Ashraf A. Elshami

Treatment of experimental human mesothelioma using adenovirus transfer of the herpes simplex thymidine kinase gene.

ObjectiveThe authors demonstrate the ability of an adenovirus vector expressing the herpes simplex thymidine klnase (HSV tk) gene to treat human malignant mesothelioma growing within the peritoneal cavity of severe combined immunodeficient (SCID) mice. Background DataIntroduction of the HSV tk gene into tumor cells renders them sensitive to the antiviral drug ganciclovir (GCV). This approach has been used previously to treat experimental brain tumors. Although malignant mesothelioma is refractory to current therapies, its localized nature and the accessibility of the pleural space make it a potential target for a similar type of in vivo gene therapy using adenovirus. MethodsAn adenovirus containing the HSV tk gene (Ad.RSV tk) was used to transduce mesothelioma cells in vitro. These cells were then injected into the flanks of SCID mice. Ad.RSV tk was also injected directly into the peritoneal cavity of SCID mice with established human mesothelioma tumors. Mice were subsequently treated for 7 days with GCV at a dose of 5 mg/kg. ResultsMesothelioma cells transduced in vitro with Ad.RSV tk formed nodules when injected in the subcutaneous tissue. These tumors could be eliminated by the administration of GCV, even when as few as 10% of cells were transduced to express HSV tk (bystander effect). Administration of Ad.RSV tk into the peritoneal space of animals with established multifocal human mesothelioma followed by GCV therapy resulted in the eradication of macroscopic tumor in 90% of animals and microscopic tumor in 80% of animals when evaluated after 30 days. The median survival of animals treated with Ad.RSV tk/GCV was significantly longer than that of control animals treated with similar protocols.

Retinoids augment the bystander effect in vitro and in vivo in herpes simplex virus thymidine kinase/ganciclovir-mediated gene therapy

Metabolic cooperation via gap junctional intercellular communication (GJIC) is an important mechanism of the bystander effect in gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) ‘prodrug’ system. Since retinoids have been reported to increase GJIC by induction of connexin expression, we hypothesized that these compounds could be used to augment the HSVtk/GCV bystander effect. Addition of all-trans retinoic acid increased GJIC in tumor cell lines, augmented expression of connexin 43, and was associated with more efficient GCV-induced in vitro bystander killing in cells transduced with HSVtk via either retrovirus or adenovirus vectors. This augmentation of bystander effect could also be seen in vivo. HSVtk-transduced tumors in mice treated with the combination of GCV and retinoids were significantly smaller than those treated with GCV or retinoids alone. These results provide evidence that retinoids can augment the efficiency of cell killing with the HSVtk/GCV system by enhancing bystander effects and may thus be a promising new approach to improve responses in gene therapy utilizing the HSVtk/GCV system to treat tumors or vascular restenosis.

The role of immunosuppression in the efficacy of cancer gene therapy using adenovirus transfer of the herpes simplex thymidine kinase gene

ObjectiveTo determine whether the immune system limits or improves the therapeutic efficacy of an adenovirus vector expressing the herpes simplex thymidine kinase (HSV tk) gene in a subcutaneous tumor model. Background DataEnhanced immune reactions against tumors may be therapeutically useful. However, recent studies with adenoviral vectors show that immune responses limit the efficacy and persistence of gene expression. The effect of the immune response on cancer gene therapy with HSV tk gene delivery by an adenovirus vector followed by treatment with gancilovir is unclear. MethodsAfter adenoviral transduction of a Fischer rat syngeneic mesothelioma cell line with the HSV tk gene in vitro, subcutaneous flank tumors were established. The ability of the HSV tk/ganciclovir system to inhibit tumor growth was compared among normal Fischer rats, immunodeficient nude rats, and Fischer rats immunosuppressed with cyclosporin. ResultsHSV tk/ganciclovir therapy was more effective in nude rats and immunosuppressed Fischer rats than in immunocompetent Fischer rats. ConclusionThese results indicate that the immune response against adenovirally transduced cells limits the efficacy of the HSV tk/ganciclovir system and that immunosuppression appears to be a useful adjunct. These findings have important implications for clinical trials using currently available adenovirus vectors as well as for future vector design.

Pleural-based mesothelioma in immune competent rats: A model to study adenoviral gene transfer

BACKGROUND Despite multimodality approaches, pleural-based malignant mesothelioma remains a disease with a very poor prognosis. Novel therapeutic strategies such as gene therapy clearly are needed to improve the survival of patients with this neoplasm. To aid in the evaluation of new treatment strategies, animal models that closely mimic human disease are required. This article describes the establishment of a pleural-based model of malignant mesothelioma in immune-competent Fischer rats. METHODS Via a modified left anterior lateral thorocotomy, a syngeneic malignant mesothelioma cell line, called II-45, was placed into the pleural cavity of Fischer rats. RESULTS Placement of II-45 cells into the pleural cavity of Fischer rats results in a model of pleural mesothelioma that closely resembles the disease seen in patients and is highly reproducible, with animals dying within 1 month. We also demonstrate the feasibility of adenoviral-mediated gene transfer to normal mesothelial cells lining the pleural cavity, as well as to malignant cells deep within the substance of pleural-based malignant mesothelioma. CONCLUSIONS The model described here offers the opportunity to study a variety of new treatment modalities, especially somatic gene transfer, against pleural-based malignant mesothelioma in an immune competent setting.

Differential sensitivity of thoracic malignant tumors to adenovirus-mediated drug sensitization gene therapy

Malignant mesothelioma may prove to be an attractive candidate for somatic gene therapy with replication-deficient recombinant adenovirus transfer of a toxic, or drug sensitization gene. Transfer of the herpes simplex thymidine kinase type I gene (HSVtk), followed by exposure to the acyclic nucleoside drug ganciclovir, has been shown to be an effective tumor cell killing system. To study generalized applicability, we tested a number of thoracic malignant cell lines for their sensitivity to gancyclovir after infection with an adenoviral vector containing the HSVtk gene (Ad.RSVtk). Using the concentration of gancyclovir required to kill 50% of the cells (IC50) as a measure of sensitivity, we detected variable sensitivity among cell lines, with mesothelioma most sensitive (IC50 = 0.075 to 2.8 mumol/L gancyclovir), and non-small-cell carcinoma lines having an intermediate sensitivity (IC50 = 1.5 to 100 mumol/L). In contrast, an ovarian carcinoma line was extremely resistant (IC50 > 2000 mumol/L). To study the possible mechanisms for these differences, we studied cell lines with regard to their ability to be infected with an adenoviral vector containing a marker gene (Ad.CMVlacZ) and expression of the vitronectin receptor alpha v (an integrin cell adhesion molecule shown to be required for adenovirus internalization after initial binding). We found that the degree of lacZ transduction correlated with HSVtk sensitivity, whereas vitronectin receptor expression did not, suggesting that differences in initial viral binding ability, rather than internalization, may explain the sensitivity differences seen in vitro.