Anticancer Gene Transfer for Cancer Gene Therapy

Gene therapy vectors are among the treatments currently used to treat malignant tumors. Gene therapy vectors use a specific therapeutic transgene that causes death in cancer cells. In early attempts at gene therapy, therapeutic transgenes were driven by non-specific vectors which induced toxicity to normal cells in addition to the cancer cells. Recently, novel cancer specific viral vectors have been developed that target cancer cells leaving normal cells unharmed. Here we review such cancer specific gene therapy systems currently used in the treatment of cancer and discuss the major challenges and future directions in this field

Oncolytic Viruses for Cancer Therapy: Overcoming the Obstacles

Targeted therapy of cancer using oncolytic viruses has generated much interest over the past few years in the light of the limited efficacy and side effects of standard cancer therapeutics for advanced disease. In 2006, the world witnessed the first government-approved oncolytic virus for the treatment of head and neck cancer. It has been known for many years that viruses have the ability to replicate in and lyse cancer cells. Although encouraging results have been demonstrated in vitro and in animal models, most oncolytic viruses have failed to impress in the clinical setting. The explanation is multifactorial, determined by the complex interactions between the tumor and its microenvironment, the virus, and the host immune response. This review focuses on discussion of the obstacles that oncolytic virotherapy faces and recent advances made to overcome them, with particular reference to adenoviruses

Dissection of human papillomavirus type 33 L2 domains involved in nuclear domains (ND) 10 homing and reorganization

AbstractWe have recently shown that the minor capsid protein L2 of human papillomavirus type 33 (HPV33) recruits the transcriptional repressor Daxx into nuclear domains (ND) 10 and causes the loss of the transcriptional activator Sp100 from these subnuclear structures (Florin et al., 2002b). In order to dissect L2 domains involved in nuclear translocation, ND10 homing, loss of Sp100, and recruitment of Daxx, a detailed deletion mutagenesis of L2 was performed. Using immunofluorescence and green fluorescent protein fusions, we have identified two nuclear localization signals (NLS) in the central and C-terminal part of L2, respectively, homologous to previously identified NLS in HPV6B L2 (Sun et al., 1995). We mapped the ND10 localization domain to within a 30 amino acid peptide in the C-terminal half of L2. L2-induced attraction of Daxx into ND10, coimmunoprecipitation of L2 and Daxx, as well as induction of the loss of Sp100 from ND10 require an intact ND10 localization domain. This domain contains conserved PXXP motives characteristic of some protein/protein interacting domains. Our data also suggest that the Daxx/L2 interaction may be the driving force for L2 accumulation in ND10

Assessment and development of adenovirus type 11 for cancer therapy

PhDThe efficacy of oncolytic virotherapy is influenced by the interactions between the tumour, virus and host immunity. The first generation of oncolytic adenoviruses, based on serotype 5 (Ad5), has achieved limited success in clinical trials. Its shortcomings include the downregulation and inaccessibility of its receptor, the Coxsackie and adenovirus receptor (CAR) in cancer cells, high prevalence of neutralising antibodies and hepatotoxicity. In contrast, Ad11 binds to CD46 and other receptor(s) but its potential as an oncolytic virus remains to be explored. A panel of human cancer cell lines were found to express higher levels of CD46 than CAR. However, not all cell lines were more sensitive to Ad11-mediated cytotoxicity in vitro compared to Ad5. Treatment of Ad5-insensitive PC-3 human prostate cancer xenografts with Ad11 resulted in significant reduction in tumour growth, but not Ad11-insensitive MIA PaCa-2 human pancreatic cancer xenografts. Virus attachment and nuclear entry of Ad11 were significantly better than Ad5 even in cells that were insensitive to Ad11 killing. In these cells, however, Ad11 E1A mRNA levels were much lower than those of Ad5, producing a negative effect on viral DNA amplification, structural protein synthesis, progeny production and cell killing. Cells that were sensitive to Ad11 cytotoxicity showed higher levels of E1A mRNA. The region upstream of Ad5 E1A demonstrated higher transcription-enhancing activity than the corresponding region of Ad11. Two Ad11 mutants were constructed in which E1A was under the control of the Ad5 E1A promoter and enhancer-promoter, respectively. With the latter virus, improved oncolytic potency was observed. It was superior to Ad11 and also to Ad5 in many cancer cell lines, and was as effective as Ad5 in the MIA PaCa-2 xenograft model. Therefore, Ad11 with the Ad5 E1A enhancerpromoter should be used as a backbone for the future development of potent and tumour-specific oncolytic Ad11 mutants

Normal self proteins as targets for tumour specific cytotoxic T lymphocytes (CTL)

Many tumour cells express high levels of proteins which are present in normal cells at lower levels. Such proteins might be targets for immunotherapy if lymphocytes with specificity for these proteins are present in the repertoire and can discriminate between cells expressing high or low levels of the target protein. The first step in exploring the possibility of targeting immunotherapy to normal self proteins is therefore to determine whether lymphocytes specific for molecules overexpressed in tumours exist and can be activated to recognize specifically cells expressing high levels of the proteins. It was possible to stimulate murine cyclin D1 or mdm2 specific cytotoxic T lymphocytes (CTL) by in vivo priming of mice with recombinant vaccinia virus expressing the proteins. The in vitro conditions used for restimulation of in vivo primed CTL were found to have a profound effect on the magnitude of the CTL response observed in vitro. In addition, in vitro priming of CTL from naïve spleens with peptide pulsed dendritic cells identified a mdm2 derived peptide, mdp441, which could stimulate CTL capable of recognising endogenously processed mdm2. These peptide specific CTL were of high avidity in contrast to most of the peptide specific CTL stimulated with other self peptides. The mdp441 peptide was not recognised by mdm2 specific CTL generated by in vivo immunisation with mdm2 protein. The results show that tolerance to self proteins is not absolute and that it is possible to stimulate CTL to recognize endogenously processed self protein either by in vivo immunisation with recombinant vaccinia virus expressing the self protein or by in vitro priming of naive responder cells with peptide pulsed antigen presenting cells. These results have implications for immunotherapy of human cancers

Regulation of Vaccinia Virus Replication: a Story of Viral Mimicry and a Novel Antagonistic Relationship Between Vaccinia Kinase and Pseudokinase

Poxviruses employ sophisticated signaling pathways that thwart cellular defense mechanisms and simultaneously ensure viral factors are modulated properly. Yet, our understanding of these complex signaling networks are incomplete. For example, the vaccinia B1 kinase plays a vital role in inactivating the cellular antiviral factor BAF, and is suggested to orchestrate other pathways. B1 is highly conserved among poxviruses and exhibits a remarkable degree of similarity to VRKs, a family of cellular kinases, suggesting that the viral enzyme has evolved to mimic VRK activity. Indeed, B1 and VRKs have been demonstrated to target a shared substrate, the DNA binding protein BAF, elucidating a signaling pathway important for mitosis and the antiviral response. Our research further characterized the role of B1 during vaccinia infection to gain novel insights into its regulation and integration with cellular signaling pathways. We began by constructing and characterizing the first B1 deletion virus (ΔB1). Then using this virus, we tested the hypothesis that cellular VRKs can complement B1 function, and discovered a VRK2 role in facilitating DNA replication in the absence of B1. Study of the VRK2 mechanism revealed that B1 and VRK2 mediate DNA replication via an additional pathway that is BAF independent. We also utilized the ΔB1 virus in an experimental evolution assay to perform an unbiased search for suppressor mutations and identify novel pathways involving B1. Interestingly, our characterization of the adapted viruses reveals that mutations correlating with a loss of function of the vaccinia B12 pseudokinase provide a striking fitness enhancement to this virus. Next, B12 characterization showed a nuclear localization, unique for poxvirus proteins, that is related to its repressive function. Our data indicate that B12 is not a global repressor, but inhibits vaccinia replication in the absence of the B1 kinase. The mechanism of B12 partially depends on suppression of BAF antiviral activity. However, the parallel B12 pathway to restrict virus replication is less clear. Together, our studies of B1 and B12 present novel evidence that a paralogous kinase-pseudokinase pair can exhibit a unique epistatic relationship in a virus, and orchestrate yet-to-be-discovered nuclear events during infection. Advisor: Matthew S. Wieb

Mechanisms of Host-Range Function of Vaccinia Virus K1L Gene: a Dissertation

The KIL gene of vaccinia virus encodes for a host range protein; in the absence of which, the virus is unable to grow in certain cell lines (RK-13 and some human cell lines). KIL function can be complemented in RK-13 cells by the cowpox host range gene product CP77 despite a lack of homology between the two proteins except for ankyrin repeats. We investigated the role of ankyrin repeats ofthe K1L gene in the host-range restriction of growth in RK-13 cells. The growth of a recombinant vaccinia virus, with the K1L gene mutated in the most conserved ankyrin repeat, was severely impaired as evidenced by lack of plaque formation and reduction in viral titers. Infection of RK-I3 cells with the mutant recombinant vaccinia virus resulted in total shutdown of both cellular and viral protein synthesis early in infection, indicating that the host restriction mediated by the ankyrin repeat is due to a translational block. A comparison of the cellular localization of the K1L wild type and mutated forms showed no difference, as both localized exclusively in the cytoplasm of RK-I3 cells. We also investigated the interaction of the vaccinia virus K1L protein with cellular proteins in RK-13 cells and co-immunoprecipitated a 90 kDa protein identified as the rabbit homologue of human ACAP2, a GTPase-activating protein with ankyrin repeats. Our result suggests the importance of ankyrin repeat for host-range function of K1L in RK-13 cells and identifies ACAP2 as a cellular protein which may be interacting with K1L

Inactivation of pathogens on food and contact surfaces using ozone as a biocidal agent

This study focuses on the inactivation of a range of food borne pathogens using ozone as a biocidal agent. Experiments were carried out using Campylobacter jejuni, E. coli and Salmonella enteritidis in which population size effects and different treatment temperatures were investigate

A New Inhibitor of Apoptosis from Vaccinia Virus and Eukaryotes

A new apoptosis inhibitor is described from vaccinia virus, camelpox virus, and eukaryotic cells. The inhibitor is a hydrophobic, multiple transmembrane protein that is resident in the Golgi and is named GAAP (Golgi anti-apoptotic protein). Stable expression of both viral GAAP (v-GAAP) and human GAAP (h-GAAP), which is expressed in all human tissues tested, inhibited apoptosis induced by intrinsic and extrinsic apoptotic stimuli. Conversely, knockout of h-GAAP by siRNA induced cell death by apoptosis. v-GAAP and h-GAAP display overlapping functions as shown by the ability of v-GAAP to complement for the loss of h-GAAP. Lastly, deletion of the v-GAAP gene from vaccinia virus did not affect virus replication in cell culture, but affected virus virulence in a murine infection model. This study identifies a new regulator of cell death that is highly conserved in evolution from plants to insects, amphibians, mammals, and poxviruses

Gene Therapy Applications to Cancer Treatment

Over the past ten years significant advances have been made in the fields of gene therapy and tumour immunology, such that there now exists a considerable body of evidence validating the proof in the principle of gene therapy based cancer vaccines. While clinical benefit has so far been marginal, data from preclinical and early clinical trials of gene therapy combined with standard therapies are strongly suggestive of additional benefit. Many reasons have been proposed to explain the paucity of clinical responses to single agent vaccination strategies including the poor antigenicity of tumour cells and the development of tolerance through down-regulation of MHC, costimulatory, signal transduction, and other molecules essential for the generation of strong immune responses. In addition, there is now evidence from animal models that the growing tumour may actively inhibit the host immune response. Removal of the primary tumour prior to T cell transfer from the spleen of cancer bearing animals, led to effective tumour cell line specific immunity in the recipient mouse suggesting that there is an ongoing tumour-host interaction. This model also illustrates the potential difficulties of clinical vaccine trials in patients with advanced stage disease