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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.

Despite advances in surgical and adjuvant therapy, the prognosis for malignant gliomas remains dismal. This gloomy scenario has been recently brightened by the increasing understanding of the genetic and biological mechanisms at the basis of brain tumor development. These findings are being translated into innovative therapeutic approaches, including gene therapy, virotherapy, and vaccination, some of which have already been experimented in clinical trials. The advantages and disadvantages of all these different therapeutic modalities for malignant gliomas will be critically discussed, providing perspective for future investigations.

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.

Taking advantage of the proximity of bowel mucosa to luminal bacteria, we have attempted to deliver a therapeutic gene to the colonic mucosa by oral administration of an invasive and non-pathogenic Escherichia coli. E. coli diamenopimelate (dap) auxotroph, harboring plasmid pGB2Omegainv-hly, express the inv gene from Yersinia pseudotubercolosis that confers the ability to invade nonprofessional phagocytic cells and the hly gene from Listeria monocytogenes that allows expression of lystreriolysin O, a perforin cytolysin able to perfore phagosomal membranes. This bacterial vector invades and transfers functional DNA to epithelial cells in vitro. We have shown that this strain carrying a therapeutic gene (pC1OmegaTGF-beta1) can significantly reduce the severity of experimental colitis in mice. However, as a consequence of mucosal barrier disruption during colitis, vector-specific mRNA transcripts could be recovered from the colon and also from extra-colonic tissues. We therefore replaced the constitutive CMV promoter in pC1OmegaTGF-beta1 by the inflammation-inducible interleukin-8 promoter generating plasmid pC1OmegaTGF-beta1IND. Plasmid-specific TGF-beta1 mRNA transcripts were detectable in mouse CMT-93 epithelial cells incubated with E. coli BM2710/pGB2Omegainv-hly carrying pC1OmegaTGF-beta1IND following exposure to inflammatory cytokines. Furthermore, the transcripts were detectable only within inflamed tissues and the therapeutic effects were comparable to those in animals treated with E. coli BM2710/pGB2Omegainv-hly+pC1OmegaTGF-beta1. In summary, engineered enteric bacteria can efficiently deliver in vivo therapeutic genes to the intact intestinal mucosa and regulation expression of the therapeutic gene by an inflammation-inducible promoter prevents its dissemination during colitis.

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.

Background/aims: Gene therapy might be a promising therapeutic approach for advanced hepatocellular carcinoma (HCC) not amenable to any effective traditional treatment. The aim of the study was to evaluate the in vitro and in vivo efficacy of combined gene therapy of HCC with two different MoMLV-derived retroviral vectors, an MFG- and a LXSN-derived vector, both containing HSV-TK and hIL-2., Results: In vitro experiments on HCC cells showed efficient killing of transduced cells and efficient bystander effect after ganciclovir (GCV) treatment, with higher antitumor activity when the MFG-based vector was used. In vivo studies in a murine syngenic model of HCC demonstrated that treatment with GCV led to complete regression of tumors composed of transduced cells and regression of distant non-transduced tumors. Tumor transduction and efficacy of treatment was also demonstrated after in vivo delivery of vectors. Microarray analysis of tumor samples in mice receiving gene therapy showed up-regulation of genes involved in immune response and signal transduction., Conclusions: We demonstrated the in vitro and in vivo efficacy of combined retroviral-mediated gene therapy for HCC, with significant systemic therapeutic efficacy in vivo.

Several viruses have been engineered for gene therapy applications, and the specific properties of each viral vector have been exploited to target a variety of inherited and acquired diseases. Preclinical and clinical studies demonstrated that viral vectors are highly versatile tools capable of efficient transfer of foreign genetic information into almost all cell types and tissues. Gene therapy applications depend on vector characteristics, such as host range, cell- or tissue-specific targeting, genome integration, efficiency and duration of transgene expression, packaging capacity, and suitability for scale-up production. This review discusses the advances in the development of viral vectors, with particular emphasis on how knowledge of virus biology has been exploited to design a variety of vectors with improved safety characteristics and efficiency, potentially suitable for a large number of gene therapy applications.

Thyroid carcinomas are suitable targets for gene therapy because they can be highly lethal on one hand, while being susceptible to specific tumour targeting on the other hand. Several gene therapy modalities have been evaluated so far in experimental models of thyroid cancer, including tumour suppressor gene replacement, oncogene inhibition, suicide gene therapy, immunotherapy, antiangiogenesis, and viral oncolysis. All of these strategies have shown promising results, but clinical studies are lacking. Based on the clinical experience achieved in a pilot study in patients with advanced thyroid cancer and on clinical results in other types of solid cancer, it is suggested that combined gene therapy approaches, as well as multimodality therapeutic regimens, including gene therapy and conventional treatments, should be pursued to achieve clinically significant results.

The field of cancer gene therapy is in continuous expansion, and technology is quickly moving ahead as far as gene targeting and regulation of gene expression are concerned. This review focuses on the endocrine aspects of gene therapy, including the possibility to exploit hormone and hormone receptor functions for regulating therapeutic gene expression, the use of endocrine-specific genes as new therapeutic tools, the effects of viral vector delivery and transgene expression on the endocrine system, and the endocrine response to viral vector delivery. Present ethical concerns of gene therapy and the risk of germ cell transduction are also discussed, along with potential lines of innovation to improve cell and gene targeting.

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.

Gene therapy may be an effective approach to thyroid carcinoma refractory to conventional treatment. A transcriptionally targeted retroviral vector for gene therapy of thyroid carcinomas was generated replacing the viral enhancer with the enhancer sequence of the human thyroglobulin (TG) gene, yielding a chimeric long-terminal repeat. The TG enhancer was used to drive the expression of either a reporter gene (beta-galactosidase) or two therapeutic genes, i.e. the prodrug-activating enzyme thymidine kinase of herpes simplex virus (HSV-TK) and human IL-2, separated by an internal ribosome entry site. The corresponding vector having an unmodified long-terminal repeat was used as control. The targeted vector allowed selective transgene expression and cell killing in differentiated thyroid tumor cells but not in anaplastic thyroid carcinoma cells and nonthyroid cells, as demonstrated by quantitative RT-PCR and cytotoxicity assays. Nude mice injected with tumor cells underwent near complete or complete regression of tumors transduced with the control vector after ganciclovir treatment. On the other hand, infection with the thyroid-specific vector led to regression only of TG-expressing tumors. In addition, tumors expressing human IL-2 showed significant growth retardation, compared with nontransduced tumors while exhibiting signs of necrosis and presence of an inflammatory infiltrate. However, HSV-TK/IL-2 plus ganciclovir was significantly more efficient than HSV-TK/IL-2 alone in eradicating tumor masses. Our results indicate that replacement of viral enhancer with TG enhancer confers selectivity of transgene expression in thyroid cells. Thus, the combined thyroid-specific expression of two therapeutic genes (cytokine and suicide genes), although a safe tumor-targeted treatment, would allow an increased anticancer effect.

Gene therapy of cancer has become a major interest of medical research since more than 60% of the ongoing gene therapy protocols today involve cancer patients. To increase the therapeutic index of cancer gene therapy, targeting strategies have been developed to ensure that the expression of therapeutic genes is restricted exclusively to the tissue of interest. An attractive approach lies in the possibility to control the expression of therapeutic genes at the transcriptional level by the introduction of tissue-specific or tumor-specific enhancers/promoters offers. We have developed transcriptionally targeted vectors for gene therapy of solid tumors, including malignant gliomas and epithelial thyroid tumors. The choice of these tumor types relies on their clinical impact, ie, morbidity and mortality, the lack of effective conventional therapeutic strategies, and the ability of these tumors to express tissue/tumor-specific genes, whose transcriptional control elements (enhancer/promoter) may be used for achieving selective transgene expression. Here we report our clinical and preclinical experience in gene therapy of brain and thyroid tumors, and review the literature published on this topic.

Gene therapy for endocrine diseases represents an exciting new type of molecular intervention that may be a curative one. Endocrine disorders that might be treated by gene therapy include monogenic diseases, such as GH deficiency and hypothalamic diabetes insipidus, and multifactorial diseases, such as diabetes mellitus, obesity and cancer. Premises seem promising for endocrine tumours, but many combined approaches of cell and gene therapy are foreseeable also for other endocrine disorders. This review outlines the principles of gene therapy, describes the endocrine disorders that might take advantage of gene transfer approaches, as well as the gene therapy interventions that have already been attempted, their major limitations and the problems that remain to be solved.