TPT on day 5. Each patient received both schedules on an alternating basis every 3 weeks. Sequential dose escalation of TPT or PTX-24 without and with G-CSF resulted in five dosage permutations of TPT/PTX (mg/ m2): 0.75/135 without G-CSF and 0.75/135, 1.25/135, 1.50/135, and 1.25/170 with G-CSF. RESULTS: Twenty-two patients received 109 courses of therapy. Dose-limiting myelosuppression consistently occurred at the first TPT/PTX-24 dose level (0.75/135 mg/m2) in the absence of G-CSF support. Although the addition of G-CSF resulted in reduced rates of complicated neutropenia, the incidences of dose-limiting neutropenia and thrombocytopenia were unacceptably high after the doses of either TPT or PTX-24 were increased. Paired analysis showed similar hematologic toxicities between the two sequences of drug administration. The pharmacologic behavior of both TPT and PTX-24 was not altered by drug sequencing. Major antitumor responses occurred in 40% of patients with measurable and assessable disease, including 45% and 9% of patients with potentially cisplatin-sensitive and -resistant tumors, respectively. CONCLUSION: The recommended doses of TPT on a daily times-five schedule combined with PTX-24 in these patients were 0.75 mg/m2/d and 135 mg/m2, respectively, with G-CSF support. Although this dose of PTX has significant single-agent activity in ovarian cancer, the dose of TPT is much lower than the TPT dose at which single-agent activity has been observed. Due to the inability to administer near relevant single-agent doses of both drugs in combination, as well as the requirement for G-CSF support, further evaluations of this regimen in women with refractory or recurrent ovarian cancer are necessary before it can be recommended for previously treated patients in this setting.