Simon D. Scott

Murine herpesvirus 68 is genetically related to the gammaherpesviruses Epstein-Barr virus and herpesvirus saimiri

Short nucleotide sequence analysis of seven restriction fragments of murine herpesvirus 68 (MHV-68) DNA has been undertaken and used to determine the overall genome organization and relatedness of this virus to other well characterized representatives of the alpha-, beta- and gammaherpesvirus subgroups. Nine genes have been identified which encode amino acid sequences with greater similarity to proteins of the gammaherpesvirus Epstein-Barr virus (EBV) than to the homologous products of the alphaherpesviruses varicella-zoster virus and herpes simples virus type 1 or the betaherpesvirus human cytomegalovirus. In addition, the genome organization of MHV-68 is shown to have an overall collinearity with that of the gammaherpesviruses EBV and herpesvirus saimiri. In common with these viruses, dinucleotide frequency analysis of MHV-68 coding sequences reveals a marked reduction in CpG dinucleotide frequency thus implicating a dividing cell population as the site of latency in vivo.

Gene Sequence and Mapping Data from Marek's Disease Virus and Herpesvirus of Turkeys: Implications for Herpesvirus Classification

Purified DNAs from Mareks disease virus (MDV) and the herpesvirus of turkeys (HVT) were randomly sheared and cloned into the M13 bacteriophage. Two-hundred and ten MDV and 130 HVT clones were sequenced to give representative samples of the genome sequences. The predicted amino acid sequences from these gammaherpes-viruses were compared to known sequences from other herpesviruses using computer analysis. Thirty-five MDV and 24 HVT genes were identified by comparison with varicella-zoster virus (VZV), an alphaherpesvirus. However, only 14 MDV and seven HVT genes, giving generally lower homology scores, were found by comparison with Epstein-Barr virus (EBV), a gammaherpesvirus, indicating that MDV and HVT sequences bear greater similarity to VZV than to EBV sequences. A number of sequences were mapped by hybridizing labelled M13 clones to Southern blots of restriction fragments of MDV or HVT DNA. The results were consistent with the MDV and HVT genomes being collinear with VZV.

A novel magnetic approach to enhance the efficacy of cell-based gene therapies

Attempts have been made to use various forms of cellular vectors to deliver therapeutic genes to diseased tissues like malignant tumours. However, this approach has proved problematic due to the poor uptake of these vectors by the target tissue. We have devised a novel way of using magnetic nanoparticles (MNPs) to enhance the uptake of such ‘therapeutically armed’ cells by tumours. Monocytes naturally migrate from the bloodstream into tumours, so attempts have been made to use them to deliver therapeutic genes to these sites. However, transfected monocytes injected systemically fail to infiltrate tumours in large numbers. Using a new in vitro assay for assessing monocyte extravasation, we show that the ability of transfected human monocytes to migrate across a human endothelial cell layer into a 3D tumour spheroid is markedly increased when cells are pre-loaded with MNPs and a magnetic force is applied close to the spheroid. Furthermore, systemic administration of such ‘magnetic’ monocytes to mice bearing solid tumours led to a marked increase in their extravasation into the tumour in the presence of an external magnet. This new magnetic targeting approach could be used to increase the targeting, and thus the efficacy, of many cell-based gene therapies in vivo.

Development of synthetic promoters for radiation-mediated gene therapy.

Exposure of cells to ionising radiation results in the activation of specific transcriptional control (CArG) elements within the early growth response 1 (Egr1) gene promoter, leading to increased gene expression. As part of a study investigating the potential use of these elements in radiation-controlled gene therapy vectors, we have incorporated their sequences into a synthetic gene promoter and assayed for the ability to induce expression of a downstream reporter gene following irradiation. In vector-transfected MCF-7 breast adenocarcinoma cells, the synthetic promoter was more effective than the wild-type Egr1 counterpart in up-regulating expression of the reporter gene after exposure to a single 5 Gy dose, and equally effective as the wild-type in U87-MG glioma cells. The level of gene expression achieved using the synthetic promoter was dependent on the inducing radiation dose for both U87-MG and MCF-7 cells, being maximal at 3 Gy and decreasing at 5 and 10 Gy. Furthermore, induction could be repeated by additional radiation treatments. The latter indicates that up-regulation should be additive during fractionated radiotherapy schedules. To demonstrate the potential clinical benefit of such an approach, the synthetic promoters were also shown to drive expression of the herpes simplex virus thymidine kinase gene, leading to enhanced cell killing in the presence of the prodrug ganciclovir (GCV) when compared with cells treated with radiation alone. Our results demonstrate that the synthetic promoter is responsive to low doses of ionising radiation and therefore isolated CArG elements function as radiation-mediated transcriptional enhancers outside their normal sequence context. The continued development and optimisation of such radiation-responsive synthetic promoters is expected to make a valuable contribution to the development of future radiation-responsive vectors for cancer gene therapy.

Novel chimeric gene promoters responsive to hypoxia and ionizing radiation.

Despite being an adverse prognostic factor in radiotherapy, hypoxia represents a physiological difference that can be exploited for selective cancer gene therapy. In this study gene therapy vectors responsive to both hypoxia and ionizing radiation (IR) were developed. Gene expression was regulated by novel, synthetic promoters containing hypoxia responsive elements (HREs) from the erythropoietin (Epo), the phosphoglycerate kinase 1 (PGK1) and the vascular endothelial growth factor (VEGF) genes, and IR-responsive CArG elements from the early growth response (Egr) 1 gene. All chimeric promoters could be activated by hypoxia and/or IR-treatment, and selectively control marker gene expression in human T24 bladder carcinoma and MCF-7 mammary carcinoma cells. Importantly, enhancers containing combinations of HREs and CArG elements were able to respond to both triggering treatments, with the Epo HRE/CArG combination proving to be the most responsive and robust. The Epo HRE/CArG enhancer could effectively control a suicide gene therapy strategy by selectively sensitizing hypoxic and/or irradiated cells expressing the enzyme horseradish peroxidase (HRP) to the prodrug indole-3-acetic acid (IAA). These data indicate that the use of such chimeric promoters may effectively regulate therapeutic gene expression within the tumor microenvironment in gene therapy strategies aimed at addressing the problem of hypoxia in radiotherapy.

How to overcome (and exploit) tumor hypoxia for targeted gene therapy

Tumor hypoxia has long been recognized as a critical issue in oncology. Resistance of hypoxic areas has been shown to affect treatment outcome after radiation, chemotherapy, and surgery in a number of tumor sites. Two main strategies to overcome tumor hypoxia are to increase the delivery of oxygen (or oxygen‐mimetic drugs), and exploiting this unique environmental condition of solid tumors for targeted therapy. The first strategy includes hyperbaric oxygen breathing, the administration of carbogen and nicotinamide, and the delivery of chemical radiosensitizers. In contrast, bioreductive drugs and hypoxia‐targeted suicide gene therapy aim at activating cytotoxic agents at the tumor site, while sparing normal tissue from damage. The cellular machinery responds to hypoxia by activating the expression of genes involved in angiogenesis, anaerobic metabolism, vascular permeability, and inflammation. In most cases, transcription is initiated by the binding of the transcription factor hypoxia‐inducible factor (HIF) to hypoxia responsive elements (HREs). Hypoxia‐targeting for gene therapy has been achieved by utilizing promoters containing HREs, to induce selective and efficient transgene activation at the tumor site. Hypoxia‐targeted delivery and prodrug activation may add additional levels of selectivity to the treatment. In this article, the latest developments of cancer gene therapy of the hypoxic environment are discussed, with particular attention to combined protocols with ionizing radiation. Ultimately, it is proposed that by adopting specific transgene activation and molecular amplification systems, resistant hypoxic tumor tissues may be effectively targeted with gene therapy. J. Cell. Physiol. 197: 312–325, 2003© 2003 Wiley‐Liss, Inc.

A radiation-controlled molecular switch for use in gene therapy of cancer.

Ionising radiation induces the expression of a number of radiation-responsive genes and there is current interest in exploiting this to regulate the expression of exogenous therapeutic genes in gene therapy strategies for cancer. However, the radiation-responsive promoters used in these approaches are often associated with low and transient levels of therapeutic gene expression. We describe here a novel radiation-triggered molecular switching device based on promoter elements from the radiation-responsive Egr-1 gene and the cre-LoxP site-specific recombination system of the P1 bacteriophage. Using this system, a single, minimally toxic dose of radiation induced cre-mediated excision of a lox-P flanked stop cassette in a silenced expression vector and this resulted in amplified levels of CMV-promoter-driven expression of the exogenous tumour-sensitising gene, HSV-tk. This strategy could be used in combination with targeted delivery and tumour-specific promoters to elicit the tumour-targeted and prolonged expression of a variety of tumour-sensitising genes and provide an unprecedented level of control and tumour selectivity.

Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy.

We have been developing synthetic gene promoters responsive to clinical doses of ionizing radiation (IR) for use in suicide gene therapy vectors. The crucial DNA sequences utilized are units with the consensus motif CC(A/T)6GG, known as CArG elements, derived from the IR-responsive Egr1 gene. In this study we have investigated the parameters needed to enhance promoter activation to radiation. A series of plasmid vectors containing different enhancer/promoters were constructed, transiently transfected into tumor cells (MCF-7 breast adenocarcinoma and U-373MG glioblastoma) and expression of a downstream reporter assayed. Results revealed that increasing the number of CArG elements, up to a certain level, increased promoter radiation-response; from a fold-induction of 1.95 ± 0.17 for four elements to 2.74 ± 0.17 for nine CArGs of the same sequence (for MCF-7 cells). Specific alteration of the core A/T sequences caused an even greater positive response, with fold-inductions of 1.71 ± 0.23 for six elements of prototype sequence compared with 2.96 ± 0.52 for one of the new sequences following irradiation. Alteration of spacing (from six to 18 nucleotides) between elements had little effect, as did the addition of an adjacent Sp1 binding site. Combining the optimum number and sequence of CArG elements in an additional enhancer was found to produce the best IR induction levels. Furthermore, the improved enhancers also performed better than the previously reported prototype when used in in vitro and in vivo experimental GDEPT. We envisage such enhancers will be used to drive suicide gene expression from vectors delivered to a tumor within an irradiated field. The modest, but tight expression described in the present study could be amplified using a molecular ‘switch’ system as previously described using Cre/LoxP. In combination with targeted delivery, this strategy has great potential for significantly improving the efficacy of cancer treatment in the large number of cases where radiotherapy is currently employed.

Analysis of p53 status in tonsillar carcinomas associated with human papillomavirus

Tonsillar squamous cell carcinomas (a total of 14) were examined both for the presence of human papillomavirus (HPV) DNA and for p53 alterations. General primer-mediated HPV polymerase chain reaction (GP-PCR) revealed the presence of HPV DNA in 12/14 cases. Subsequent typing by HPV type-specific PCR and sequence or hybridization analysis of GP-PCR products revealed DNA from HPV 16 in seven cases, from HPV 33 in two cases, and from HPV 7, HPV 16/33 and HPV 33/59 each in a single case. p53 immunohistochemistry performed on nine HPV containing tonsillar carcinomas using polyclonal serum CM-1 showed elevated p53 levels in four cases. These included 3/5 HPV 16 containing carcinomas and the HPV 33/59 containing carcinoma. Analysis of p53 mutations using denaturing gradient gel electrophoresis (DGGE) of GC-clamped PCR products of exons 5 to 8 showed p53 gene alterations in 3/13 cases, including 2/11 HPV positive cases and 1/2 HPV negative cases. The alterations included a silent point mutation within exon 8 of an HPV 16 containing carcinoma, a 1 bp deletion within exon 8 of an HPV 33 containing carcinoma, and a missense mutation within exon 7 of one of the HPV negative carcinomas. There was evident discrepancy between p53 immunohistochemistry and gene analysis. Four HPV containing cases showing elevated p53 levels did not reveal the presence of exon 5 to 8 alterations affecting the amino acid code, suggesting the presence of mutations occurring in other exons or non-mutational p53 stabilization. The data indicate that HPV and elevated p53 can coexist in tonsillar carcinomas and that despite the low frequency of p53 mutations the presence of HPV is not exclusively related to the absence of mutated p53.

Hypoxia- and radiation-activated Cre/loxP 'molecular switch' vectors for gene therapy of cancer

Although a significant negative prognostic factor, tumor hypoxia can be exploited for gene therapy. To maximize targeting within the tumor mass, we have developed synthetic gene promoters containing hypoxia-responsive elements (HREs) from the erythropoietin (Epo) gene as well as radiation-responsive CArG elements from the early growth response (Egr) 1 gene. Furthermore, to achieve high and sustained expression of the suicide gene herpes simplex virus thymidine kinase (HSVtk), our gene therapy vectors contain an expression amplification system, or ‘molecular switch’, based on Cre/loxP recombination. In human glioma and breast adenocarcinoma cells exposed to hypoxia and/or radiation, the HRE/CArG promoter rapidly activated Cre recombinase expression leading to selective and sustained HSVtk synthesis. Killing of transfected tumor cells was measured after incubation with the prodrug ganciclovir (GCV; converted by HSVtk into a cytotoxin). In vitro, higher and more selective GCV-mediated toxicity was achieved with the switch vectors, when compared with the same inducible promoters driving HSVtk expression directly. In tumor xenografts implanted in nude mice, the HRE/CArG-switch induced significant growth delay and tumor eradication. In conclusion, hypoxia- and radiation-activated ‘molecular switch’ vectors represent a promising strategy for both targeted and effective gene therapy of solid tumors.