Review article: gene therapy, recent developments and future prospects in gastrointestinal oncology.

BACKGROUND: Gene therapy consists of the introduction of genetic material into cells for a therapeutic purpose. A wide range of gene therapy vectors have been developed and used for applications in gastrointestinal oncology. AIM: To review recent developments and published clinical trials concerning the application of gene therapy in the treatment of liver, colon and pancreatic cancers. METHODS: Search of the literature published in English using the PubMed database. RESULTS: A large variety of therapeutic genes are under investigation, such as tumour suppressor, suicide, antiangiogenesis, inflammatory cytokine and micro-RNA genes. Recent progress concerns new vectors, such as oncolytic viruses, and the synergy between viral gene therapy, chemotherapy and radiation therapy. As evidence of these basic developments, recently published phase I and II clinical trials, using both single agents and combination strategies, in adjuvant or advanced disease settings, have shown encouraging results and good safety records. CONCLUSIONS: Cancer gene therapy is not yet indicated in clinical practice. However, basic and clinical advances have been reported and gene therapy is a promising, new therapeutic approach for the treatment of gastrointestinal tumours.

Targeting sodium/iodide symporter gene expression for estrogen-regulated imaging and therapy in breast cancer.

Expression of the sodium iodide symporter (hNIS) has been detected in breast cancer tissue, but frequently, not at the levels necessary to mediate (131)I accumulation. Transducing the hNIS gene into breast cancer cells with adenovirus could be a tractable strategy to render breast cancer susceptible to radioiodide therapy. We constructed the replication-incompetent virus, AdSERE, in which an estrogen-responsive promoter directs the expression of hNIS. In vitro, we demonstrate that AdSERE mediates hNIS expression and iodide uptake in ER+ breast cancer cells. In vivo, we show that AdSERE-infected ER+ tumors can be imaged due to tracer accumulation; in addition, AdSERE in combination with therapeutic doses of (131)I suppresses tumor growth.

Uveal vs. cutaneous melanoma. Origins and causes of the differences

Melanoma is a malignant tumour derived from melanocytes (dendritic cells originated from the neural crest and capable to produce melanin synthesis) that could be established on the skin or less frequently on the uvea. The cellular origin from both kind of melanoma seems to be the same but the melanocytes migrates to the epithelia for cutaneous melanoma, while for uveal melanoma, they migrate to mesodermic tissues. Despite the common origin, both melanomas show extreme differences in their metastatic potential, clinical response to treatments, immune response and genetic alterations. We will describe some of those differences in this review (AU)

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Non-invasive genetic imaging for molecular and cell therapies of cancer.

Gene therapy is a very attractive strategy in experimental cancer therapy. Ideally, the approach aims to deliver therapeutic genes selectively to cancer cells. However, progress in the improvement of gene therapy formulations has been hampered by difficulties in measuring transgene delivery and in quantifying transgene expression in vivo. In clinical trials, endpoints rely almost exclusively on the analysis of biopsies, which provide limited information. Non-invasive monitoring of gene delivery and expression is a very attractive approach as it can be repeated over time in the same patient to provide spatiotemporal information on gene expression on a whole body scale. Thus, imaging methods can uniquely provide researchers and clinicians the ability to directly and serially assess morphological, functional and metabolic changes consequent to molecular and cellular based therapies. This review highlights the various methods currently being developed in preclinical models.

Gene therapy in head and neck cancer: a review.

Gene therapy for cancer is a rapidly evolving field with head and neck squamous cell cancer being one of the more frequently targeted cancer types. The number of clinical trials in the UK is growing and there is already a commercially available agent in China. Various gene therapy strategies along with delivery mechanisms for targeting head and neck cancer are reviewed.

Non-invasive genetic imaging for molecular and cell therapies of cancer

Gene therapy is a very attractive strategy in experimental cancer therapy. Ideally, the approach aims to deliver therapeutic genes selectively to cancer cells. However, progress in the improvement of gene therapy formulations has been hampered by difficulties in measuring transgene delivery and in quantifying transgene expression in vivo. In clinical trials, endpoints rely almost exclusively on the analysis of biopsies, which provide limited information. Non-invasive monitoring of gene delivery and expression is a very attractive approach as it can be repeated over time in the same patient to provide spatiotemporal information on gene expression on a whole body scale. Thus, imaging methods can uniquely provide researchers and clinicians the ability to directly and serially assess morphological, functional and metabolic changes consequent to molecular and cellular based therapies. This review highlights the various methods currently being developed in preclinical models (AU)

SPECT/CT imaging of oncolytic adenovirus propagation in tumours in vivo using the Na/I symporter as a reporter gene.

Oncolytic adenoviruses have shown some promise in cancer gene therapy. However, their efficacy in clinical trials is often limited, and additional therapeutic interventions have been proposed to increase their efficacies. In this context, molecular imaging of viral spread in tumours could provide unique information to rationalize the timing of these combinations. Here, we use the human sodium iodide symporter (hNIS) as a reporter gene in wild-type and replication-selective adenoviruses. By design, hNIS cDNA is positioned in the E3 region in a wild-type adenovirus type 5 (AdIP1) and in an adenovirus in which a promoter from the human telomerase gene (RNA component) drives E1 expression (AdAM6). Viruses show functional hNIS expression and replication in vitro and kinetics of spread of the different viruses in tumour xenografts are visualized in vivo using a small animal nano-SPECT/CT camera. The time required to reach maximal spread is 48 h for AdIP1 and 72 h for AdAM6 suggesting that genetic engineering of adenoviruses can affect their kinetics of spread in tumours. Considering that this methodology is potentially clinically applicable, we conclude that hNIS-mediated imaging of viral spread in tumours may be an important tool for combined anticancer therapies involving replicating adenoviruses

La actividad de la Caspasa-1 como gen sensibilizador a radio y quimioterapia es independiente de las vías de JNK y p38 / Caspase-1 as a radio and chemosensitizer regardless of JNK and p38 pathways

El efecto citotóxico de las drogas antitumorales es producido mediante la inducción de apoptosis. Esta observaciónimplica la posibilidad de que los factores que afecten la activación de caspasas pueden ser determinantesimportantes como sensibilizantes a los tratamientos antitumorales. Aquí, examinamos el efecto de la sobreexpresiónde caspasa-1 en la respuesta a la quimio y radioterapia. La expresión de la caspasa-1 mediada porun vector adenoviral fue capaz de matar directamente a las células y de sensibilizar las restantes a cisplatino oradiación gamma in vitro. En células HeLa transfectadas establemente con caspasa-1, la sensibilización a cisplatinofue debida a una amplificación en la vía mitocondrial de apoptosis inducida por cisplatino pero esteefecto es independiente del estado de p53, JNK o p38 en la célula

The cytotoxic effect of anticancer drugs has been shown to involve induction of apoptosis. This observationraises the possibility that factors affecting caspase activation might be important determinants as anticancerdrug sensitivity. Ectopic expression of caspase-1 has been shown to trigger apoptosis. Here, we examine theeffect of caspase-1 over-expression on the response to chemotherapy and radiotherapy. Caspase-1 expressionmediated by an adenoviral vector was able to kill directly cells and to sensitize the remaining cells to cisplatinor ã-radiation in vitro. In HeLa cells stably transfected with caspase-1, sensitisation to cisplatin was due to anamplification of the cisplatin-induced mitochondrial apoptotic pathway activation but this effect is independentof p53, JNK or p38 status

Dominant negative inhibitors of signalling through the phosphoinositol 3-kinase pathway for gene therapy of pancreatic cancer.

BACKGROUND: Ras signalling is frequently aberrant in pancreatic cancer so that there is constitutive activation of the phosphatidylinositol 3-kinase (PI3K) and AKT/protein kinase B pathway, as well as the RAF/MEK/ERK pathway. AIMS: In the present study we investigated the role of the PI3K/AKT pathway in malignant transformation of pancreatic cancer cells. METHODS: A genetic approach was used to interfere with signal transduction in vitro and in vivo. RASN17, a dominant negative mutant of RAS, was applied to inhibit the PI3K/AKT pathway upstream of PI3K. The regulatory p85beta subunit of PI3K and the negative regulator PTEN were utilised to inhibit the pathway at the level of PI3K, and AAA-AKT, a dominant negative mutant of AKT was employed to interfere with PI3K/AKT signalling at the level of AKT. RESULTS: Antiproliferative, proapoptotic, and anticancer effects were documented, showing that inhibition of the PI3K pathway in these cell lines suppresses tumour cell growth in vitro and reduces growth in nude mice. CONCLUSIONS: The PI3K/AKT pathway represents a potential therapeutic target for pancreatic cancer, and gene therapy may be one approach to produce selective inhibition.

Potential therapeutic applications of recombinant, invasive E. coli.

An invasive Escherichia coli expressing the inv gene from Yersinia pseudotuberculosis was used as a vector for protein delivery to mammalian epithelial cells. Upon incubation with beta1-integrin-expressing mammalian cells, the bacteria are internalized, allowing bacteria-encoded proteins to function from within the mammalian cell. These bacteria are eventually processed in the host phagosome where they are destroyed. Expression of listeriolysin O from Listeria monocytogenes in the bacterium and its subsequent release into the phagosome triggers the breakdown of the membrane, allowing the release of the bacterial content into the cytosol of host cells. Using this vector, we demonstrate delivery of a gene and intact, functional proteins into mammalian cells in which beta1-integrin is expressed and accessible. At a ratio of bacteria/mammalian cells compatible with the survival of the mammalian cells, protein delivery can be observed in the entire cell population in vitro, while gene transfer is far less efficient. Protein delivery can also be achieved in vivo in mouse tumour models and can be detected at least 96 h after inoculation. Functional, natural E. coli proteins are delivered in the process and can provide therapeutic benefit in vivo, when associated with prodrugs. This therapeutic effect is associated with infiltration of neutrophils, eosinophils, macrophages and to a lesser extent dendritic cells in the tumour mass.

Use of suicide genes for cancer gene therapy: study of the different approaches.

Cancer is a disease of high incidence for which conventional treatments are not necessarily effective. There is a need for the development of new alternative strategies. Among them, suicide gene therapy has been developed. In this approach, a gene encoding for a protein toxic under particular conditions is delivered to the target cells, resulting in their death. Although this approach has been in development for a long time, new combinations with other gene therapy areas, such as selective replicative viruses, tumour targeting, or conventional treatments such as chemo- or radiotherapy, are currently being tested. This review will summarise some of these approaches.

Transient foamy virus vector production by adenovirus vectors.

The genome of the prototype foamy virus (PFV) has been introduced into an adenoviral/PFV hybrid vector and tested for stable in vitro gene transfer. Three different adenoviruses are used to encode: (i) the PFV structural genes gag and pol (Ad-GagPolDeltaPacI); (ii) the PFV structural gene env (Ad-Env); and (iii) the PFV vector genome (Ad-MD9) encoding the transgene (the enhanced green fluorescent protein (eGFP) gene). Following cotransduction by the three adenoviruses, the target cells become transient PFV vector-producing cells, resulting in the in situ release of recombinant PFV at a titre of up to 10(3) vector particles/ml, which can then infect surrounding cells, leading to stable integration of the expression cassette. Stable eGFP expression, observed for up to 60 days (11 passages) in cells transduced with all three adenoviral vectors, was shown by PCR to be the result of PFV integration. In contrast, cells transduced with only the adenovirus encoding the PFV vector genome showed a marked decrease in eGFP expression by passage 2 (16 days post-transduction) and did not contain integrated PFV vector. In short, this paper describes the production of a hybrid vector capable of high in vitro transduction and stable transgene expression using adenovirus and PFV vectors.

Cellular characterization of the tropism of recombinant adenovirus for the adrenal glands.

BACKGROUND: Recombinant adenoviruses are widely used in gene therapy clinical trials. A particular tropism for the adrenal glands has been reported but the precise cellular base for this tropism has not been determined. MATERIALS AND METHODS: Recombinant adenoviruses were injected intravenously into Balb/c nu/nu or C57BL/6 mice. Seventy-two hours later, the animals were sacrificed and the adrenal glands and livers collected. The glands were sectioned and analyzed using immunohistochemical methods to detect adenoviral epitopes and transgene expression. Total RNA were extracted from the liver and adrenal glands of some animals and subjected to real-time RT-PCR. RESULTS: The only cell type infected in the adrenal glands of Balb/c nu/nu or C57BL/6 mice is the adrenocortical cells in the zona fasciculata. Quantitatively, the relative level of gene expression in the adrenal gland is comparable but lower than that measured in the liver. CONCLUSIONS: Systemic injection of recombinant adenovirus could be used as a procedure to restore adrenal steroidogenesis in clinical gene therapy protocols. In addition, our study suggest that adrenal dysfunction should be considered when criteria are established to assess the safety of gene therapy formulations administered systemically.

A recombinant E. coli vaccine to promote MHC class I-dependent antigen presentation: application to cancer immunotherapy.

We have examined the potential of recombinant Escherichia coli expressing listeriolysin O (LLO) to deliver tumour antigens to dendritic cells (DCs) for cancer immunotherapy. Using OVA as a model tumour antigen, we have shown in murine DCs that E. coli expressing cytoplasmic LLO and OVA proteins can deliver the OVA K(b)-restricted epitope SIINFEKL for MHC class I presentation. In contrast, when E. coli expressing OVA alone were used, MHC class II presentation of the OVA 323-339 I-A(b)-restricted peptide was predominant. When injected in vivo, DCs pulsed with E. coli expressing LLO and OVA induced production of cytotoxic T-lymphocytes capable of lysing an OVA-expressing melanoma cell line (B16-OVA) and resulted in suppression of tumour growth following challenge with B16-OVA. Immunisation of mice by direct injection of E. coli LLO/OVA provided a more potent anti-tumour response, resulting in complete protection in 75% of mice. Injection of live bacteria was not necessary as immunisation with paraformaldehyde-fixed E. coli LLO/OVA provided an even stronger anti-tumour response against B16-OVA. Altogether, our data highlight the potential of this system as a novel and efficient strategy for tumour immunotherapy.

Human foamy virus integrase fails to catalyse the integration of a circular DNA molecule containing an LTR junction sequence.

The presence of closed circular forms of the linear DNA genome of human foamy virus (HFV) has not been established. The ability of the HFV integrase (IN) to catalyse the integration of these circular forms (termed 2 long terminal repeat (LTR) circles) was investigated, with a view to producing a novel hybrid vector. To this end, a construct was made containing, in addition to the enhanced green fluorescent protein (eGFP) marker gene, the last 27 bp of the 3' U5 LTR region of HFV fused to the first 28 bp of the 5' U3 LTR, the latter representing a 2LTR circle. Marker gene expression following transfection of both 293 and 293T cells indicated that the level of integration was not significantly increased by the HFV IN. Moreover, correctly integrated provirus-like forms of the input plasmid could not be detected by PCR. Taken together, these results show that the HFV IN is not able to integrate a circular molecule containing an LTR junction and, hence, the technique is not exploitable as a tool to produce hybrid vectors for gene therapy.

An element in intron 1 of the CFTR gene augments intestinal expression in vivo.

The elements controlling the complex developmental and tissue-specific expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene lie outside the basal promoter region and have not been characterized. We previously identified a tissue-specific DNase I hypersensitive site (DHS) in intron 1 (185 + 10 kb) of the CFTR gene. Here we show that removal of the core element abolishes the activity of this DHS in transient transfection assays of reporter/enhancer gene constructs. We then compared expression from a 310 kb yeast artificial chromosome (YAC) that contains the entire CFTR gene with expression from the same YAC from which the DHS element had been deleted. Stable transfection of a human colon carcinoma cell line showed that transcription from the deleted YAC was reduced by approximately 60%. In transgenic mice, deletion of the intron 1 DHS had no effect on expression in the lung, but reduced expression in the intestine by approximately 60%. Thus, the regulatory element associated with the intron 1 DHS is tissue-specific and is required for normal CFTR expression levels in the intestinal epithelium in vivo.

Herpes simplex virus thymidine kinase/ganciclovir-induced cell death is enhanced by co-expression of caspase-3 in ovarian carcinoma cells.

There is a need to enhance the efficacy of genetic prodrug activation therapy using herpes simplex virus thymidine kinase (tk) and ganciclovir (GCV) following disappointing results in early clinical trials. tk/GCV has been shown to lead to the activation of caspase-3, a potent executor of apoptosis. We demonstrate that co-expression of pro-caspase-3 with tk/GCV leads to enhanced cell death in ovarian carcinoma cells in vitro. Following transfection with recombinant adenoviral vectors encoding tk, GCV treatment leads to greater cell death in pro-caspase-3-expressing clones of SKOV3 and IGROV1 than control cells, as well as more rapid activation of caspase-3 and more rapid cleavage of PARP. Flow cytometry suggests that there is a greater degree of S-phase block in the pro-caspase-3-expressing clones than in control cells following treatment with tk/GCV. None of these effects is seen following transfection with a control adenovirus that does not encode tk. The increased cell death, early caspase-3 activation and PARP cleavage, and flow cytometric changes seen in pro-caspase-3-expressing cells can be partially inhibited by treatment with benzyloxycarbonyl-val-ala-asp fluoromethylketone, a synthetic caspase inhibitor. Our data suggest that co-expression of pro-caspase-3 may lead to a significant enhancement of the efficacy of tk/GCV therapy.

Gene therapy for carcinoma of the breast: Genetic toxins.

Gene therapy was initially envisaged as a potential treatment for genetically inherited, monogenic disorders. The applications of gene therapy have now become wider, however, and include cardiovascular diseases, vaccination and cancers in which conventional therapies have failed. With regard to oncology, various gene therapy approaches have been developed. Among them, the use of genetic toxins to kill cancer cells selectively is emerging. Two different types of genetic toxins have been developed so far: the metabolic toxins and the dominant-negative class of toxins. This review describes these two different approaches, and discusses their potential applications in cancer gene therapy.

Analysis of a DNase I hypersensitive site located -20.9 kb upstream of the CFTR gene.

The cystic fibrosis transmembrane conductance regulator gene (CFTR) shows a tightly regulated pattern of expression with spatial and temporal control. The regulatory elements achieving this appear to lie outside the basal promoter of the gene. We previously identified DNase I hypersensitive sites (DHSs) at -79.5 kb and -20.5 kb with respect to the CFTR translational start site which may contain important regulatory elements. We have now investigated further the DHS at -20.5 kb to evaluate its potential function in the regulation of CFTR expression. Finer mapping revealed that the DHS lies at -20.9 kb. Deletion of the DHS from a 310-kb yeast artificial chromosome (YAC) containing the human CFTR gene has shown that this site may be responsible for about 60% of wild-type levels of transcription from the YAC transgene when expressed in Caco2 cells. DNase I footprinting showed several regions of protection within the -20.9 kb region with nuclear extracts from Caco2 cells, but not with extracts from lymphoblastoid cells, which do not show the DHS. Matches to several transcription factor-binding sites were found, but supershift analysis with specific antibodies did not identify the transcription factors involved. Two purine/pyrimidine mirror repeat elements within the -20.9-kb DHS were shown not to adopt non-B-DNA conformations. Thus, we provide evidence for a role for the -20.9 kb DHS in the transcriptional regulation of the CFTR gene, although the mechanisms mediating this effect remain unclear.