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We performed a clinical trial of gene therapy in patients with recurrent glioblastoma multiforme based on retroviral vector-mediated combined delivery of interleukin-2 and thymidine kinase of herpes simplex virus (HSV)-1. Treatment consisted of intratumor injection of retroviral vector-producing cells, followed by intravenous ganciclovir administration. The primary endpoints of the study were the evaluation of the safety of the treatment and demonstration of transduction of tumor cells. Here, we report details on the clinical protocol and the methods used.

Following our pilot clinical study of combined IL-2/HSV-TK gene therapy for recurrent glioblastoma multiforme (GBM), we extended the protocol to a larger population of patients and evaluated safety, feasibility, and biological activity of treatment. A total of 12 patients received intratumor injection of retroviral vector-producing cells (RVPCs), followed by intravenous ganciclovir (GCV). Treatment was well tolerated with only minor adverse events. Transduction of tumor cells was demonstrated in tumor biopsies. A marked and persistent increase of intratumor and plasma Th1 cytokine levels was demonstrated after RVPC injection. At magnetic resonance imaging evaluation, two patients had a partial response (including a patient showing disappearance of a distant noninjected tumor mass), four had a minor response, four had stable disease, and two had progressive disease. The 6- and 12-month progression-free survival rates were 47 and 14%, respectively. The 6- and 12-month overall survival rates were 58 and 25%, respectively. In conclusion, the results of our clinical protocol of gene therapy for recurrent GBM, based on combined delivery of a suicide and a cytokine gene, demonstrate that intratumor injection of RVPCs was safe, provided effective transduction of the therapeutic genes to target tumor cells, and activated a systemic cytokine cascade, with tumor responses in 50% of cases.

This study represents the first report of gene therapy for anaplastic thyroid carcinoma, one of the most aggressive solid tumors in humans. Two patients with end-stage anaplastic thyroid carcinoma were treated by direct intratumor injection of retroviral vector producer cells followed by ganciclovir. The retroviral vector carried the human IL-2 gene and the suicide gene thymidine kinase of herpes simplex virus type 1. Treatment was safe and associated with only mild adverse events. Transduction of tumor cells and production of T helper type 1 cytokines was demonstrated in tumor biopsies. Gene therapy led also to a marked increase in T helper type 1 cytokine expression in peripheral blood mononuclear cells. Radiological evaluation of injected tumor masses demonstrated local tumor necrosis.

Objective and Design: Based on our clinical experience with combined gene therapy of glioblastoma, we developed a retroviral vector expressing two therapeutic genes (i.e. thymidine kinase of herpes simplex virus, HSV-TK, and interleukin-2, IL-2) and evaluated its efficiency in vitro and in vivo., Methods: Expression of therapeutic genes in transduced thyroid carcinoma cells was analyzed by real-time RT-PCR. Ganciclovir sensitivity of infected cells was assessed in vitro in thyroid carcinoma cell lines and in vivo in nude mice bearing xenografted thyroid cancers. The combined effect of IL-2/HSV-TK was compared with the effect of IL-2 alone., Results: Expression of therapeutic genes was higher in differentiated than in anaplastic thyroid carcinoma cells. Ganciclovir treatment led to dose- and time-dependent killing of transduced cells in vitro. A bystander effect was demonstrated by using mixtures of infected and non-infected cells. In vivo studies showed a significant reduction of growth and the presence of an inflammatory infiltrate in transduced thyroid tumors expressing IL-2 alone, as compared with non-infected tumors. By using the retroviral vector expressing IL-2/HSV-TK, treatment with ganciclovir led to complete eradication of anaplastic tumors and a >80% reduction of the size of differentiated thyroid carcinomas. Histological analysis of tumor specimens showed extensive necrosis and inflammatory cell infiltrates. The combination of IL-2/HSV-TK plus ganciclovir was significantly more efficient than IL-2 alone in eradicating tumor masses. The bystander effect was also obtained in vivo., Conclusions: These findings demonstrate the feasibility and efficiency of a combined immunomodulating and suicide gene therapy approach for thyroid carcinomas.

Retrovirus-mediated gene therapy is a particularly attractive approach for glioblastoma multiforme (GBM), given the poor prognosis of this tumour and its localized proliferation in post-mitotic tissue. In this study we assessed, for the first time in humans, the therapeutic potential of a newly designed bicistronic Moloney vector (pLIL-2-TK), combining the expression of a suicide gene (thymidine kinase, tk) with an immunomodulatory gene (human interleukin 2, IL-2). Evidence of transgene activity in the treated tumours is presented.

Gene-based therapeutic strategies for cancer mainly include augmentation of immunotherapeutic and chemotherapeutic approaches. In this study we report the design and functional assay of a novel bicistronic Moloney-based retroviral vector expressing human interleukin-2 (IL-2) and herpesvirus thymidine kinase (tk) through a cap-dependent translation and an internal ribosome entry site (IRES)-regulated translation, respectively. This construct has the potential for allowing combination of cytokine and suicide gene therapy, especially in areas such as the brain, composed of post-mitotic cells refractory to transduction by type C retroviral vectors. Accordingly, human glioma cells were used as targets for gene transfer after selecting a packaging cell clone that produced a reasonable titer of recombinant virus and expressed high levels of IL-2 and tk transcripts. Although transduction efficiency was reduced in glioma cells as compared with murine NIH 3T3 cells, transgene expression was effectively achieved. Transduced glioma cells were sensitive to ganciclovir and secreted around 1000 U/ml IL-2 in the culture supernatants. Simultaneous production of IL-2 and tk in vivo by genetically treated tumor cells would hopefully potentiate the effect of gangiclovir-induced metabolic suicide, possibly by boosting the immune response associated with tumor debulking or by amplifying the bystander response.