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Volume 8 - 2004


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1-8

Combination of Optison with ultrasound and electroporation increases albumin and thrompoietin transgene expression whilst elongation factor promoter prolongs its duration

Author(s): Dr. Nagy Habib,

Abstract: Optison can increase their expression. Gene Therapy and Molecular Biology Vol 8, page 1 Hypoalbuminaemia and thrombocytopaenia are two clinical problems frequently encountered in patients with chronic liver failure or cancer following treatment with chemotherapy. The current study was designed to assess the magnitude and duration of thrombopoietin and albumin transgene expression hoping to increase the production of albumin and platelets. Immunocompetent and immunocompromised (nude) mice were injected intramuscularly with plasmids expressing either human serum albumin or human thrombopoietin. The therapeutic expression cassette of the plasmids was driven by either CMV or elongation factor 1-alpha promoters respectively. In order to achieve muscle specific expression both gene constructs included the myosin light chain enhancer. The experiment was conducted in a group of mice which were injected with the transgene plasmid either in normal saline or plasmid followed by electroporation, ultrasound, optison and a combination of all three to increase transgene expression. The result showed that plasmids with the CMV promoter induced the highest transgenic expression lasting for one week whilst plasmids with the elongation factor 1-alpha promoter produced a weaker expression lasting for a longer and more stable duration of expression up to 3 months in both immunocompetent and nude mice. The combination of electroporation and ultrasound with Optison provided the highest transgene expression. We concluded that it would be possible to increase albumin and platelets production by an intramuscular injection of plasmids expressing human albumin and thromopoietin. A combination of electroporation and ultrasound with

Keywords: Du Cane Road, London W12 ONN, UK



9-18

Adeno-associated virus (AAV) vector-mediated liver- and muscle-directed transgene expression using various kinds of promoters and serotypes

Author(s): Dr. Hiroaki Mizukami,

Abstract: AAV vectors have become a practical choice for gene transfer into a variety of tissues in vivo. For the purpose of supplemental gene therapy, liver and skeletal muscles are the major targets of transduction. Recently, vector serotypes and their tissue specificity is an important issue for successful gene transfer. Although the potential utility of each serotype has been announced, a systematic comparison has not been fully made. In addition, the choice of a suitable promoter is also critical. In this study, we investigated the level of the transgene product by comparing different serotypes as well as promoters. We utilized murine erythropoietin (mEpo)-expressing or LacZ-expressing AAV vectors and transferred them into immune-competent mice. For the muscle-directed expression, serotype 1 showed the highest Epo expression followed by 5, 2, 3 and 4. As for the promoter analysis in the liver, the CAG promoter achieved the highest Epo concentration, followed by CMV, PGK, and EF-1α. Further comparison in the liver with different serotypes utilizing the CAG promoter revealed that serotype 5 showed the highest level of expression. These results will aid in the design of an optimal AAV vector structure, especially for the systemic delivery of transgene products.

Keywords: dependovirus, serotype, erythropoietin, promoters, muscle, liver



19-30

Signaling pathways and polyomavirus oncoproteins: Importance in malignant transformation

Author(s): Dr. Kamel Khalili,

Abstract: Treatment of a particular cancer, whether by gene therapy or by small molecule inhibitors, is predicated upon a knowledge of the signal transduction pathways that became dysregulated as the tumor developed and cells underwent malignant transformation. Usually cancer is thought of as a disease that progresses through the gradual accumulation of multiple successive genetic “hits” leading from normal cells to fully malignant metastatic tumors in a fashion such as that described for the colorectal adenoma-carcinoma sequence. These hits include mutations that activate oncogenes, knockouts of anti-oncogenes (tumor suppressors) and events that increase mutation rates or destabilize the genome. Polyomaviruses are small DNA viruses that encode proteins that promote cell transformation in culture, induce tumors in experimental animals and have been found in association with some human cancers. The small genome size of the polyomaviruses means that a few proteins must perform many tasks. There is evidence that these multifunctional viral proteins can provide several “hits” at once to cells and thus circumvent certain steps that are necessary for malignant transformation during non-viral tumorigenesis. In this review, the biological and pathological properties of polyomavirus proteins are discussed. The relevance of these is underscored by the growing evidence for the involvement of polyomaviruses in some human cancers. Polyomavirus gene-specific therapeutic strategies might be considered in these cancers since ablation of the function of viral oncoproteins would be expected to reverse multiple molecular events involved in malignant transformation.

Keywords: Signaling pathways, polyomavirus, oncoproteins, Large-T antigen, pRB, p53, p300, IRS-1, β-Catenin, p185/p193, Chaperone activity, Small-t, Agnoprotein



31-44

Radioprotective antioxidant gene therapy: Potential mechanisms of action

Author(s): Dr. Joel S. Greenberger,

Abstract: The plasmid/liposome (PL) delivery of the manganese superoxide dismutase (MnSOD) transgene to lung, esophagus, oral cavity, bladder, or delivery of the same transgene by viral vectors including Herpes virus, adenovirus, and retrovirus to cells in culture or to intestine have been demonstrated to confer cellular and tissue resistance to ionizing iradiation damage including apoptosis. In contrast, transgene mediated expression of other antioxidant transgenes including Cu/ZnSOD, metallothionine, glutathione peroxidase, was ineffective. Delivery of protein by liposomes containing MnSOD, lazaroid, or vitamin E was also ineffective for normal tissue radioprotection. Radioprotective MnSOD transgene protection has been shown to be specific to localization of protein to the mitochondrial membrane in that deletion of the mitochondrial localization signal from the transgene was associated with cytoplasmic overexpression and was not radioprotective. Addition of the mitochondrial localization to cytoplasmic Cu/ZnSOD transgene, localization of transgene product to the mitochondria and conferred radioprotection. The biochemical mechanism of MnSOD radioprotection has been shown to be associated with stabilization and an increase in intracellular thiol and antioxidant pool size at the level of the mitochondrial membrane suggesting that overexpression of MnSOD stabilizes cellular antioxidant reserves, and that its antioxidant reserve capacity is associated with cellular and tissue radioprotection. In three orthotopic tumor model systems (Lewis lung carcinoma at the carina, SCC-VII murine floor of the mouth tumors in C57BL/6J mice, and mediastinal sarcomas in C3H/HeJ mice), tumor radioprotection was not detected under conditions of MnSOD-PL protection of the associated normal tissue: lung, oral cavity/oropharynx, and esophagus respectively. Reduced ability of many tumors to handle increased hydrogen peroxide bioavailability, which is the catabolite of MnSOD dismutation of superoxide, has been reported to synergize with ionizing iradiation to enhance tumor killing. In this regard, radiosensitization of oral cavity and lung tumor cells in vitro has been demonstrated with MnSOD-PL transfection. The data suggest that differences between normal tissue and tumor management of oxidative stress may be exploited by MnSOD-PL gene therapy to facilitate simultaneous normal tissue radioprotection and tumor radiosensitization.

Keywords: Manganese Superoxide Dismutase, Antioxidant, Ionizing iradiation, Gene Therapy, Antioxidant Pool Size



45-59

Huntington’s disease: pre-clinical models to therapy using genetically-modified immunoisolated cells and lentiviral vectors

Author(s): Dr. Dwaine F. Emerich,

Abstract: Summary Huntington’s disease (HD) is a devastating genetic disorder. Despite the absence of effective therapy, there has been an explosion in interest for developing treatment strategies aimed at lessening or preventing the neuronal death that occurs in this disease. In large part, the renewed interest in neuroprotective strategies has been spurred by our increasing understanding of the genetic and molecular events that drive the underlying neuropathology of HD. This escalating understanding of the biological underpinnings of HD is derived from several convergent sources represented by investigators with clinical, genetic, molecular, physiological and neurobehavioral backgrounds. The diversity of data being generated has, in turn, produced a unique time in HD research where an impressive number of potential therapeutics are coming to the forefront. This review outlines the use of intracerebrally delivered CNTF using immunoisolated, genetically-modified cells and lentiviral vectors. Each approach has advantages and disadvantages but both have produced impressive pre-clinical demonstrations of neuronal preservation in models of HD. These benefits are highlighted in the context that HD is a neurodegenerative disorder in which genetic detection provides a clear and unequivocal opportunity for neuroprotection.

Keywords: Intracerebral delivery, CNTF, Huntington’s disease, gene therapy, Neuropathology, neuroprotection



61-74

Histone deacetylase inhibitors: promising candidates for chemotherapeutic drugs

Author(s): Dr. Ricky W. Johnstone,

Abstract: Despite advances in the molecular understanding of tumorigenesis and tumor cell apoptosis, the efficacy of chemotherapeutic treatment has not significantly improved over the last decades. Conventional treatment regimens suffer from a small therapeutic window and are often associated with severe side effects. Recent data suggest that a novel class of compounds, the histone deacetylase inhibitors (HDAC inhibitors), displays potent cytotoxicity towards tumor cells with low or negligible effects on untransformed cells. In addition to causing cell cycle arrest and/or differentiation and programmed cell death in tumor cells, they can also inhibit tumor angiogenesis and display immunosuppressive properties. Excitingly, various HDAC inhibitors compounds show synergy with other anti- cancer drugs and are involved in early clinical trials or pre-clinical development.The mechanism of action of HDAC inhibitors has not been completely elucidated. They induce histone hyperacetylation associated with transcriptional modulation of a set of genes. Treatment of malignant cells with HDAC inhibitors has been reported to elicit upregulation of the cell cycle inhibitor p21(WAF1), and induction of apoptosis most likely is coincident with the cleavage and activation of the proapoptotic Bcl-2 family member Bid. In addition, reactive oxygen species seem to play an important role in HDAC inhibitor-mediated cell death.This article summarizes what is currently known about the molecular and systemic sequelae of HDAC inhibitor treatment and focuses on recent progress regarding potential applications for cancer treatment as stand-alone or combination therapy.

Keywords: Histone deacetylase inhibitors, Chromatin and histone, Cyclic peptides, Short-chain fatty acids, Benzamides, Ketones, apoptosis



75-84

Successful lipofectin mediated transduction of a pIRES-fl-tk gene in human breast carcinoma: effective inducing the expansion as well as increasing the activity of dendritic cells

Author(s): Dr. Xuetao Pei,

Abstract: A novel approach to the treatment of cancer is based on combining the gene therapy and DC (dendritic cell) immunotherapy. In this study, DC were induced and expanded from cord blood and PBMC (peripheral blood mononuclear cell). The two kinds of DC exhibited the capacity to stimulate the proliferation of allogeneic T lymphocytes. We further constructed a recombinant vector carrying herpes simplex virus thymidine kinase gene (tk), internal ribosome entry site (IRES), Flt-3 ligand gene (fl) and transfected it into the human breast carcinoma cell line MCF-7. The supernatant of MCF-7/FL-IRES-TK cells could stimulate the proliferation of CD34+ cells from cord blood and increase the rate of CD1a+ cells (DC) in the presence of GM-CSF, TNF-·.In vitro experiment demonstrated dose-dependent cell killing by a transduction of the HSV-tk gene followed by GCV (ganciclovir) treatment. We found that GCV treatment of HSV-tk-fl expressing cell line induced apoptosis. DC contacting with apoptotic cells can further stimulate the proliferation of T cells. These experiments suggested HSV-tk/GCV system is not only a tumor vaccine, but also a method to enhance the DC function. Taken together, our findings therefore indicated that MCF-7/FL-IRES-TK could provide apoptotic bodies to DC as well as secrete FL to generation DC.

Keywords: dendritic cell, suicide gene, flk-2/flt-3 ligand; apoptosis, gene-therapy



85-90

Recombinant adenoviruses and adenovirus penton vectors: from DNA transfer to direct protein delivery into cell

Author(s): Dr. Pascal Fender,

Abstract: Recombinant adenovirus (rAd) is one of the most frequently used vectors with 171 human gene therapy trials involving 644 patients in 2002. Its success stems mainly from the capacity of rAd to efficiently penetrate various cell lines or tissues and from the ease of producing large amounts of this vector on industrial scale. Recently, in parallel to gene transfer by viral vectors, a new technology aiming to deliver proteins rather than genetic material in human cells has emerged, using small vehicle peptides often derived from viral proteins. In the first part of this review, I summarize the different types of rAd vectors used for gene transfer, their advantages and their limits. In the second part, I summarize the main vectors used for direct protein delivery into human cells and I show how an amazing nanoparticle called “adenovirus dodecahedron” can be used for this emerging therapeutic area.

Keywords: Recombinant adenoviruses, DNA transfer, Adenovirus pentons, dodecahedra, Protein delivery, Transduction peptides



91-102

Development of constitutive and inducible self- inactivating lentiviral vectors and their application in cardiovascular gene transfer

Author(s): Dr. Wayne A. Marasco,

Abstract: Regulatable lentiviral vectors are among the newest gene transfer tools that are being used increasingly for basic research. They are being explored for clinical applications as well. In this study, we conducted investigations that were aimed toward improving the biosafety and the efficiency of our previously described one piece tetracycline- autoregulatable lentiviral vector (Ogueta et al, 2001). The new genetically modified regulatable vectors are now self- inactivating (SIN) minimal vectors and contain the central polypurine track (cPPT) region and the Woodchuck Hepatitis Virus posttranscriptional regulatory element (WPRE) for increased transgene expression. Newly designed lentiviral vectors were tested both in vitro and in an in vivo rat model of myocardial gene transfer. The in vitro results show that while all three generations of the lentiviral vectors respond to doxycycline control in both 293T and HUVEC cells, increased green fluorescence protein (GFP) expression in the presence of WPRE element is only observed in 293T cells, suggesting a tissue/cell type dependent effect of the WPRE. In vivo experiments in rats showed that positive LacZ/GFP gene transfer was accomplished by direct myocardial injection and that LacZ gene expression was regulatable by doxycycline. The success of direct myocardial gene transfer with one piece tetracycline-autoregulatable lentiviral vector adds an attractive gene transfer tool to the field of cardiovascular gene delivery.

Keywords: lentiviral vectors, tetracycline regulated expression, auto-regulation, cardiovescular gene transfer, WPRE



103-114

Myxoma virus tropism in human tumor cells

Author(s): Dr. Joanna Sypula, Fuan Wang, Yiyue Ma, John Bell and Grant McFadden,

Abstract: Myxoma virus is a species-specific poxvirus that causes myxomatosis in European rabbits but is nonpathogenic in other vertebrate species, including man. We show here that myxoma virus productively infects the majority (15/21) of human tumor cell lines tested from the NCI-60 reference collection. To assess for the potential involvement of virus host range genes, we screened several candidate gene knockout mutants of myxoma virus for permissiveness in these tumor cells. We observed that one particular myxoma virus variant, deleted in the ankyrin- repeat host range gene M-T5, was uniquely defective for replication in most of the human tumor cells that were permissive for the wild-type virus. Myxoma virus, therefore, exhibits specific tropism in a broad spectrum of human tumor cells and thus has the potential to be exploited as a novel oncolytic virus candidate.

Keywords: Myxoma virus tropism, X-gal staining, β-galactosidase, vMyxlac and vMylacT5- replication, viral gene expression, myxoma infection



115-124

Intracrine signaling of phospholipid mediators, PAF and LPA, via cognate nuclear G protein-coupled receptors

Author(s): Dr. Sylvain Chemtob,

Abstract: Platelet-activating factor (PAF) and lysophosphatidic acid (LPA) are ubiquitous lipid mediators that play important roles in inflammation, cardiovascular homeostasis and immunity and are also known to modulate gene expression of specific pro-inflammatory genes. The mechanism of action of these phospholipids is thought to be primarily dependent on their specific plasma membrane receptors belonging to the superfamily of G protein-coupled receptors (GPCR). However, increasing evidence suggest the existence of a functional intracellular GPCR population. It has been suggested that immediate effects are mediated by cell surface receptors whereas long-term responses are mediated by intracellular receptors. PAF and LPA1 receptors localize at the cell nucleus of cerebral microvascular endothelial cells of newborn pig, rat hepatocytes and cells overexpressing each receptor, and stimulation of isolated nuclei reveal biological functions, including transcriptional regulation of major genes, namely cylooxygenase-2 and inducible nitric oxide synthase. This mini review focuses on the nuclear localization and signaling of GPCRs recognizing PAF and LPA phospholipids as ligands. Theories on how nuclear PAF and LPA1 receptors activate gene transcription and nuclear localization pathways are discussed. Intracrine signaling for lipid mediators uncover novel pathways to elicit their effects; moreover, intracellular GPCRs constitute a distinctive mode of action for gene regulation.

Keywords: PAF, LPA, G proteins, receptors, signaling, localization, gene transcription, caveolae, endothelial cells



125-136

Epigenetic and gene therapy in cardiovascular diseases: an appraisal

Author(s): Dr. José Marin-Garcia,

Abstract: Modifications of DNA and its nuclear environment (i.e. chromatin) may underlie cardiovascular diseases (CVD) and aging. Epigenetic alterations in chromatin (e.g. histone acetylation) have been directly implicated in the modulation of myocardial gene expression in progressive CVD, including cardiac hypertrophy. Cytosine methylation which can lead to gene inactivation has also been linked to increased levels of homocysteine, an important CVD disease risk factor. Advances in the identification of genes affected in CVD has lead to improved therapies either by the use of gene replacement and/or gene suppression (silencing) methodologies. Preclinical studies have shown that therapeutic gene transfer can provide beneficial results in treating heart failure, myocardial protection, hypertension, hypertrophy, cardiac arrhythmias and myocarditis as well as in disorders of the vascular wall where drug therapy has often proved to be of limited value. While early phases of clinical gene therapy trials for CVD have shown promising results in particular with therapeutic angiogenesis and restenosis treatment, the development of improved vectors, methods of delivery, and the acquisition of safety and toxicity data are critically needed before the use of these therapies can be indicated in a clinical setting. In this review, we will survey the current information available on the role of epigenetic modifications in CVD, and examine the present status and prospects for clinical use of epigenetic and gene therapy.

Keywords: Epigenetics; Gene therapy; Cardiovascular disease; DNA methylation



137-146

Computational approaches to study oncolytic virus therapy: insights and challenges

Author(s): Dr. Dominik Wodarz,

Abstract: The paper reviews computational models for analyzing the use of replicating oncolytic viruses as therapeutic agents against cancers. The paper highlights viral and host paramters which are crucial for success, and discusses how virus strains can be optimized in order to achieve maximial remission of cancers. The models consider three mechansisms by which oncolytic virus therapy could work: (i) virus-mediated killing of tumor cells. (ii) Induction of immune responses against the virus which can kill infected tumor cells. (iii) Induction of tumor specific immune responses following the release of stimulatory signals as a result of the virus infection. The models further give rise to insights into how virus variants should be tested in vitro in order to determine their therapeutic potential.

Keywords: oncolytic virus therapy, virus-specific CTL, virus infection, tumor-specific CTL



147-155

E2F-1 cancer gene therapy

Author(s): Dr. Kelly M. McMasters,

Abstract: Malignant cells frequently have disruption of normal apoptosis pathways due to mutation or dysfunction of apoptosis-related genes. Reconstitution of the apoptotic pathways represents a major strategy for cancer therapy. In this regard, adenoviral vector-mediated p53 cancer gene therapy was first used in human clinical trials. However, the disadvantage of p53 gene therapy is that cancers with a wild-type p53 gene do not respond well to p53 gene therapy, and other mechanisms of p53 resistance are often present in cancer cells. In recent years, E2F-1 has gained increased attention as a target for cancer gene therapy. Transduction of E2F-1 into tumor cells has demonstrated significant anti-tumor effect in vitro and in vivo, and in a wide spectrum tumor types regardless of p53, RB, P16 and p73 status. Furthermore, transduction of a very low dose of E2F-1 adenovirus into tumor cells can dramatically sensitize cells to some chemotherapeutic drugs in mouse models of cancer, indicating a new chemosensitization strategy. This may play an important role in chemoresistant tumors such as melanoma and sarcoma. In this review, we shall discuss the function of E2F-1, mechanisms of apoptosis, preclinical studies, as well as the promise and limitations of E2F-1 cancer gene therapy.

Keywords: E2F-1, cancer gene therapy, apoptosis, cell cycle, chemogene therapy, adenovirus



157-161

Is the MLC1/3 promoter / enhancer the right system to generate skeletal muscle specific transgenic animals?

Author(s): Dr. Thomas Walther,

Abstract: To document the role of iNOS expression for skeletal muscle alterations as observed in patients with chronic heart failure, the aim of this study was to generate skeletal muscle specific iNOS transgenic animals. The complete coding cDNA was inserted into a vector using the MLC-1/3 promoter/enhancer, which was used for pronuclear microinjection into 223 oocytes. PCR and Southern blot analysis of tail biopsies of 37 founder animals demonstrated that none was positive for the transgene. RT-PCR analysis of bovine oocytes for the expression of myosin light chain-1 revealed a positive amplification product. These results demonstrate for the first time that MLC-1 is already expressed in oocytes thus preventing the generation of transgenic animals using the MLC-1/3 promoter in conjunction with a toxic gene like iNOS.

Keywords: chronic heart failure, skeletal muscle, transgenic animal, promoter activity



163-174

ROS mediate signaling crosstalk between NF-κB and JNK

Author(s): Dr. Hiroyasu Nakano,

Abstract: Activation of NF-κB inhibits apoptosis by upregulating various anti-apoptotic genes, such as c-FLIP, Bcl-xL, A1/Bfl- 1, and X chromosome-liked inhibitor apoptosis (XIAP). However, the molecular mechanism by which NF-κB inhibits apoptosis is not fully understood. In contrast, activation of c-Jun N-terminal kinase (JNK) promotes apoptosis in a context-dependent manner. Recent studies showed that one of the anti-apoptotic functions of NF-κB is to downregulate JNK activation, and this function is mediated by two NF-κB-inducible genes, GADD45β and XIAP. More recently, NF-κB activation inhibits TNFα-induced accumulation of reactive oxygen species (ROS) that mediates prolonged JNK activation and necrotic cell death. In this review, we will focus on the signaling crosstalk between NF-κB and JNK cascades via ROS.

Keywords: NF-κB, c-Jun N-terminal kinase (JNK), reactive oxygen species (ROS), apoptosis, anti-apoptotic genes, necrosis



175-180

Growth factor receptors: targets for gene therapy and immunotherapy for cancer treatment

Author(s): Dr. Shulin Li,

Abstract: Angiogenesis has a crucial role in the invasion, growth and metastasis of solid tumors. Known angiogenic growth factors derived from tumors and vascular cells include VEGF, FGF, Ang-1, Ang-2, EGF, IGF and PDGF. Angiogenic growth factors bind to receptors with tyrosine kinase activity (RTK). These RTKs are overexpressed or mutated in a variety of human cancers, therefore they represent a potential target for anti-cancer therapy. Recent advances in the fields of gene therapy and molecular biology can be applied to design new vaccines and antitumor therapies for the treatment of cancer. In this review, the use of RTKs for anti-angiogenic immunotherapy and anti- angiogenic gene therapy is described, followed by a comparison among several gene delivery methods.

Keywords: angiogenesis, cancer treatment, delivery systems, gene therapy, Growth factor receptors, growth factors, immunotherapy



181-192

Regulatory sequences of the H19 gene in DNA based therapy of bladder cancer

Author(s): Dr. Patricia Ohana,

Abstract: The objective of the present study was to develop novel DNA based therapy strategies for bladder cancer. We detected a high expression level of the H19 gene in murine and human bladder carcinoma tissues compared to nearly undetectable levels in the surrounding normal tissues. On the basis of these findings we constructed a plasmid in which H19 regulatory sequences drove the expression of the diphtheria toxin A gene. This plasmid was introduced by intravesical instillation into the bladders of rats with bladder carcinoma (orthotopic model) and into the bladders of two human patients suffering recurrent superficial transitional cell carcinoma, refractory to all commonly used treatments. Very significant tumor growth inhibition was observed in the rat bladder tumors after two intravesical injections of 50 μg of DTA-H19 toxin vector as compared to control animals. Nearly complete ablation of the tumor was determined by video imaging in the two human patients after treating once a week with 2 mg of DTA-H19 plasmid for a total of 9 weeks. Not even a trace of the plasmid could be detected in the bloodstream of the patients. This observation strongly indicates the safety of our treatment. These observations may be the first step of a major breakthrough in the treatment of human bladder carcinoma.

Keywords: H19 regulatory sequences; transitional bladder carcinoma; diphtheria toxin A; intravesical vector instillation



193-200

Efficient expression of Gag protein by recombinant Modified Vaccinia Ankara Virus (MVA) with HIV-1 Indian Subtype C gagprotease gene segments

Author(s): Dr. Pradeep Seth,

Abstract: The AIDS epidemic in the developing world particularly in India represents a major threat where the infections are due to non-B clades of HIV-1. Most human immunodeficiency virus (HIV) vaccines currently under development are based on clade B strains of HIV-1. Since in India clade C is the predominant strain of HIV-1, it is imperative to develop an effective vaccine candidate from the locally circulating HIV strains. We describe here the development of recombinant Modified Vaccinia Ankara (MVA) expressing gag protein of HIV-1 Indian subtype C. Plasmid transfer vector, pSC 65, was used to transfer gag-protease gene segment of HIV-1 subtype C strain 49587 to MVA and gene segment was placed under the control of synthetic early late promoter (PE/L). The recombinant MVA was selected by BrdU/X-gal selection. The recombinant MVA was found to express gag protein in BHK-21 cells and the expression was evaluated by p24 antigen capture ELISA, immunoblotting, immunoflourescence and formation of virus-like-particles (VLPs) in infected cells by electron microscopy. The construct showed stable and high expression of HIV-1 gag gene in eukaryotic cells.

Keywords: HIV-1, Subtype C, DNA vaccine, MVA



201-212

Mechanims and possibilities of liposomal gene transfer to affect dermal and epidermal regeneration using the IGF-I cDNA construct

Author(s): Dr. Marc G Jeschke,

Abstract: The use of wounds as a model is very attractive for multiple reasons. The skin and wounds are easy accessible to monitor and determine physiologic and molecular responses to treatment. In addition the clinical entity of patients with acute, e.g. burn and trauma wounds, and chronic wounds, e.g. diabetic, autoimmune, arterial and venous wounds are steadily increasing. Thus, studies defining molecular mechanisms of the complex cascade of wound healing can be used several fold. The examined treatment can be translated into other organs and cell systems and at the same time new therapeutic approaches that affect dermal and epidermal regeneration can be studied. Others and we have shown that administration of growth factors represent a new therapeutic strategy to enhance tissue regeneration. The clinical application of growth factors in the form of proteins, particularly in acute and chronic wounds, has been shown to be of little benefit. Therefore new delivery systems and therapeutic strategies needed to be developed to improve dermal and epidermal regeneration, one of which is gene therapy. Essential for the success of gene therapy is the vector. Out of many vectors we chose for our model the liposomal vector, for its non- immunogenicity and non-viral components. From in-vitro to in-vivo we investigated the possibilities and biological and genetic responses of liposomal gene transfer using the insulin-like growth factor-I (IGF-I) cDNA. This review discusses the success, potential and limitations of liposomal gene transfer to improve tissue regeneration by using the IGF-I cDNA.

Keywords: Wound healing, liposomal gene transfer, IGF-I cDNA construct; dermal and epidermal regeneration



213-219

Xenograftic tumor models in mice for cancer research, a technical review

Author(s): Dr. Bingliang Fang,

Abstract: The transplantation and growth of human tumor xenografts in immunodeficient mice is frequently used in preclinical studies to test the efficacy of various anticancer agents. Subcutaneous models are most commonly used because of the simple methods of implanting tumor cells and monitoring tumor growth. A major criticism of subcutaneous models, however, is the unrealistic growing condition of the local skin environment. Alternatively, direct intravascular or intraorgan injections can be used to produce metastatic or orthotopic tumor models under more realistic and clinically applicable conditions. Despite the apparent widespread use of these models, detailed technical information on establishing these tumor models is difficult to find in the literature. Therefore, the purpose of this article is to provide the technical details for establishing useful tumor models in mice.

Keywords: tumor model, mouse, metastasis, cancer cell line, SOI



221-230

The optimal molecular design of polymeric drug carriers and its application for renal drug targeting

Author(s): Dr. Yasuo Tsutsumi,

Abstract: Renal disease is a serious health problem which is on the increase in the world. Over time such conditions necessitate dialysis and may require a kidney transplant. However these therapies are expensive and do not restore normal health. Therefore, new therapeutic strategies must be developed for treating patients with renal disease. Drugs such as steroids have been used to prevent the progression of renal disease, but they produce toxicity because of their wide distribution in the body. The development of a renal delivery system that selectively carriers drugs to the kidneys is a promising approach for limiting tissue distribution and controlling toxicity. To overcome the problems associated with conventional therapies, bioactive proteins have been conjugated with water-soluble polymeric carriers. Conjugated bioactive protein with polymeric carriers regulate the tissue distribution of bioactive proteins, resulting in a selective increase in its desirable therapeutic effects, and a decrease in undesirable side effects. However, for further enhancement of the therapeutic potency and safety of conjugated bioactive proteins, more precise control of the in vivo behavior of each protein is necessary for selective expression of their therapeutic effect. Recently, we reported that the poly(vinylpyrrolidone-co-dimethyl maleic acid) [PVD] was selectively distributed into the kidneys after intravenous injection and it was conjugated with the amino groups of drugs. The conjugates demonstrated high accumulation and retention in the kidneys without any adverse toxicity. In this review, with reference to our recent studies, we propose that bioconjugation with the appropriate polymeric modifier of PVD can be a potential therapeutic agent for various renal diseases.

Keywords: conjugation, PEGylation, polyethylene glycol, polyvinylpyrrolidone, dimethylmaleic acid, protein therapy



231-240

Ovine atadenovirus as a vector for gene transfer and vaccination

Author(s): Dr. Peter Löser,

Abstract: The use of adenoviruses as gene transfer vectors has become widespread within the last two decades. The commonly used vectors are based on human adenovirus type 2 and type 5 (hAdV-2 and hAdV-5) of subgroup C. Although these vectors are very efficient in transferring foreign genes into a variety of cell types, their use as gene transfer vehicles for clinical application in humans is compromised under some conditions due to wide-spread pre-existing immunity to these viruses in the population (Flomenberg et al, 1995; Horwitz et al, 1996; Chirmule et al, 1999). Therefore, and to allow for repeated vector administration into the same individual, vectors have been derived from less common human adenoviruses (Kass-Eisler et al, 1996; Mastrangeli et al, 1996; Vogels et al, 2003). Alternatively, gene transfer vectors based on adenoviruses of non-human origin have been developed and successfully tested in several animal models (Both et al, 2002a; Löser et al, 2002a). They combine the many favorable features of adenoviruses as gene transfer vectors with the lack of natural immunity to these viruses in humans. Here we review the features and utility of a novel vector based on ovine atadenovirus type 7 (OAdV) which has been developed for transient gene delivery purposes over the last several years.

Keywords: ovine atadenovirus, OAdV, gene transfer vector, vaccination



241-258

Protein modification by cyclopentenone prostaglandin addition: biological actions and therapeutic implications

Author(s): Dr. Dolores Pérez-Sala,

Abstract: Cyclopentenone prostaglandins (cyPG) are naturally occurring eicosanoids which are generated by the spontaneous dehydration of other PG. The cyPG of the A series arise from the dehydration of PGE. The cyPG of the J series, such as 15-deoxy-∆12,14-PGJ2 (15d-PGJ2), arise from the dehydration of PGD2. CyPG have been detected in vivo in body fluids and at sites of chronic and acute inflammation. However, the mechanisms underlying their generation, the levels attained in vivo and their pathophysiological significance are still not completely elucidated. CyPG display varied biological activities in cellular models including antiviral and antitumoral effects, induction of stress response and apoptosis. Anti-inflammatory and protective roles of 15d-PGJ2 against several kinds of tissue injury have been recently evidenced in various animal models. This has led to propose the use of cyPG as therapeutic agents. The mechanism of action of cyPG may be multiple. Some cyPG, like 15d-PGJ2, may act as a ligands of the nuclear receptor PPARγ. In addition, cyPG possess an α,β-unsaturated carbonyl group in the cyclopentane ring which may form adducts with cellular proteins or with glutathione, leading to a modulation of protein function and to changes in the cellular redox status. The modification of cellular proteins by addition of cyPG is arising as an important mechanism in the anti-inflammatory and anti-proliferative effects of these compounds. Several proteins have been identified which can be targets for this modification including several components of the pro- inflammatory transcription factors NF-κB and AP-1, proteins involved in the cellular response to oxidative or electrophile-induced stress, and H-Ras proteins. The identification of novel protein targets for modification by cyPG and the functional and structural characterization of this type of modification will contribute to the understanding of the mechanisms of action of cyPG and the assessment of their therapeutic potential.

Keywords: prostaglandins, cyclooxygenase, posttranslational modification, inflammation



259-290

Intraparenchymal delivery of GDNF towards a treatment for Parkinson’s disease

Author(s): Dr. Nikunj K. Patel,

Abstract: Parkinson’s disease (PD) is a neurodegenerative disease characterised by the progressive loss of neural dopaminergic (DA) neurons. Although symptomatic therapies to substitute for the missing neurotransmitter dopamine (DA) are efficient at the early stages of the disease, the goal is to find alternative therapies, which could protect DA neurons from the degenerative process. GDNF promotes recovery of the injured nigrostriatal DA system and improves motor function in both rodent and nonhuman primate models of PD, although intracerebral administration is necessary because of the limited penetration into the brain tissue from either the blood or the CSF. The powerful neuroprotective and neurorestorative properties of GDNF seen in preclinical studies suggest that trophic factors may play an important role in treating PD, and intraparenchymal delivery of GDNF may represent a new treatment option. Intraparenchymal delivery in animal studies is effective whether by bolus injection, by chronic infusion using a pump, by implantation of genetically engineered cell line releasing GDNF (ex vivo gene therapy) or by infecting the brain with live replication-deficient viral particles engineered to deliver GDNF (in vivo gene therapy). We have studied the effects of direct striatal chronic infusion of GDNF in 5 PD patients and noted significant symptomatic improvements and positron emission tomography (PET) scans of 18F- DA uptake showed a significant increase in putamen DA storage after 24 months, suggesting a direct effect of GDNF on DA function. This study warrants careful examination of GDNF as a treatment for PD, and the clinical application of other methods of delivery.

Keywords: Parkinson’s disease; Animal model; Neurotrophic factor; Glial cell-line derived neurotrophic factor; Cell transplantation; Cell encapsulation; Gene therapy; Viral vectors



291-306

Human papillomavirus-associated cervical cancer: Prophylactic and therapeutic vaccines

Author(s): Dr. Hildegund C.J. Ertl,

Abstract: Cervical cancer remains a leading cause of cancer-related mortality in reproductive age women. Although effective screening programs have decreased the incidence of cervical cancer in developed countries, they are too expensive for comprehensive use in developing countries. Since 60-80% of cervical cancers are associated with human papillomavirus (HPV)-16 and HPV-18 infections, antigens encoded by these viruses are obvious targets for prophylactic and therapeutic vaccines. Prophylactic vaccines aim to reduce the incidence of cervical cancer by preventing virus infection through induction of virus neutralizing antibodies against capsid proteins. Therapeutic vaccines are designed to induce cellular immune responses able to clear persistent infections with HPV or to eradicate cells transformed by HPV’s oncoproteins. A number of candidate prophylactic and therapeutic HPV vaccines have been developed and tested in animal models and are now approaching clinical trials.

Keywords: human papillomavirus, vaccine, cervical cancer



307-318

Gene transfer to human oral cancer cells via non- viral vectors and HSV-tk/ganciclovir-mediated cytotoxicity; Potential for suicide gene therapy

Author(s): Dr. Krystyna Konopka,

Abstract: Head and neck squamous cell carcinoma (HNSCC) afflicts about half a million people worldwide every year and has a high rate of mortality. Some gene therapy strategies for the treatment of HNSCC have been explored in animal models. Because of concerns with the use of viral vectors in gene therapy we examined whether cationic lipid-DNA complexes could be used to deliver effectively the Herpes Simplex Virus thymidine kinase (HSV-tk) "suicide gene" to human oral cancer cells as a primer to in vivo studies. Transfection of HSC-3, H357, H413 and H376 cells was optimized using β-galactosidase (β-gal)-expressing plasmids and four transfection reagents: Fugene, Escort, Lipofectamine 2000, and GenePORTER. Fugene and Escort+Transferrin (TF) provided the highest efficiency of transfection, with about 40% and 10% of HSC-3 cells positive for β-gal, respectively. The delivery of the HSV-tk gene to HSC-3 cells by Fugene or Escort+TF, followed by ganciclovir (GCV) treatment for 8 days, resulted in >80% cytotoxicity, determined by the Alamar Blue assay. In contrast, less than 5% of H357, H413 and H376 cells was positive for β-gal staining. Nevertheless, in H357 cells about 80% and 50% cytotoxicity were observed with Fugene and Escort+TF, respectively. In H413 cells, the viability was 40% with Fugene and 20% with Escort+TF, while in H376 cells it was reduced to 60% only with Fugene. Thus, cytotoxicity in the presence of GCV was observed despite the very low efficiency of transfection, in these cell lines. Lipid-DNA complexes may be useful for suicide gene therapy of oral squamous cell carcinoma (OSCC) cells since a high percentage of GCV-specific cell death can be achieved despite the low efficiency of transfection, possibly due to the “bystander effect”, and since these non-viral vectors have a favorable safety profile.

Keywords: transfection; HSV-tk gene; squamous cell carcinoma cells; non-viral vectors; β-galactosidase; ganciclovir



319-326

Phosphorothioated CpG Oligonucleotide induced hemopoietic changes in mice

Author(s): Dr. Pradeep Seth,

Abstract: visceral leishmaniasis and HIV infection. Gene Therapy and Molecular Biology Vol 8, page 319 Bacterial DNA and the synthetic CpG-oligodeoxynucleotides (ODNs) derived thereof have attracted attention because they activate cells of the adaptive immune system (lymphocytes) and the innate immune system (macrophages). They induce a Th1 biased immune response upon activation of the immune cells. In this paper we addressed whether unmethylated phosphorothioated CpG ODN (for example 1826 CpG-ODNs) affected hemopoiesis. We observed an overall Th1 dominant response upon in-vitro stimulation of naïve splenocytes with 1826-ODN. Immunizing mice with immunostimulatory CpG motifs led to transient splenomegaly, with a maximum increase of spleen weight at 4 weeks post immunization. Thereafter the splenomegaly regressed. The induction of splenomegaly by CpG-ODNs was dose-dependent with the maximum spleen weights recorded at the 250 μg immunizing dosage of 1826-ODN. In addition, the splenomegaly was also associated with dose dependent extramedullary hemopoiesis and reactive follicular hyperplasia in the spleens and lymph nodes, which could be of therapeutic relevance particularly in patients with life threatening chronic and persistent infectious diseases like

Keywords: CpG motifs; 1826-ODN; Splenomegaly; Hemopoiesis



327-334

Development of HIV-1 subtype C Gag based DNA vaccine construct

Author(s): Dr. Pradeep Seth,

Abstract: Recently, the success of genetic immunization as a novel means to induce protective immunity has been demonstrated. DNA vaccines mimic antigen presentation closely to the natural history of viral infection. This is particularly relevant in infectious diseases where-in cell mediated immunity plays a larger role in protection, such as HIV-1 infection. In this paper we present the work done towards development of a gag based DNA immunogen for local circulating HIV-1 subtype C viruses in India. Gag gene was cloned under the control of CMV promoter in a mammalian expression plasmid vector. The other main features of the expression cassette in the construct pJWgagprotease49587 are bovine growth hormone polyadenylation signal and a t-PA leader signal. The construct was confirmed for expression in vitro by various means, p24 antigen capture assay, immunoblotting and electron microscopy. The TEM studies on transiently transfected COS-7 cells showed the presence of virus like particles (VLPs) as a consequence of gene expression from the construct pJWgagprotease49587. This finding is the first report of VLPs for a subtype C based gag construct. We expect that this construct will be able to prime a good immune response when used in in-vivo mice studies owing to the formation of virus like particles from the construct in vitro.

Keywords: gag, DNA vaccine, CMV promoter, Virus like particles (VLPs)



335-342

Targeting retroviral vector entry by host range extension

Author(s): Dr. Barbara S.Schnierle,

Abstract: The dream of vectorologists is a vector with magic bullet properties. This conceptual breakthrough in gene therapy would be a gene transfer vector that could be systemically applied, allowing targeted gene transfer into a predetermined cell type. The host range of a retroviral vector is determined by the interaction between the viral envelope glycoprotein and the retrovirus receptor on the surface of the host cell. Here are summarized current efforts to engineer the envelope glycoprotein of ecotropic murine leukemia virus, which does not infect human cells, in order to extend its host range and accomplish gene delivery in a highly specific manner.

Keywords: murine leukemia virus, targeting, vector, envelope, virus entry, host range



343-350

Role of the Brn-3a and Brn-3b POU family transcription factors in cancer

Author(s): Dr. David S. Latchman,

Abstract: Brn-3a and Brn-3b are closely-related POU family transcription factors both of which play an important role in the nervous system. However, both these factors were originally isolated from a neuroblastoma cell line and their expression has been shown to be altered in several different human cancers. Interestingly, functional studies have shown that Brn-3b has a growth-stimulating effect in neurobastomas, whereas Brn-3a has a growth-inhibiting effect. Similarly, Brn-3b is over-expressed in human breast cancers and stimulates their growth. However, Brn-3a is strongly over-expressed in human cervical cancer and stimulates cervical tumour growth by activating expression of the human papilloma virus E6 and E7 oncogenes which are essential for development of this tumour. Hence, these closely-related factors play critical but distinct roles in different human cancers.

Keywords: Brn-3a, Brn-3b, POU family transcription factors, neuroblastoma, Ewing's sarcoma, breast cancer, cervical cancer



351-360

Angiogenic gene therapy in the treatment of ischemic cardiovascular diseases

Author(s): Dr. Tamer A. Malik,

Abstract: Encouraging preliminary data suggest that gene therapy may soon be an option for the treatment of patients with advanced coronary artery disease that is not amenable to conventional treatment. A critical consideration in developing cardiovascular gene transfer as a therapy is the ability to deliver the vector, viral or plasmid, to the desired tissue in a safe fashion. Attempts at developing non-viral direct DNA therapy delivered through the intravenous route are currently underway and with the use of advanced technology the possibility of making gene therapy a simple outpatient procedure does not seem out of the realm of possibility. Several clinical trials are currently underway that should help characterize the risk–benefit profile of various products, the optimal dose that should be administered, and the patient population likely to derive greatest benefit.

Keywords: VEGF, FGF, HGF, Retrovirus, Adenovirus, Adeno-associated virus, Plasmids, Liposomes, MRI, AGENT trials, VEGF trials



361-368

Targeting Myc function in cancer therapy

Author(s): Dr. Peter J. Hurlin,

Abstract: The development of novel therapeutic strategies to improve the survival rate of patients with cancer requires a better understanding of the critical events that underlie the origins and progression of tumors. The Myc family of transcription factors play important normal roles in regulating cell proliferation and their deregulated or elevated expression is one of the most common features of cancer cells. Here, we review mechanisms thought to underlie Myc-dependent tumor formation and discuss possible strategies for disrupting the oncogenic activity of Myc family proteins.

Keywords: Myc, Max, Mnt, apoptosis



369-384

Transfection pathways of nonspecific and targeted PEI-polyplexes

Author(s): Dr. Salvador F. Aliño,

Abstract: Polyethyleneimine (PEI) based vectors have become in an important vehicle for nonviral gene transfer. However, despite their extensive use and efficacy in the transfection of several cellular models both in vitro and in vivo, the mechanism by which they transfect cells has not been fully elucidated, and controversy remains over the interpretation of some apparently contradictory findings. A review is made of the studies on PEI polyplexes, focusing on PEI polyplex transfection properties (as physico-chemical characteristics important for transfection) and the mechanistic findings of PEI polyplex transfection comprising cell membrane binding with nonspecific and targeted–PEI polyplexes, the putative internalization pathways (such as the proton sponge hypothesis), the nuclear bioavailability of the transported nucleic acid, and other relevant issues such as the influence of polyplex size in vitro upon transfection activity.

Keywords: PEI-polyplexes, transfection, DNase degradation, Interactions, cell surface, cell culture medium, specificity, efficacy, cell internalization, Endosome trafficking, proton-sponge effect, Cytoplasm transport, nuclear accession, dissociation



385-394

c-myc: a double-headed Janus that regulates cell survival and death

Author(s): Dr. Ivana Scovassi,

Abstract: A paradox for cancer biology is represented by the fact that some oncogenes, including c-myc, provide an advantage to cancer cells by stimulating uncontrolled proliferation while, at the same time, they exert a pro-apoptotic activity. The prominent roles of c-myc and the relevance of phosphorylation and subcellular compartmentalization of c-Myc protein are described in this review, which focuses also the possible strategies to modulate (i.e. up- and down- regulate) the c-myc level. The gene expression targeted approach of c-myc modulation as anticancer therapeutic treatment is discussed.

Keywords: Antisense, apoptosis, cancer, c-myc, phosphorylation, TFO



395-402

DNA-based vaccine for treatment of intracerebral neoplasms

Author(s): Dr. Terry Lichtor,

Abstract: Antigenic differences between normal and malignant cells of the cancer patient form the rationale for clinical immunotherapeutic strategies. Because the antigenic phenotype of neoplastic cells varies widely among different cells within the same malignant cell-population, immunization with a vaccine that stimulates immunity to the broad array of tumor antigens expressed by the cancer cells is likely to be more efficacious than immunization with a vaccine for a single antigen. A vaccine prepared by transfer of DNA from the tumor into a highly immunogenic cell line can encompass the array of tumor antigens that characterize the patient’s neoplasm. Poorly immunogenic tumor antigens, characteristic of malignant cells, can become strongly antigenic if they are expressed by highly immunogenic cells. A DNA-based vaccine was prepared by transfer of genomic DNA from a breast cancer that arose spontaneously in a C3H/He mouse into a highly immunogenic mouse fibroblast cell line, where genes specifying tumor-antigens were expressed. The fibroblasts were modified in advance of DNA-transfer to secrete an immune augmenting cytokine and to express allogeneic MHC class I-determinants. In an animal model of breast cancer metastatic to the brain, introduction of the vaccine directly into the tumor bed stimulated a systemic cellular anti-tumor immune response and prolonged the lives of the tumor-bearing mice.

Keywords: Gene Therapy, Breast Cancer, Brain Tumors, Tumor Vaccine



403-412

The involvement of H19 non-coding RNA in stress:Implications in cancer development and prognosis

Author(s): Dr. Suhail Ayesh,

Abstract: The H19 gene is an imprinted gene expressed from the maternal allele. It is known to function as an RNA molecule, cDNA microarray hybridization was used in an attempt to identify novel kinases participating in cellular response to hypoxia and serum deprivation. The expression of H19 RNA was examined in embryonic cells (Human amniocytes) that normally express H19 RNA basal level. At low serum (0.1% FCS) medium or hypoxia: 100μM CoCl2; or both: without serum (0.1% FCS) and 100μM CoCl2 for 16hr the fold increase of H19 RNA expression was: 1.9 ±0.11, 1.73 ± 0.2 and 2.0 ± 0.18 folds respectively. Significant increase in expression and induced (up) expression of certain genes were observed in TA31 cell line that highly expresses H19 RNA. Using the human cDNA atlas microarray, we detected differentially expressed genes modulated by the presence of H19 RNA in certain conditions: serum deprivation, hypoxia and both serum deprivation and hypoxia which may resemble the stress conditions in cancer. Some of the key genes that had increased or induced (up) expression mainly in serum deprivation are: CDK2, FGFR1, IRAK, JNK1, uPAR and PRK2. In hypoxia the key genes are PKC-ζ, cot-proto oncogene, PKC-α, FAK and MEK2. In serum deprivation and hypoxia these genes are: Tie2, JNK2, ERK2 and VEGFR1. Using Atlas Array and observing the genes that had increased or induced (up) expression, a good indication for certain genes and pathways was found to be involved in tumor progression and angiogenesis. The major angiogenesis genes include FGFR1, VEGF, TIE2, uPA, and PKC-ζ. Other signal molecules associated with the invasive and migratory potential include JNK2, uPAR and FAK.

Keywords: Human cDNA expression assay, Bladder carcinoma cell lines, serum deprivation, hypoxia, Angiogenesis



413-422

PSA promoter-driven conditional replication- competent adenovirus for prostate cancer gene therapy

Author(s): Dr. Yi Lu,

Abstract: A conditional, replication-competent adenovirus (AdPSAE1) carrying the adenoviral E1 region under the control of a prostate specific antigen (PSA) promoter was generated in an effect to target the prostate for cancer gene therapy. The anti-prostate tumor efficacy and specificity of AdPSAE1 were examined in vitro and in vivo in prostate and nonprostate cancer models. In vitro at multiplicity of infection (moi) of 1, AdPSAE1 effectively killed the human prostate cancer cell lines PPC-1 and LNCaP, but had no effect on nonprostate cancer cells including the human bladder cancer cell line RT4, human breast cancer cell line MCF-7, and rat gliosarcoma cell line 9L. As a control, an adenovirus expressing the ß-galactosidase transgene under the control of the same PSA promoter (AdPSAlacZ) was used in parallel in all experiments. The in vivo tissue-specific expression driven by this PSA promoter was examined in a xenograft tumor model. Intratumoral injection of AdPSAlacZ resulted in PSA promoter-driven expression of lacZ in xenograft tumors in nude mice derived from human prostate cancer PPC-1 cells, but not in tumors derived from human bladder cancer RT4 cells. Intratumoral injection of AdPSAE1 effectively inhibited in vivo growth (61.8% reduction in tumor size) of xenograft PPC-1 prostate tumors compared to untreated or AdPSAlacZ treated tumors. Conversely, intratumoral injection of AdPSAE1 had no effect on the growth of xenograft RT4 bladder tumors when compared to untreated control group. These results indicate that prostate- targeted conditional replication-competent adenoviruses may be useful in gene therapy of prostate cancer.

Keywords: adenovirus, PSA, E1, replication-competent, prostate cancer



413-422

PSA promoter-driven conditional replication- competent adenovirus for prostate cancer gene therapy

Author(s): Dr. Yi Lu,

Abstract: A conditional, replication-competent adenovirus (AdPSAE1) carrying the adenoviral E1 region under the control of a prostate specific antigen (PSA) promoter was generated in an effect to target the prostate for cancer gene therapy. The anti-prostate tumor efficacy and specificity of AdPSAE1 were examined in vitro and in vivo in prostate and nonprostate cancer models. In vitro at multiplicity of infection (moi) of 1, AdPSAE1 effectively killed the human prostate cancer cell lines PPC-1 and LNCaP, but had no effect on nonprostate cancer cells including the human bladder cancer cell line RT4, human breast cancer cell line MCF-7, and rat gliosarcoma cell line 9L. As a control, an adenovirus expressing the ß-galactosidase transgene under the control of the same PSA promoter (AdPSAlacZ) was used in parallel in all experiments. The in vivo tissue-specific expression driven by this PSA promoter was examined in a xenograft tumor model. Intratumoral injection of AdPSAlacZ resulted in PSA promoter-driven expression of lacZ in xenograft tumors in nude mice derived from human prostate cancer PPC-1 cells, but not in tumors derived from human bladder cancer RT4 cells. Intratumoral injection of AdPSAE1 effectively inhibited in vivo growth (61.8% reduction in tumor size) of xenograft PPC-1 prostate tumors compared to untreated or AdPSAlacZ treated tumors. Conversely, intratumoral injection of AdPSAE1 had no effect on the growth of xenograft RT4 bladder tumors when compared to untreated control group. These results indicate that prostate- targeted conditional replication-competent adenoviruses may be useful in gene therapy of prostate cancer.

Keywords: adenovirus, PSA, E1, replication-competent, prostate cancer



423-430

A platform for constructing infectivity-enhanced fiber-mosaic adenoviruses genetically modified to express two fiber types

Author(s): Dr. Marianne G. Rots,

Abstract: Adenoviruses type 5 have been successfully exploited as gene transfer vectors and numerous vectorological improvements have contributed to increasing efficiency and specificity of adenoviral gene therapy. Despite these improvements, inefficient gene transfer still is an important limitation and is, at least in part, due to the low expression of the primary receptor (CAR) on target cells. Combining two different fiber types (the fiber of Ad5 for CAR-dependent uptake and the fiber of Ad3 for CAR-independent uptake) on an Ad5-based capsid would increase the options for improvement of specificity and efficiency. In this study, we present an approach to engineer fiber- mosaic adenoviruses by cloning the fiber of Ad3 into the Ad5 genome under the control of the Major Late Promoter using native splicing signals. Such fiber-mosaic viruses were efficiently rescued using conventional 293 cells and demonstrated good infection profiles. Pre-incubation with recombinant fiber knob (either derived from Ad5 or Ad3) indicated different mechanisms of entry for the fiber-mosaic viruses. The introduction of an additional entry pathway can be further exploited to overcome low infection efficiency due to low CAR expression. In addition, the technology will be of value in increasing the specificity of adenoviral gene therapy since this approach allows the incorporation of two different retargeting ligands per capsid. Such infectivity enhancement will also prove powerful in the context of replicative agents.

Keywords: gene therapy, adenovirus, fiber, infectivity enhancement



431-438

Internal ribosome entry sites in cancer gene therapy

Author(s): Dr. Caroline G. Lee,

Abstract: Cancer gene therapy is a promising treatment modality. Strategies in cancer gene therapy include tumor-directed therapy (e.g. the delivery of suicide, immunomodulatory, anti-angiogenic, apoptotic genes or oncolytic viruses or genes to reinstate tumor suppressor activity) and host-directed therapy (e.g. the delivery of genes encoding factors that enhance the antigen presenting function of dendritic cells or protect the patient against myelosuppression). As cancer, a complex disorder, often results from several defective genes, efficacy of cancer gene therapy can be improved by a combination approach whereby several different genes are targeted simultaneously. Of several methods to effect co-expression of multiple genes, the employment of internal ribosome entry sites (IRES) represents a promising approach. This review examines the various preclinical and clinical studies employing IRESs for cancer gene therapy, as well as properties of various IRESs that could be exploited for cancer gene therapy.

Keywords: cancer gene therapy, Tumor-directed therapy, Host-directed therapy, Internal ribosome



439-450

The pathway of uptake of SV40 pseudovirions packaged in vitro: from MHC class I receptors to the nucleus

Author(s): Dr. Michael M. Gottesman,

Abstract: SV40 vectors packaged in vitro are an efficient delivery system in vitro and in vivo using plasmids up to 17.7 kb, with or without SV40 sequences. Using confocal microscopy, we followed the pathway of SV40 pseudovirions in human lymphoblastoid cells, which are rich in MHC I receptors, using fluorescence-tagged DNA and an antibody against the main capsid protein, VP1. The wild-type SV40 virus as well as the pseudovirions enter the cells after binding to MHC I. However, the MHC I route is not the only way that SV40 pseudovirions enter cells. From the cell surface, the vectors progress through the Golgi to the ER, where they are unpackaged. Only the reporter DNA proceeds to the nucleus; VP1 remains at the ER. Results indicate that some of the reporter DNA, carried by these vectors, is trapped in the ER. Delivery of DNA plasmids which harbor nuclear localization sequences, such as the enhancer of wild-type SV40 or the cPPT sequence from the HIV-1 virus upstream from the GFP cDNA, did not improve GFP expression. However, improved expression from the EGFP reporter gene carried by SV40 vectors was achieved using the histone deacetylase inhibitor, TSA.

Keywords: Gene delivery; SV40 in vitro packaging; pathway of SV40 pseudovirions; MHC I receptors



451-464

The importance of PTHrP for cancer development

Author(s): Dr. Jürgen Dittmer,

Abstract: Parathyroid hormone-related protein (PTHrP) is expressed by many cells and usually acts as an autocrine, paracrine and/or intracrine factor to play numerous roles in embryonic development and normal physiology. Evidence has been accumulated suggesting that PTHrP may also serve important functions in tumor development. PTHrP has the potential to cause humoral hypercalcaemia of malignancy and is able to induce local osteolysis which facilitates growth of tumor cells that have metastasized to bone. Furthermore, PTHrP has been shown to stimulate proliferation as well as invasiveness of cancer cells and to protect cancer cells from apoptosis. In this review, I summarize the current knowledge about the role of PTHrP in cancer development and about the factors that control PTHrP expression in cancer.

Keywords: PTHrP for cancer development, cancer proliferation, invasion, metastasis, apoptosis, osteolysis, ets transcription factors, regulating factors



465-474

Gene-based vaccines for immunotherapy of prostate cancer - lessons from the past

Author(s): Dr. Milcho Mincheff,

Abstract: Gene-based vaccination in its current mode of application is effective in breaking tolerance to a self- or tumor- associated antigen, but the response is narrow and restricted to few of the potential epitopes due to immunodominance. In cancer, immunodominance carries the risk of inefficient immune surveillance due to loss of MHC alleles or point mutations in the recognized sequences. We have found that a T cell response to sub-dominant epitopes can be primed with transfected dendritic cells in which the newly expressed antigen is purposefully targeted for proteasomal degradation. Beginning in May 1998, we performed a phase I/II clinical trial for immunotherapy of prostate cancer that targeted the prostate-specific membrane antigen (PSMA). The primary objective of the study was to determine the safety of the described vaccines after repeated intradermal injections (Mincheff et al., 2000a; Mincheff et al., 2000b), since using PSMA as a target could be seriously offset by the development of autoimmunity (Gilboa, 1999b; Overwijk and Restifo, 2000). So far, six years since the study has begun, no patient has experienced any short- or long-term side effects, including anti-DNA antibody. Twenty-nine patients from this random population were treated solely by immunotherapy. Eighteen of them had biochemical recurrence following radical prostatectomy and eleven responded to the therapy with a PSA drop exceeding 50% of pre-therapy value. Patients with advanced disease and distant metastases were not influenced by the immunotherapy despite the fact that they all showed signs of T cell immunity towards PSMA. We found, however, that the post-vaccination T cell response was directed against only two of the potential 4 PSMA epitopes that had high affinity for binding. At least in vitro, priming with one of our vaccines led to a poly-epitope response. Unfortunately, even in such instances, consequent exposure to poly-epitope expressing dendritic cells during re- immunization led to selection of an immunodominant clone. To alleviate immunodominance and decrease tumor evasion due to loss of antigenic determinants, a poly-epitope T cell response would need to be maintained. Ensuring such a cytotoxic T cell response, therefore, would require either construction of separate epitope encoding vectors for boosting, an approach with limited therapeutic application, or identifying conditions during boosting that would restrict immunodominance. CD4 T cell depletion, GITR-L signaling or CTLA-4 all show promise in achieving this goal.

Keywords: PSMA, Gene-based vaccine, immunodominance, CTLA-4



475-486

An erythroid-specific chromatin opening element increases β-globin gene expression from integrated retroviral gene transfer vectors

Author(s): Dr. Christopher H. Lowrey,

Abstract: Gene therapy strategies requiring long-term high-level expression from integrated genes are currently limited by inconsistent levels of expression. This may be observed as variegated, silenced or position-dependent gene expression. Each of these phenomena involve suppressive chromatin structures. We hypothesized that by actively conferring an open chromatin structure on integrated vectors would increase transgene expression. To test this idea we used a 100bp element from the β-globin locus control region (LCR) which is able to independently open local chromatin structure in erythroid tissues. This element includes binding sites for GATA-1, NF-E2, EKLF and Sp-1 and is evolutionarily conserved. We constructed a series of MSCV-based vectors containing the β-globin gene driven by a minimal β-globin promoter with combinations of the HSFE and LCR derived enhancer elements. Pools of MEL clones containing integrated vectors were analyzed for chromatin structure and β-globin gene expression. The HSFE increased the extent of nuclease sensitive chromatin over the promoters of the constructs. The most effective vector included tandem copies of the HSFE and produced a 5-fold increase in expression compared to the promoter alone. These results indicate that the HSFE is able to augment the opening of β-globin promoter chromatin structure and significantly increase gene expression in the context of an integrated retroviral vector.

Keywords: chromatin structure, β-globin, retrovirus, DNase I hypersensitive site, locus control region



487-494

Decreased tumor growth using an IL-2 amplifier expression vector

Author(s): Dr. David T. Harris,

Abstract: The success of gene therapy relies on sufficient gene expression in the target tissue. The application of non-viral vectors, such as plasmid DNA, is limited by low in vivo transfection efficiency compared to viral vectors. Strategies to enhance gene transcription should augment target gene expression and make the vector more efficient. In the present study we describe a transcription factor- based amplifier strategy to enhance transgene expression. Our data showed that compared to CMV promoter driven IL-2 expression, expression of TAT in the same plasmid downstream of the HIV LTR significantly enhanced the expression level of IL-2 (up to 20-fold). Gene-modification of murine B16 melanoma with the amplifier IL-2 expression vector resulted in decreased tumor growth and prolonged animal survival in vivo.

Keywords: Cancer, interleukin-2, amplifier vector, gene therapy



495-500

Multiple detection of chromosomal gene correction mediated by a RNA/DNA oligonucleotide

Author(s): Dr. Giuseppe Rainaldi,

Abstract: Chimeric RNA/DNA oligonucleotide (RDO)-mediated gene correction of a single base mutation in a gene of an eukaryotic cell is still a controversial strategy. To better define the potential and applicability of this strategy, new systems, that allow to detect RDO-mediated gene correction in the chromosomal DNA of human cells, are needed. Here, we developed a construct containing hygromycin resistance mutant gene fused to the EGFP gene as target for correction. HeLaS3 cells were transfected with the fusion gene and clones, which had integrated one or two copies of the mutated fusion gene, were isolated and expanded. These cells were transfected with a RDO with a mismatch at the position 336 of the bacterial hygromycin resistance gene. If the gene correction occurs, the expression of both hygromycin resistance and EGFP genes is recovered. The RFLP and FACS analysis demonstrated that hygromycin resistance phenotype was due to the correction of the mutation.

Keywords: chimeric RNA/DNA oligonucleotide, gene correction, chromosomal target, HeLa cells, HygB/EGFP fusion gene.



501-508

Nitric oxide and endotoxin-mediated sepsis: the role of osteopontin

Author(s): Dr. Philip Y. Wai,

Abstract: Septic shock continues to be a life threatening complication of systemic infection despite advances in the clinical care of these patients. The incidence of severe sepsis in critically ill patients has increased annually by 8.7% and mortality rates are excessive, ranging from 30%-60%. Nitric oxide plays a central role in the molecular biology and biochemistry of septic shock. In endotoxin-mediated sepsis and septic shock, pro-inflammatory cytokines are elaborated and inducible nitric oxide synthase is systemically expressed in multiple cell types. The sustained production of nitric oxide in high concentration regulates multiple cellular and biochemical functions. Multiple studies have investigated the role of nitric oxide synthase antagonists in the treatment of septic shock in both animal models of endotoxemia and human clinical trials. However, cumulative data from these studies have not provided definitive evidence for a survival benefit in the use of these agents in humans. While the signalling pathways that activate iNOS expression or activity are well characterized, little is known about the endogenous molecular determinants that decrease NO. In this regard, osteopontin, recently identified as an intrinsic regulator of iNOS expression in endotoxin-stimulated macrophages, represents a novel target in the understanding of nitric oxide pathobiology in sepsis. The purpose of this review is to discuss the S-nitrosylation of heterogeneous ribonucleoprotein A/B in the transcriptional regulation of osteopontin in nitric oxide- mediated sepsis.

Keywords: osteopontin, hnRNP A/B, sepsis, endotoxin, LPS, nitric oxide



509-514

Feasibility to delineate distribution of solution injected intraprostatic using an ex-vivo canine model

Author(s): Dr. Louis L. Pisters,

Abstract: We sought to identify an injection scheme and amount of solution injected resulting in optimal distribution of an injected solution into the prostate and to determine whether magnetic resonance (MR) imaging is suitable for evaluating intraprostatic distribution of an injected solution. Freshly excised canine prostates mounted in gelatin were injected under ultrasound guidance with a standard volume (3 ml) of 1:10 dilution of gadolinium DTPA (Gd- DTPA) and a 1:10 dilution of 1% methylene blue in phosphate-buffered saline. Three different schemes were used: three-core, 10-core, and 20-core injection schemas. The prostates were subsequently imaged by MR imaging. After imaging, samples were fixed in formalin, sectioned transversely, and digitally photographed. The distributions of injected solution on photographs and MR images were compared. Findings on MR images correlated well with photographic findings. Regions of injected solution were generally seen as hyperintense on the T1-weighted images. A 20-core injection scheme distributed the injected solution better than a three-core or 10-core scheme. A 20-core injection scheme resulted in optimal distribution within the prostate of injected methylene blue–Gd-DTPA solution. MR imaging may be useful for imaging the distribution of solution injected into the prostate.

Keywords: prostate, gadolinium, magnetic resonance imaging, gene therapy



515-522

ER stress and the JNK pathway in insulin resistance

Author(s): Dr. Hideaki Kaneto,

Abstract: The endoplasmic reticulum (ER) is an organelle which synthesizes various secretory and membrane proteins. These proteins are correctly folded and assembled by chaperones in the ER. During stressful conditions such as upon an increase in the misfolded protein level, the chaperons become overloaded and the ER fails to fold and export newly synthesized proteins, leading to ER stress. Under diabetic conditions ER stress is induced and the JNK pathway is subsequently activated, which is involved in the insulin resistance. Increase of ER stress and activation of the JNK pathway interferes with insulin action. In reverse, reduction of ER stress and suppression of the JNK pathway in obese diabetic mice markedly improve insulin resistance and ameliorate glucose tolerance. Taken together, increase of ER stress and subsequent activation of the JNK pathway play a crucial role in the progression of insulin resistance found in diabetes and thus could be a potential therapeutic target for diabetes.

Keywords: diabetes JNK pathway, ER stress, insulin resistance



523-538

Molecular insight into human heparanase and tumour progression

Author(s): Dr. Andreas J Kungl,

Abstract: The human heparanase is a key enzyme in tumour vascularisation and metastasis. Here we review the current molecular knowledge on this protein and present a model of its active domain.

Keywords: human heparanase, angiogenesis, tumour progression, metastasis, angiogenic factors, molecular modeling



539-546

Two dimensional gel electrophoresis analyses of human plasma proteins. Association of retinol binding protein and transthyretin expression with breast cancer

Author(s): Dr. Prof. Lotfi Chouchane,

Abstract: The identification of markers for either early diagnosis, treatment response or for survival of breast cancer is of critical importance. The plasma carries an archive of important histological information whose determination may help to improve early disease detection. Using two dimensional gel electrophoresis and protein sequencing we investigated the changes in protein expression profiles derived from analysis of plasma from healthy Tunisian women and patients with breast carcinoma. We have found an association between retinol binding protein, transthyretin expression and breast cancer. The levels of acute phase proteins known to accompany both acute and chronic inflammatory disorders comprising haptoglobin, serum amyloid P, apolipoprotein A1, α1-antitrypsin and α1-acidic glycoprotein were also intimately associated with this neoplastic disease.

Keywords: Two dimensional gel electrophoresis. Breast cancer. Acute phase proteins.




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