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Volume 3 - 1999


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1-14

Routes of vector application for brain tumor gene therapy

Author(s): Dr. , Nikolai G. Rainov,

Abstract: The development of highly efficient virus and non-virus vector systems for gene transfer to and gene therapy of brain tumors has advanced to the stage of clinical trials, but has still not successfully addressed some major limiting factors, such as the inability of a single delivery modality or therapeutic transgene to target a maximum number of tumor cells in diffuse or multifocal tumors, such as human glioblastoma, and to confer eradicating cytotoxicity to the whole neoplastic mass. Moreover, the choice of vectors and the route of their administration dramatically affect both the efficiency of tumor transduction and its spatial distribution, as well as the extent of transgene expression within a brain tumor and outside it, in the surrounding tumor cell-infiltrated tissue. Three main routes of vector delivery to experimental brain tumors are reviewed in this paper: stereotactic or direct intratumoral inoculation; intrathecal and intraventricular injection; and intravascular infusion with or without modification of the blood-brain-tumor-barrier. The pros and cons of all these modes of application are discussed in respect to the specific and unique features of tumors in the central nervous system. We conclude that, at the present time, there is no ideal vector or unconditionally efficient application mode, and so the successful approaches to brain tumor gene therapy need to combine different application routes with different vectors and therapeutic genes designed to address the individual features of different tumor types. The intravascular vector delivery route, although at an early stage of development, seems to be the most pervasive and demonstrates the greatest therapeutic potential in animal experiments, but for human use it should be combined either with direct intratumoral vector injections or with CSF vector delivery.

Keywords: adenovirus, brain neoplasms, gene therapy, gene transfer, herpes simplex virus, intra-carotid delivery, liposomes, plasmids, retrovirus.



15-23

Efficient in vivo expression of a reporter gene in rat brain after injection of recombinant replication- deficient Semliki Forest virus

Author(s): Dr. , Kenneth Lundstrom,

Abstract: Recombinant replication-deficient Semliki Forest virus (SFV) expressing bacterial  -galactosidase was injected into the amygdala and striatum of male Wistar rats. Reporter gene expression was detected up to 28 days post-injection. The maximal expression levels were obtained 1-2 days post- injection. In situ hybridization studies demonstrated high expression of LacZ mRNA until day 2, but no signal was detected 4 days post-injection. No significant change in body weight and temperature, exploratory locomotor behavior and forced motor performances were observed after SFV-LacZ injections. The neuronal gene transfer with SFV vectors did not trigger any major cell toxicity.

Keywords: Semliki Forest virus; in vivo expression; rat brain; β-galactosidase



25-33

Establishment of an assay to determine adenovirus- induced endosome rupture required for receptor- mediated gene delivery

Author(s): Dr. , Renate Fuchs,

Abstract: Successful human gene therapy requires methods to transfer recombinant genes to cells efficiently. One possibility is to use adenoviral-based vectors. The entry route of adenovirus involves endocytic uptake, penetration of modified viral particles into the cytoplasm by endosome rupture, transport to the nuclear pore complex, disassembly of modified particles and import of the DNA into the nucleus. Since endosome rupture is a rate-limiting step in foreign gene expression, we developed a two-step assay to quantitative virus-mediated membrane rupture. Following endosome labeling of HeLa cells with a pH-sensitive (FITC-dextran) and pH-insensitive (Cy5-dextran) fluid- phase marker in the absence or presence of replication-defective adenovirus type 5 (Ad5), first, the pH of labeled compartments was determined by flow-cytometry of cell suspensions. When compared to control cells, the pH of labeled compartments was elevated by co-internalization of Ad5 indicating endosome lysis and penetration of the marker into the pH-neutral cytoplasm. Second, single-organelle flow analysis (SOFA) of cell homogenates of the same cells was applied to quantitate the amount of labeled as well as unlabeled vesicles in the presence of Ad5. Our results demonstrate that adenovirus internalized for 10 min into HeLa cells destroys about 30% of endosomal compartments. This assay can be applied to rapidly screen various gene delivery systems for their ability to disrupt endosomal membranes and to enter the cytoplasm.

Keywords: adenovirus entry, endosome rupture, flow cytometry



35-44

Gene regulation in Herpesvirus saimiri and its implications for the development of a novel gene therapy vector

Author(s): Dr. , Adrian Whitehouse,

Abstract: We have investigated the potential of HVS as a human gene therapy vector and found that it is capable of infecting an extremely broad spectrum of human cell lines and primary cultures with efficiencies that are at least as good as (and in many cases better than) currently available vector systems. Like others we found that the virus was capable of stably transferring a functional heterologous gene by virtue of episomal maintenance. Although transduced clones can be established in all cases, we have also been able to demonstrate low levels of virus production from these cells. This finding necessitates the development of disabled mutants for potential future clinical applications. Fundamental research carried out in this laboratory has identified the interactions between the two known transcriptional regulatory genes encoded by HVS. Overall, these results suggest that ORF50 and ORF57 are ideal essential candidate genes to delete in order to produce a replication- disabled HVS. This will provide the basis for a novel gene therapy vector which is theoretically capable of addressing the problems faced by current vector systems. 

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45-46

Regulation of papillomavirus transcription and replication; insights for the design of extrachromosomal vectors

Author(s): Dr. , Alison A. McBride,

Abstract: Laboratory of Viral Diseases, National Institutes of Allergy and Infectious Disease, National Institutes of Health, Bethesda,

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57-65

Gene transfer with adeno-associated virus 2 vectors: the growth factor receptor connection

Author(s): Dr. , Arun Srivastava,

Abstract: Adeno-associated virus 2 (AAV)-based vectors have gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy. However, there are at least two major obstacles that limit high-efficiency transduction by AAV vectors. The first relates to the extent of expression of the cellular receptor for AAV, and the second concerns the rate-limiting step of the viral second-strand DNA synthesis. With reference to the first obstacle, although the ubiquitously expressed cell surface heparan sulfate proteoglycan (HSPG) has been reported to be a receptor AAV, HSPG alone is insufficient for AAV infection, and human fibroblast growth factor receptor 1 (FGFR1) has been identified as a co-receptor for successful viral entry into the host cell. With reference to the second obstacle, a cellular protein, designated the single-stranded D-sequence binding protein (ssD-BP), phosphorylated at tyrosine residues, has been identified which plays a crucial role in viral second-strand DNA synthesis. The ssD-BP is phosphorylated by the protein tyrosine kinase activity of the human epidermal growth factor receptor (EGFR). Thus, both FGFR1 and EGFR are crucial determinants in the life cycle of AAV, and further studies on the interaction between the FGFR and EGFR may yield new insights not only into its role in the host cell but also in the optimal use of AAV vectors in human gene therapy.

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67-74

Hepatocyte-specific gene expression by a recombinant adeno-associated virus vector carrying the apolipoprotein E enhancer and  1-antitrypsin promoter

Author(s): Dr. , Torayuki Okuyama,

Abstract: An adeno-associated virus vector was constructed to express exogenous genes to the liver. The original plasmid construct carried two expression units; a neomycin resistant gene and human  1- antitrypsin cDNA under the control of hepatocyte specific transcription elements. Cells were transfected with the constructed plasmid DNA with another packaging plasmid, and recombinant adeno-associated viruses (rAAV) were then recovered after adenovirus infection. Alternatively, rAAV were recovered by transduction of DNAs of the packaging plasmid and adenovirus into pre- selected cells carrying constructed proviral DNA. When the transducing abilities were evaluated based on G418 resistant colony formation on HeLa cells, the latter method was found to give almost 10-fold more rAAV. We then isolated G418 resistant colonies and established several independent clones for the HeLa and Hepa1A cells infected with the rAAV. All of the eight clones derived from Hepa1A cells produced significant amounts of the human  1-antitrypsin protein. In contrast, none of the five clones derived from HeLa cells produced a detectable level of  1- antitrypsin. Our results suggest that liver-specific promoter and enhancer maintain the tissue specificity in the rAAV construct, and that the rAAV vector system would be useful in hepatocyte directed gene therapy.

Keywords: adeno-associated virus, liver-specific promoter, α1-antitrypsin promoter, apolipoprotein E enhancer, gene therapy



75-78

Human cytomegalovirus (HCMV) nuclease: implications for new strategies in gene therapy

Author(s): Dr. , Elke Bogner,

Abstract: Human cytomegalovirus (HCMV), one of eight human herpesviruses, can cause serious illness in neonates as well as in immunocompromised adults (Alford and Britt, 1993). Transplant and AIDS patients, e.g. may develop life- threatening diseases as a consequence of primary infection or reactivation of latent infection. Additionally, HCMV infections are also associated with congenital neurological complications in approximately 7,500 newborns annually (Alford and Britt, 1993). The current drugs are toxic and cause additional complications including drug resistance. Since present therapeutical approaches are limited new strategies are needed that may result from a better understanding of viral molecular biology.

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79-89

Application of recombinant Herpes Simplex Virus-1 (HSV-1) for the treatment of malignancies outside the central nervous system

Author(s): Dr. , Katherine L. Molnar-Kimber,

Abstract: Attenuated HSV-1 mutants are promising novel vectors for human gene therapy of cancer. In addition to their efficacy in treatment of experimental CNS tumors, HSV mutants have shown promise in treatment of extra-CNS tumors including mesothelioma, melanoma, breast cancer, epithelial ovarian carcinoma, colon carcinoma and non small cell lung carcinoma in various animal models. HSV mutants which have been partially attenuated can function as direct oncolytic agents capable of proliferating within three-dimensional tumors and causing tumor cell death. A major advantage of these replication-restricted HSV mutants is that they can selectively replicate in tumor cells and thus, potentially express transgenes in a higher percentage of the tumor cells. Alternatively, super- attenuated HSV mutants and amplicons can function as efficient vectors for gene therapy and have the ability to host large and multiple transgenes. A multi-pronged strategy for HSV-based anti-tumor therapy is currently emerging, where multi-attenuated viruses or the oncolytic HSV mutants are used as gene therapy vectors for intratumoral delivery of immunomodulatory or chemotherapy sensitizing transgenes. HSV-based tumor therapy has been reported to induce an anti-tumor immune response in some animal models. These findings may be due to the combination of co-expression of immunomodulatory molecules, immunogenic properties of the virus, necrosis of the tumor tissue and subsequent tumor antigen presentation. Thus, HSV oncolytic agents and gene therapy vectors show great potential as anti-tumor therapies. Further studies are required to test the efficacy and safety of these agents in extra-CNS malignancies.

Keywords: gene therapy, HSV-1, cancer



91-101

Transcriptional repression in cancer gene therapy: targeting HER-2/neu overexpression as an example

Author(s): Dr. , Mien-Chie Hung,

Abstract: Overexpression of the HER-2/neu oncogene has been well-documented as a frequent event in human cancers. In clinic, overexpression of HER-2/neu indicates a unfavorable prognosis and highly correlated with the low survival rate of patients associated with breast and ovarian cancers. Downregulation of the HER-2/neu gene expression in cancer cells by attenuating the promoter activity of the gene is therefore an attractive strategy to reverse the transformation phenotype induced by HER-2/neu overexpression. We have identified a number of cellular and viral transcriptional regulators, including the ets family member PEA3, the SV40 large T antigen, and the adenovirus type 5 E1A, which are able to repress the HER-2/neu gene expression. Expression of these transcriptional regulators resulted in downregulation of the HER-2/neu promoter activity and reversed the malignant phenotype of the transformed cells in vitro. These observations were followed by a series of studies to investigate whether these HER-2/neu repressors can act therapeutically as tumor suppressor genes for cancers that overexpress HER-2/neu. The growth of tumors derived from HER-2/neu-overexpressing cancer cells was inhibited by the transcriptional repressors, accompanied by decreased HER-2/neu expression in tumor cells. The results of these preclinical studies clearly indicate that transcriptional repressors which downregulate HER-2/neu can be a promising regimen for cancer treatment in a gene therapy format.

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103-112

Gene therapy targeting p53

Author(s): Dr. , John Nemunaitis,

Abstract: The product of the p53 gene plays a critical role in the regulation of cell growth. Mutations of this gene are associated with transformation to a malignant phenotype. Correction of the gene defect through transfer of a wildtype p53 gene into malignant cells, or targeting malignant cells with oncolytic viruses (ONYX-015) genetically engineered to proliferate in cells containing mutant genes has been identified as a therapeutic approach by preclinical assessment. Initial clinical trials have confirmed functional activity and expression of the transgene product in Adp53-injected malignant tissue and tumor specific viral proliferation have been observed in patients receiving intratumoral injection of ONYX-015.

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113-121

Targeted therapy of CEA-producing cells by combination of E. coli cd/HSV1-tk fusion gene and radiation

Author(s): Dr. , Xin-yao Wu,

Abstract: To enhance the specific cytotoxic effects caused by the transfer of the E. coli cytosine deaminase (cd) and HSV1-tk to CEA (carcinoembryonic antigen)-producing cells, the expression of the cd-tk fusion gene, driven by the CEA promoter, was investigated followed by treatment with 5-FC and GCV in combination with radiation. The expression vector pCEAcd-tk, based on pcDNA3, was introduced into CEA-producing cells using liposomes. In CEA-producing cells, the CEA promoter could efficiently drive the expression of the fusion suicide gene. The expression activity of the E. coli cd gene driven by the CEA promoter was about three times higher than that driven by the CMV promoter in transfected LoVo cells. A combination of 5-FC and GCV could cause higher cytotoxicity to the cells expressing CD and TK than the use of a single prodrug alone. The cytotoxic effect after combining the two prodrugs with radiation was the highest among all treatments in vitro. In vivo, the result of a subrenal capsule assay showed that the inhibition rates for 5-FC (0.5 mg/g) and GCV (0.1 mg/g) to GLC-82 cells transfected with pCEAcd-tk were 18.04% and 55.00%, respectively. A combination of the prodrugs at the same dose resulted in a 152.50% inhibition rate. In addition, the bystander effect exerted by the pCEAcd-tk/5-FC+GCV system in vitro was greater than that induced by cd/5-FC or tk/GCV alone.

Keywords: Xin-yao Wu, Ph.D. Professor, Department of Biochemistry, Sun Yat-sen University of Medical Sciences, Guangzhou, 510089, P.R. China. E-mail: xyaow@gzsums.edu.cn



123-131

Efficacy of antiherpetic drugs in combined gene/chemotherapy of cancer is not affected by a specific nuclear or cytoplasmic compartmentation of herpes thymidine kinases

Author(s): Dr. , Jan Balzarini,

Abstract: Introduction of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in tumor cells, followed by treatment of the transfected tumor cells with an antiherpes drug has shown promise in the treatment of solid tumors. We have recently shown that the HSV-1 TK fused to green fluorescent protein (GFP) was localized almost exclusively in the nuclei of HSV-1 TK-GFP fusion gene-transfected human osteosarcoma cells, due to the presence of a nuclear localization signal (NLS) at the N-terminus of the HSV-1 TK. A deletion mutant, lacking the N-terminal 34 amino acids [ (AA1-34)HSV-1 TK-GFP], was distributed throughout the cytoplasm and nucleus of transfected tumor cells. In addition, varicella-zoster virus (VZV) TK-GFP, which lacks the NLS and which is uniformly distributed in the nucleus and cytoplasm of the VZV TK-GFP gene- transfected tumor cells, could be specifically targeted to the nucleus by ligating the HSV-1 TK nuclear localization signal to the VZV TK-GFP sequence. Two pairs of osteosarcoma cell lines stably expressing HSV-1 TK-GFP or VZV TK-GFP either in the nucleus or throughout the cell were established and compared for their sensitivity to the cytostatic effects of a variety of antiherpetic nucleoside analogues. In addition, the efficacy of nucleoside analogues in contributing to the bystander effect (i.e., the killing of non-transfected tumor cells by neighbouring TK gene- transfected cells after gap junctional transfer of phosphorylated nucleoside metabolites), was evaluated using the HSV-1 TK-GFP and (AA1-34)HSV-1 TK-GFP gene constructs. From our experiments it is inferred that there is no difference in cytostatic activity of the antiherpetic 123 Degrève et al: Antiherpetic drugs in combined gene/chemotherapy of cancer nucleoside analogues against TK gene-transfected cells, whether the TK activity is solely localized in the nucleus or spread over the nucleus and cytosol. Also, the bystander killing effect of the antiviral compounds was independent of the nature of the intracellular compartment in which the HSV-1 TK-GFP fusion protein was expressed.

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133-148

Glioblastoma multiforme: molecular biology and new perspectives for therapy

Author(s): Dr. , Giorgio Palù,

Abstract: Pathogenic features of glioblastoma multiforme and of other gliomas are reviewed in the present article. Emphasis is given to those genetic alterations which are involved in oncogenesis, to the process of tumor neoangiogenesis and to the role played by the immune system in controlling neoplastic growth. Aspects which are relevant to therapeutic interventions are also dissected, and gene therapy in particular. A new gene therapy approach that combines tumor suicide, via enzyme- directed prodrug activation, and cytokine-promoted immune rejection is reported, together with results from the first application of this approach in humans.

Keywords: Therapy, gene therapy, brain tumors, gliomas, glioblastoma multiforme, molecular biology, pathogenesis, immunotherapy, neoangiogenesis, oncogenes



149-155

Gene-based vaccine strategies against cancer

Author(s): Dr. , Jong J. Kim,

Abstract: In recent years, the characterization of gene-based cancer vaccines has been an important step in the development of different treatment options for human carcinoma. These particular vaccines make use of proteins that are specifically produced at very high levels by tumor cells. These tumor-associated antigens (TAAs) are not only used in diagnostic situations, but also in the development of cancer vaccines. In this review we will focus on two well characterized TAAs, carcinoembryonic antigen (CEA) and prostate specific antigen (PSA). The two methods of in vivo delivery we will examine are recombinant vaccinia virus and nucleic acid immunization. The TAA gene can be cloned into vaccinia virus and the viral infection stimulates an adequate immune response in the host. In the case of nucleic acid immunization, DNA constructs encoding for TAAs are directly injected into the host and are taken up by its cells. The cells express the specific encoded antigen upon which the immune system acts. The effects of CEA recombinant vaccinia virus (rV-CEA) have been characterized in rodents, macaques, and humans. It was shown that the vaccine induced both humoral and cellular immune responses in mice and monkey models. In a phase I clinical trial, a CEA-specific cytotoxic T- lymphocyte response was observed. The effects of a CEA DNA vaccine were investigated in both mice and dogs and both humoral and cellular immune responses were found as well. A recombinant vaccinia virus expressing PSA was tested in rhesus monkeys and induced a PSA-specific long term cellular immune response. Experiments were also performed injecting a PSA DNA construct into both mice and rhesus monkeys. PSA-specific humoral and cellular immune responses were observed in both cases. All these experimental approaches demonstrate the efficacy and advantages of gene- based cancer vaccine strategies and support further clinical investigations.

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157-165

Rational vaccine design through the use of molecular adjuvants

Author(s): Dr. , Jong J. Kim,

Abstract: Nucleic acid immunization is an important vaccination strategy which delivers DNA constructs encoding for a specific immunogen into the host. These expression cassettes transfect the host cells, which become the in vivo protein source for the production of antigen. This antigen then is the focus of the resulting immune response. This vaccination technique is being explored as an immunization strategy against a variety of infectious diseases as well as cancer. The first generation DNA immunization experiments have shown that the DNA vaccines’ ability to elicit humoral and cellular responses in vivo in a safe and well-tolerated manner in various model systems, including humans. As we explore the next generation of DNA vaccines, our goal is to refine the current strategy to elicit more clinically efficacious immune responses. A more clinically effective vaccine may need to elicit a more specific immune response against the targeted pathogen. It would be a distinct advantage to design immunization strategies which can be “focused” according to the correlates of protection known for the particular pathogen. In order to focus the immune responses induced from DNA immunization, we have investigated the co-delivery of genes for immunologically important molecules, such as costimulatory molecules and cytokines which play critical regulatory and signaling roles in immunity. We and others have shown that the use of these molecular adjuvants could enhance and modulate immune responses induced by DNA immunogens. Co-administration of costimulatory molecules (CD80 and CD86), proinflammatory cytokines (IL-1 , TNF- , and TNF- ), Th1 cytokines (IL-2, IL-12, IL-15, and IL-18), Th2 cytokine (IL-4, IL-5 and IL-10), and GM-CSF with DNA vaccine constructs led to modulation of the magnitude and direction (humoral or cellular) of the immune responses. These studies demonstrate the potential utility of molecular adjuvant strategy as an important tool for the development of more rationally designed vaccines.

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167-177

In vivo production of therapeutic antibodies by engineered cells for immunotherapy of cancer and viral diseases

Author(s): Dr. , Marc Piechaczyk,

Abstract: Our recently developed ability to produce human monoclonal antibodies, together with that of reshaping antibody molecules, offers new tools for treating a number of human diseases. Direct injection of purified antibodies, or of antibody-related molecules, to patients would, however, not always be possible or desirable. This is especially true in the case of long-term therapies for at least two reasons. One is the high cost of antibodies certified for human use. The other is the possibility of neutralizing anti-idiotypic immune responses as a result of repeated injection of massive doses of antibody. In vivo production of therapeutic antibodies through either genetic modification of patients' cells or implantation of antibody-producing cells might overcome both of these hurdles. Several cell types suitable for use in cell/gene therapy protocols, such as skin fibroblasts, keratinocytes, myogenic cells and hepatocytes, are capable of producing monoclonal antibodies in vitro upon gene transfer. Furthermore, the grafting of engineered myogenic cells permits the long-term systemic delivery of recombinant antibodies in immunocompetent mice. Importantly, antibodies produced both in vitro and in vivo, retain the specificity and the affinity of the parental antibody and no anti-idiotypic response is detected in mice producing ectopic antibodies. Long-term systemic delivery of such antibodies into mice can also be achieved through the implantation of antibody-producing cells encapsulated into a new biocompatible material, cellulose sulphate. Importantly, no inflammation occurs at capsule implantation sites over periods as long as 10 months. Moreover, no anti-idiotypic response develops against antibodies released by encapsulated cells. Encapsulation of antibody-producing cells in immunoprotective devices should offer multiple advantages over genetic modification of patients' cells. These include protection against immune cells of treated individuals, the possibility of easy removal of implanted cells as well as that of implantation of non-autologous cells. Taken together, these observations demonstrate that long-term in vivo production and systemic delivery of monoclonal antibodies is technically feasible. Application of this technology to the treatment of various viral and autoimmune diseases as well as that of cancer is currently underway.

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179-187

Use of DNA priming and vaccinia virus boosting to trigger an efficient immune response to HIV-1 gp120

Author(s): Dr. , Dolores Rodríguez,

Abstract: To enhance the efficiency of DNA vaccines to HIV-1, we immunized BALB/c mice sequentially with a gp120 DNA vector and a recombinant vaccinia virus (VV). We have also evaluated the effect of granulocyte macrophage colony stimulation factor (GM-CSF) expression by a DNA vector on both cellular and humoral immune responses when coadministered with the gp120-encoding DNA at priming. Our results show a significant enhancement of both arms of the immune system when the DNA prime/VV boost regime is used, as compared with the immunization protocol based on priming and boosting with vector DNA. A 100-fold increase in the number of antigen-specific IFN- -secreting CD8+ T cells was observed in splenocyte cultures from mice immunized with the combined vector DNA/VV protocol. The humoral immune response is also improved in animals receiving the vector DNA/VV combined vaccine, as shown by the increase in both env-specific antibody titers and HIV-1 neutralizing activity in sera. IgG1 was the predominant isotype detected in sera from the immunized animals. This, together with the IL-4 and IFN- production in splenocyte cultures from these animals, indicated that both Th1 and Th2 responses are induced by the combined immunization approach. Coadministration of a GM-CSF-expressing DNA vector in the priming step resulted in enhanced T cell proliferation rates, irrespective of whether the booster was given with vector DNA or recombinant VV. In addition, a slight increase in the humoral immune response was also observed in animals primed with gp120 and GM-CSF-expressing plasmid and boosted with recombinant VV. These findings describe a combinatorial priming/booster immunization approach that may be effective in the control of HIV-1 infection and of other pathogens.

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189-196

Gene therapy approaches to the treatment of hemoglobinopathies

Author(s): Dr. , Ryszard Kole,

Abstract: Hemoglobinopathies such as thalassemia and sickle cell anemia are potentially amenable to gene therapy. Applicable gene therapy strategies can be divided into four categories: those that replace the faulty gene with a complete transcriptional unit, those that activate transcription of fetal hemoglobin genes, those that modify the endogenous gene itself, and those that attempt to repair the defective globin RNA transcripts transcribed from the gene. Before becoming valuable in the treatment of human patients, each of these methodologies must overcome obstacles in efficiency of delivery, level of effectiveness, and length of time the treatment remains effective.

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197-206

Intramuscular injection of plasmid DNA encoding intracellular or secreted glutamic acid decarboxylase causes decreased insulitis in the non- obese diabetic mouse

Author(s): Dr. , Alan Escher,

Abstract: Our goal is to determine whether gene vaccination can be used for the treatment of insulin- dependent diabetes mellitus (IDDM), an autoimmune disease. In this work, weanling non-obese diabetic (NOD) mice, an animal model system for the study of IDDM, received intramuscular injections of “naked” plasmid DNA encoding either intracellular or secreted human glutamic acid decarboxylase (GAD), one of the major autoantigens recognized during the onset of IDDM. Seven weeks later, each pancreas was scored for insulitis, an inflammation indicative of the disease. Mice treated with either type of gad gene-carrying plasmid showed a significant decrease in the severity of insulitis when compared to controls. These results suggest that vaccination using autoantigen- encoding genes may provide a means of treating IDDM.

Keywords: non-obese diabetic; IDDM, insulin-dependent diabetes mellitus; GAD, glutamic acid decarboxylase; GABA, gamma-aminobutyric acid; aa, amino acids.



207-221

Muscle-based tissue engineering for the musculoskeletal system

Author(s): Dr. , Johnny Huard,

Abstract: Somatic gene therapy through the transfer of genes into a particular tissue to alleviate a biochemical deficiency has emerged as a novel and exciting form of molecular medicine. Due to a number of factors, muscle tissue has emerged as a promising target for muscle based gene therapy and tissue engineering. First, many muscle groups are readily accessible and tolerate delivery by injection well. Second, muscle is composed of multinucleated, post- mitotic myofibers and may facilitate high and long term persistence of transgene expression. Third, muscle can be easily and repeatedly biopsied without compromising the health and function of human and animal subjects. Finally, muscle is very well vascularized, making systemic delivery through the bloodstream feasible. Based on these unique features of the skeletal muscle, we have described four different applications of muscle based gene therapy and tissue engineering: inherited muscle diseases, muscle injury and repair, bone healing and finally intra-articular disorders. Since the field of muscle based gene therapy and tissue engineering has expanded and matured over the last few years, we will review some hurdles facing the practical application of this technology as well as potential approaches to circumvent these limitations to eventually apply this technology to the treatment of pathologies and conditions of the musculoskeletal system.

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223-232

Helper-dependent adenoviral vectors as gene delivery vehicles

Author(s): Dr. , Manal A. Morsy, Diane M. Harvey,

Abstract: Adenoviral (Ad)-mediated in vivo gene transfer and expression is limited in part by cellular immune responses to viral- encoded proteins. In an attempt to diminish these responses, we have previously developed and described helper- dependent (HD) Ad vectors in which the viral protein coding sequences are completely deleted. These vectors provided efficient delivery, and greater safety which represents a significant advance over existing Ad vectors. In addition, the inherent enhanced insert capacity (up to ~ 37kb) allows for the insertion of large or multiple genes, including expression regulatory sequences. Several drug-regulated gene expression systems are now available for controlling target gene transcription through the use of small-molecule inducing compounds. While early experiments have demonstrated the utility of inducible systems in cell culture and transgenic mice, continued evaluation of such systems in viral gene therapy vectors should lead to discoveries and improvements which will make them amenable for use in a therapeutic context. The generation of a gene therapy approach that combines both safe and efficient vector delivery of one or multiple genes of interest and a small molecule-controlled gene expression system will provide a powerful tool for therapeutic intervention.

Keywords: adenoviral vectors, leptin, tetracycline-inducible, ob/ob mice, antiprogestin, GAL4 DNA-binding domain, gene switch, rapamycin-regulated gene expression



233-241

Gene transfer into muscle for the treatment of muscular dystrophy and haemophilia

Author(s): Dr. , Geoffrey Goldspink,

Abstract: Muscle has proven to be an appropriate expression system for genes, the product of which is required in the general circulation as well as for muscle genes per se. This review deals with the design of the gene constructs including the vectors and the regulatory elements required for optimisation of expression following introduction of the relevant cDNA by intramuscular injection. The relative merits and problems associated with each type of vector including the immunogenic responses they elicit are discussed. Duchenne muscular dystrophy is used to illustrate the problems associated with gene therapy for a disease in which a muscle protein is defective or missing whilst haemophilia is chosen as an example of how a systemic protein, Factor VIII or IX, may be produced at low constitutive levels in muscle rather than liver.

Keywords: intramuscular, gene therapy, vectors, muscular dystrophy, haemophilia



243-248

Gene therapy for arthritis

Author(s): Dr. , Raphael Hirsch,

Abstract: Gene therapy for the treatment of arthritis is developing rapidly. The ability to deliver genes to local sites of inflammation decreases the possibility of systemic side effects, making arthritis a good candidate for gene therapy. Animal models of arthritis provide a means of testing gene transfer strategies. Several issues still need to be addressed including which genes to deliver, how to deliver these genes, and how to regulate gene expression in vivo.

Keywords: Gene therapy, in vivo gene transfer, rheumatoid arthritis, inflammation, gene expression



249-256

Antisense gene therapy in the long-term control of hypertension

Author(s): Dr. , Mohan K. Raizada,

Abstract: Studies from the last two decades have established that both circulating and tissue renin-angiotensin system (RAS) are important. Their coordinated interaction is essential in the regulation of blood pressure and play a key role in the development, establishment and maintenance of hypertension. Interruption of the RAS pathway, either by preventing the formation of Ang II (i.e. ACE inhibitor) or by blocking its actions at the level of the receptor (i.e. AT1 receptor antagonists), has been shown to reduce BP and protect against target-organ injury. Since there are problems associated with pharmacological control of high blood pressure, we developed a viral gene delivery approach to target hypertension. It was our intention to try and interrupt the RAS at the genetic level in order to achieve long term control of hypertension and reversal of pathophysiology associated with the disease. In general, delivery of antisense to the AT1R was able to prevent (for up to 18 months) or reverse the elevated blood pressure, and the alterations in vascular calcium homeostasis, alterations in ion channel activity, and cardiac vascular ultrastructure. These results demonstrate that antisense gene delivery is useful in the long-term treatment of hypertension.

Keywords: AT receptor antisense, hypertension, viral vectors, cardiac and renal pathophysiology, long-term prevention



257-269

Construction and deployment of triple ribozymes targeted to multicatalytic proteinase subunits C3 and C9

Author(s): Dr. , Gary A. Clawson,

Abstract: We have been developing triple ribozyme constructs for modulating gene expression, which consist of 2 cis-acting ribozymes flanking an internal trans-acting ribozyme, which is targeted to a selected cellular RNA. The 2 cis-acting ribozymes function autocatalytically, resulting in liberation of the internal ribozyme with minimal nonspecific flanking sequences. Here, we test 2 triple ribozyme constructs targeted to the multicatalytic proteinase subunits C3 and C9. The liberated internal ribozyme is 5-20 times more active in vitro than the same ribozyme contained with a double-G mutant (which cannot undergo autocatalytic processing) or contained within nonspecific flanking vector sequences. These triple ribozymes were placed within inducible expression vectors, which were used to produce stably transfected hepatocyte cell lines. Both of the constructs distributed between nucleus and cytoplasm, produced substantial growth inhibition in the cell lines, and their effectiveness was due to their catalytic activity and not to antisense effects, as demonstrated with catalytically inactive mutants. Thus, the triple ribozyme approach appears to represent a substantial improvement over conventional ribozymes.

Keywords: antisense oligonucleotides, triple ribozyme, transfection



271-280

Development of hammerhead ribozymes for HIV-1 gene therapy: principles and progress

Author(s): Dr. , Sadhna Joshi,

Abstract: Hammerhead ribozymes are small RNA molecules that can be designed to specifically recognize and cleave a target RNA. A single ribozyme can theoretically act in a catalytic manner thus cleaving more than one molecule of its target RNA. Because of their sequence specificity, ribozymes are being developed as therapeutic agents to eliminate unwanted cellular and viral RNAs. Ribozymes are being used to inhibit human immunodeficiency virus type-1 (HIV-1) replication. Promising results have been obtained by several groups using ribozymes targeted against various sites within the HIV-1 genome. This article reviews some of the factors relevant to the design of hammerhead ribozymes with a particular focus on their application in HIV-1 gene therapy.

Keywords: Hammerhead ribozymes, HIV-1 replication, gene therapy, multimeric ribozymes, retroviral vectors, HIV-1 Env



281-291

Use of antisense oligonucleotides to study homeobox gene function

Author(s): Dr. , Kenichi Takeshita,

Abstract: Homeobox genes code for transcription factors known to be important in hematopoiesis and leukemogenesis. In order to understand the function of a homeobox gene in leukemia, we have used an antisense oligonucleotide technique to inhibit the expression of a homeobox gene, DLX7, which is expressed at high levels in some leukemia cell lines and patients. With careful design and use of the antisense oligonucleotide, we have found that the loss of DLX7 gene expression results in loss of expression of secondary genes, cell cycle arrest and apoptosis. These studies have led to an understanding of DLX7 gene function in leukemia cell growth and the identification of genes regulated by DLX7. These studies also raise the possibility that DLX7 antisense oligonucleotides may be useful in the treatment of patients with DLX7-positive leukemia.

Keywords: Homeobox genes, leukemia, antisense oligonucleotides, DLX7, apoptosis, hematopoietic differentiation



293-300

Potential application of dominant negative retinoic acid receptor genes for ex vivo expansion of hematopoietic stem cells

Author(s): Dr. , Keiya Ozawa,

Abstract: It is difficult to expand hematopoietic stem cells ex vivo by stimulation with hematopoietic cytokines, since any cytokine thus far tested induces differentiation as well as growth. It is therefore important to consider how to inhibit differentiation of hematopoietic stem cells during ex vivo culture. For this purpose, we have constructed retroviral vectors expressing dominant-negative retinoic acid receptor- (RAR ) genes. The immature hematopoietic cell lines 32D and FDCPmix, transduced with the dominant-negative RAR -expressing vectors, remained blastic or promyelocytic and continued to grow without differentiation even under the differentiation-inducing conditions. This block of differentiation could be overcome by treatment with all-trans retinoic acid, suggesting that the transduced cells still retained their differentiating ability. This leads to the possible application of dominant-negative RAR  genes for the ex vivo expansion of hematopoietic stem cells in combination with hematopoietic cytokines. For clinical applications, however, dominant-negative RAR  genes should be removed from host cells after ex vivo expansion. We have, therefore, incorporated two loxP sites on either side of the RAR gene in each vector so that removal of the integrated differentiation-blocking RAR  genes from the transduced cells could be achieved using the Cre recombinase/loxP system (reversible integration of a gene of interest). We are investigating efficient ways to introduce the Cre recombinase into host cells.

Keywords: gene therapy, hematopoietic stem cell, ex vivo expansion, retroviral vector, hematopoietic cytokine, dominant negative retinoic acid receptor, Cre recombinase, loxP, all-trans retinoic acid, differentiation block



301-310

Optimized expression of serotonin receptors in mammalian cells using inducible expression systems

Author(s): Dr. , Peter Vanhoenacker,

Abstract: Efficient expression of neurotransmitter receptor proteins in a pure and active form has become an indispensable tool for modern pharmaceutical research. Different expression systems for producing receptor proteins have been used with varying success, but the expression levels are often low or not stable over a long period of time. We evaluated different inducible expression systems for the stable, high-level expression of several serotonin receptors. Using the human interleukin-6 promoter, which is inducible by a variety of biological and chemical agents, only modest expression levels were obtained. Most likely, this is due to a down-regulation of the receptors by the inducing agents used. More successful was the type I interferon-inducible Mx promoter, with which high-level and stable expression of four different serotonin receptors was obtained for several months. Finally, the tetracycline-inducible expression system was also tested and resulted in a still higher expression, with induction levels varying from 10- tot 700-fold.

Keywords: serotonin receptors, interleukin-6 promoter, interferon, Mx promoter, tetracycline-inducible, interleukin-6, neurotransmitter receptors



311-325

Identification of a negative regulatory mechanism for the repair of U5 long terminal repeat DNA by the human immunodeficiency virus type 1 integrase DNA polymerase

Author(s): Dr. , ,

Abstract: The quasi-random integration of retroviral DNA elements into the chromosomes of infected cells is believed to proceed by a four-step mechanism. The 3'- ends of the long terminal repeats (LTRs) are processed by the endonucleolytic cleavage and removal, usually, of a GT dinucleotide (step 1); the 3'-processed DNA ends are inserted at staggered nicks in the host DNA via a DNA strand transfer reaction, simultaneously generating short gaps at the sites of insertion (step 2); the gaps are repaired by a DNA polymerase (step 3); and the 5'-ends of the viral DNA are joined to the host DNA (step 4). Human immunodeficiency virus type 1 (HIV-1) integrase was previously reported to possess enzymatic activities capable of performing at least the first 3 steps of the integration process including an intrinsic DNA - dependent polymerase activity capable of short gap repair (Acel et. al., 1998 J. Virol. 72: 2062-2071). In the present study, the behavior of the integrase DNA polymerase was examined in a DNA end-repair assay in which the frequency of polymerization on 3'-processed HIV-1 U5 LTRs was examined. The frequency of polymerization was negatively regulated by the 5'-AC sequence comprising the 2-nucleotide template and by the sequence of the adjacent conserved 5'TG/CA dinucleotide. Mutations within these DNA elements of the LTR enhanced the polymerization frequency on 2-nucleotide templates between 3- and 100-fold. In most cases, the integrase DNA polymerase added only one nucleotide to 3'-processed LTRs even though the DNA was comprised of 2-nucleotide template-primers. This level of regulation was controlled by a DNA binding and/or zinc finger domain in the integrase protein. By contrast, the integrase DNA polymerase behaved in a processive manner with homopolymeric pyrimidine templates, extending nascent DNA chains up to at least 20 nucleotides, whereas DNA polymerization with an oligo dA template exhibited a lower processivity of 1-7 nucleotides. The results suggest a model whereby an interaction between integrase and specific DNA elements in the HIV-1 LTRs prevents the repair of 3'-processed LTRs by the integrase DNA polymerase. Drugs with the ability to alter this regulatory aspect of integrase DNA polymerase function and thus induce repair of processed LTRs are predicted to block integration of HIV-1 DNA, and thus have potentially lethal consequences for HIV-1 replication.

Keywords:



327-345

Molecular mechanisms of viral transcription and cellular deregulation associated with the HTLV-1 Tax protein

Author(s): Dr. , Brian A. Lenzmeier,

Abstract: It is estimated that between 10 and 20 million people worldwide are infected with the human T- cell leukemia virus, type I (HTLV-I). Since HTLV-I is associated with a variety of human diseases, including an aggressive lymphoproliferative disorder named adult T-cell leukemia, infection by HTLV-I has become increasingly recognized as an important public health concern. Malignant transformation associated with HTLV-I infection is linked with the synthesis of a virally-encoded protein called Tax. In this review, we will highlight our current understanding of Tax protein function, both in its role as an activator of HTLV-I transcription and deregulator of cellular homeostasis. It is widely believed that Tax deregulation of cellular gene expression and cell cycle progression accounts for the pathogenicity associated with HTLV-I infection.

Keywords: HTLV-I, human T-cell leukemia virus type-I; ATL, adult T-cell leukemia; TSP/HAM, tropical spastic paraparesis/HTLV-1 associated myopathy; LTR, long terminal repeat; CREs, cyclic AMP response elements; CREB, cAMP- response element binding protein; EMSA, el



347-354

What does acetylcholinesterase do in hematopoietic cells?

Author(s): Dr. , Bernard.J. Jasmin,

Abstract: Acetylcholinesterase (AChE) is an essential component of cholinergic synapses since it hydrolyzes acetylcholine released from presynaptic nerve terminals. However, it is well documented that AChE is also expressed in a variety of non-cholinergic tissues including hematopoietic cells. Despite the recent progress made in our understanding of the molecular mechanisms regulating expression of AChE, our knowledge of the precise function of this enzyme in hematopoietic cells still remains limited. Previous work has led to the notion that AChE may be involved in myelodysplastic syndromes as well as acute myeloid leukaemias since it may regulate hematopoiesis by acting as a tumor suppressor gene. In addition, recent studies have further demonstrated the involvement of AChE in the proliferation of multipotent stem cells, as well as in the mechanisms leading to apoptosis in cells undergoing erythroid and megakaryocytic differentiation. In this review, we first present an overview of the cellular and molecular biology of AChE and then, focus more specifically on the expression of AChE in hematopoietic cells. Finally, we also discuss the recent evidence linking AChE expression and the proliferative capacity of these cells. A better understanding of the functional significance of AChE in hematopoietic cells may be relevant for the future design of novel therapeutic strategies against proliferative disorders of hematopoietic tissues.

Keywords: acetylcholinesterase, cholinergic synapses, hematopoietic cells, tumor suppressor, myeloid leukaemia, apoptosis, proliferative disorders



355-371

The ETS-domain transcription factors: lessons from the TCF subfamily.

Author(s): Dr. , Andrew D. Sharrocks,

Abstract: The ternary complex factors (TCFs) represent a subfamily of ETS-domain transcription factors. In recent years, several significant advances have been made in our understanding of their function at the molecular level. Many aspects of TCF function are conserved with other ETS-domain proteins and with transcription factors from other families, therefore these studies contribute more generally to our understanding of transcription factor function. Structural and biochemical studies have furthered our understanding of protein-DNA recognition by the TCFs. Furthermore, significant insights into the regulation of TCF activity by phosphorylation have been achieved. The TCFs have developed as a paradigm for nuclear targets of MAP kinase pathways and further advances have been made into understanding the specificity of kinase action towards the TCFs. The TCFs are known to bind to multiple protein partners which have roles in up- and down-regulating their activity, recruitment to DNA and transmission of transcriptional activation and repression signals. Finally, our understanding of the role of TCFs in specific biological processes is starting to become clearer, with roles defined in immediate-early gene regulation in a neuronal context.

Keywords: ETS-domain, ternary complex factor, TCF, transcription factors, MAP kinases.



373-378

Transcriptional activation of the ras oncogenes and implications of BRCA1 in the cell cycle regulation through p53 checkpoint

Author(s): Dr. , D.A. Spandidos,,

Abstract: Aberrant expression of ras genes has been recognized in several human cancers and is associated with the development of the disease. Thus, revealing the mechanisms that regulate the expression of ras genes is critical for understanding their role in the process of tumorigenesis. Transcriptional regulation of the H-ras gene, involves nuclear factors recognizing elements in the promoter region of the gene and hormones; so far, a glucocorticoid response element and a p53 element have been identified. Alternative splicing and specific methylation patterns may regulate the expression of ras genes as well. Altered expression of ras genes has been detected in a variety of human tumours. Differential expression of the ras family genes in breast cancer has shown overexpression of all three members of ras genes. A significant correlation of overexpression of ras genes and stage of the disease was also observed suggesting that aberrant expression of the ras genes may be an initial event in breast cancer oncogenesis. Overexpression of Ras p21 protein has been detected in human endometrial and ovarian tumours, due to elevated p53 protein binding on the p53 element of the c-H-ras gene, suggesting that p53 protein participates in the development of human gynecological neoplasias through aberrant transcriptional regulation of the H-ras proto-oncogene. Investigation of the BRCA1 expression levels in relevance with the expression levels of p53, mdm-2 and p21WAF1/CIP1 genes, implicated in cell cycle progression, revealed combined alterations of these genes in sporadic breast cancer specimens, indicating that loss of function of BRCA1 may arrest the cell cycle through p53 checkpoint.

Keywords: ras genes, transcriptional regulation, p53, steroid hormone receptors,BRCA1, cell cycle regulation.



379-385

Nuclear receptor coactivators as potential therapeutical targets: the HATs on the mouse trap

Author(s): Dr. , Arndt Benecke,

Abstract: The recent past has seen an immense burst in our understanding of nuclear receptor (NR) signaling. Key achievements have been the structure determination of the ligand binding domain and the identification of coregulators which mediate the transcriptional effects of NRs. Both types of studies have now converged on the description of the NR coactivator interface at the atomic level, which, together with the elucidation of the structure of two coactivator related histone acetyl transferases (HATs) points towards previously unknown targets for drug design potentially leading to novel types of non-ligand antagonists of NR function.

Keywords: histone acetyl transferase, retinoic acid receptor, nuclear receptor, drug discovery, transcriptional regulation, coactivator



387-395

High mobility group protein HMGI-C: a molecular target in solid tumor formation

Author(s): Dr. , Erik Jansen,

Abstract: The high mobility group protein HMGI-C is a non-histone chromosomal protein characterized by its capacity to bind in the narrow minor groove of AT-rich DNA. It is thought to act as a so-called architectural transcription factor and to modulate the expression of target genes through the formation of stereospecific complexes on the regulatory regions of these targets. Towards defining its function, there is now increasing evidence for a critical role of HMGI-C in the regulation of cell growth and proliferation. A direct role for HMGI-C in tumorigenesis has been demonstrated by the finding that the HMGI-C gene on human chromosome 12q15, is rearranged in a variety of solid tumors, resulting in ectopic expression. In lipomas, the LPP gene on chromosome 3q27-28 is the preferential translocation partner. It was shown to encode a novel, proline-rich, LIM domain containing protein and HMGI-C/LPP fusion transcripts have been identified in lipomas. Moreover, in overexpression experiments, the tumor-derived aberrant HMGI-C protein products are able to induce neoplastic transformation. In complementary studies, it was shown that targeted inactivation of Hmgi-c in mice disrupts both pre- and postnatal growth, resulting in the pygmy phenotype. When cultured in vitro, pygmy mouse-derived embryonic fibroblasts display a lower growth rate. In addition, anti-sense HMGI-C expression has been shown to inhibit retrovirally induced transformation.

Keywords: solid tumor formation; high mobility group I proteins; LPP



397-412

Replication of simple DNA repeats

Author(s): Dr. , Maria M. Krasilnikova,

Abstract: This chapter presents an overview of studies on the replication of simple DNA repeats conducted in our laboratory during the last seven years. The recent massive increase in available DNA sequences has led to the clear understanding that natural DNAs, particularly in eukaryotes, are extraordinarily enriched in different repeats (Schroth and Ho, 1995; Cox and Mirkin, 1997). This leads to an obvious question: what are the biological functions (if any) of these repeated elements? This problem is currently the subject of very intense studies in many laboratories all over the world. We came to this question after we realized that many repeated DNA sequences constitute a major obstacle to DNA polymerization in vitro (Dayn et. al., 1992; Samadashwily et. al., 1993; Samadashwily and Mirkin, 1994; Krasilnikov et. al., 1997). Subsequently, we found that several such repeats attenuate DNA replication in vivo as well (Samadashwily et. al., 1997; Krasilnikova et. al., 1998). Based on our data, we conclude that there are at least three mechanisms by which different repeats inhibit replication. We believe that this may reflect a potentially important role of repeated DNA as punctuation marks for major genetic processes in DNA texts. Repeat-caused replication attenuation might also contribute to the mechanisms of repeat length polymorphism seen in many human diseases.

Keywords: DNA repeats, inverted repeats, DNA replication, repeat length polymorphism, replication attenuation



413-422

Separation of the DNA replication and transactivation activities of EBNA1, the origin binding protein of Epstein-Barr virus

Author(s): Dr. , Lori Frappier,

Abstract: During latent infection of human B-lymphocytes, Epstein-Barr virus (EBV) genomes are stably maintained as DNA episomes that replicate once per cellular S phase. The replication and segregation of the EBV episomes requires the latent origin of replication, oriP, and one viral protein, Epstein-Barr Nuclear Antigen 1 (EBNA1). EBNA1 also activates the transcription of other latent viral genes and some cellular genes. EBNA1 fulfills all of its functions by directly interacting with EBV sequences, but the functional role of EBNA1 residues outside of the DNA binding domain is not well understood. We have explored the contribution of EBNA1 regions to transactivation activity. Our results indicate that the C-terminal acidic tail of EBNA1, that was previously implicated in transactivation, is not required for this function. Rather, the transactivation activity resides primarily in an internal arginine-rich region (amino acids 325-376) that was previously shown to mediate interactions at a distance between DNA-bound EBNA1 molecules as well as interactions with at least two cellular factors. An EBNA1 mutant, lacking amino acids 325-376, supports the transient replication of oriP plasmids at wild type levels but is severely impaired for transcriptional enhancement. Therefore, our results indicate that the replication and transactivation functions of EBNA1 can be separated.

Keywords: Epstein-Barr virus, Epstein-Barr nuclear antigen 1 (EBNA1), DNA replication, origin of replication, transactivation, activation of transcription, transactivation domain.



423-435

The activation of the chicken lysozyme locus in development is a cooperative process

Author(s): Dr. , Constanze Bonifer,

Abstract: The chicken lysozyme gene is a marker for the myelomonocytic lineage of the hematopoietic system. In early experiments we demonstrated that correct activation of the chicken lysozyme locus in macrophages of transgenic mice requires the complete set of cis-regulatory elements. Different cis-elements are activated at distinct developmental stages and their chromatin structure is differentially remodelled. We have shown that the early onset of transcriptional activation of the chicken lysozyme locus is entirely dependent on enhancer elements which are structurally activated early in development (-6.1 kb and -3.9 kb early enhancers). However, the structural reorganization of the early enhancers requires the presence of promoter sequences. We concluded from these experiments that the early enhancers and the promoter cooperate in order to activate the lysozyme locus. Subsequently, we performed experiments aimed at elucidating the cis-regulatory requirements of chromatin rearrangement at the early enhancers. The -6.1 kb enhancer is well characterized at the molecular level and all transcription factors contributing to its activity in transfection studies are known. We have placed this element into a new sequence context on the lysozyme locus by deleting extended flanking regions and analyzed this construct in transgenic mice. Surprisingly, its chromatin rearrangement ability as judged from DNaseI hypersensitive site formation was impaired. We conclude from this experiment that the cooperation of enhancer core and flanking sequences is necessary for enhancer activity. We hypothesize that all sequences of a gene locus serve a purpose in the developmental control of its activation.

Keywords:



437-445

Mechanisms involved in regulation of the estrogen- responsive pS2 gene

Author(s): Dr. , Ann M. Nardulli,

Abstract: Estrogen is a hormone of critical importance in the development and maintenance of normal reproductive tissues and has been implicated in initiation of mammary carcinogenesis. Estrogen's actions are mediated through an intracellular estrogen receptor (ER), which interacts with estrogen response elements (EREs) to bring about changes in transcription of estrogen-responsive genes. Although it is clear that the ER-ERE interaction is a critical link in the chain of events that lead to transcription activation, the mechanisms by which transcriptional changes occur remain unclear. We present evidence that ERE sequence and ligand act as allosteric modulators of ER conformation and that these conformational changes most likely play a role in regulating transcription of estrogen-responsive genes.

Keywords: ER, estrogen receptor; EREs, estrogen response elements; DMS, dimethylsulfate; LMPCR, ligation mediated polymerase chain reaction



447-453

Biological function of the USF family of transcription factors

Author(s): Dr. , Michèle Sawadogo,

Abstract: USF is a family of ubiquitous transcription factors that are structurally related to the Myc oncoproteins and also share with Myc a common DNA-binding specificity. While the structure and DNA-binding properties of the USF transcription factors are well characterized, their biological function is only beginning to emerge. Experiments in cultured cells suggest that USF can antagonize the activity of Myc in cellular proliferation and transformation. The phenotype of USF- deficient mice indicates an additional and essential role of USF in embryonic development as well as pleiotropic functions in adult animals.

Keywords:



455-464

The role of chromatin in the establishment of enhancer function during early mouse development

Author(s): Dr. , Sadhan Majumder,

Abstract: In mammals, enhancers are believed to stimulate transcription from RNA polymerase II promoters primarily by relieving their chromatin-mediated repression. Interactions responsible for enhancer function are developmentally acquired. Factors responsible for this repression are not present in the paternal pronuclei of 1-cell embryos, making them impervious to enhancer function. Although such chromatin-mediated repression is observed in oocytes, maternal pronuclei of 1-cell embryos, and the 2-cell embryos, the enhancer function first appears in 2-cell embryos, a stage in development that corresponds to the beginning of major zygotic gene expression (ZGE). The lack of enhancer function prior to 2-cell stage is also not due to the absence of specific activation proteins, but appears to be due to the absence of an essential coactivator activity. The coactivator activity first appears in 2-cell embryos, and is required for enhancers driven by different classes of transcription factors. The absence of the coactivator activity and the corresponding enhancer function appears to be unique to oocytes and 1-cell embryos, suggesting that it provides a safeguard against premature activation of genes prior to ZGE.

Keywords: transcription, enhancer function, chromatin, mammalian embryonic development



465-474

Molecular mechanisms that regulate hyaluronan synthesis

Author(s): Dr. , Paraskevi Heldin,

Abstract: Hyaluronan is an ubiquitous glycosaminoglycan found in almost all tissues of the body in vertebrates and in the extracellular capsule of certain pathogenic bacteria. Hyaluronan is biologically active and affects cell migration and proliferation. The amount of hyaluronan in the extracellular matrix increases during inflammation, wound healing and certain forms of cancer. Hyaluronan is synthesized by a membrane-bound enzyme which uses UDP-sugar nucleotides precursors (UDP-glucuronic acid and UDP-N-acetylglucosamine). Three mammalian hyaluronan synthase genes (HAS1, HAS2 and HAS3) have been identified. The encoded proteins share 56-71% sequence similarities. The synthesis of hyaluronan is carefully regulated in a cell specific manner. Certain growth factors and cytokines lead to an increased activity of existing synthase molecules as well as induce the synthesis of new enzyme molecules.

Keywords: Hyaluronan, hyaluronan synthase, HAS1, cytokine, extracelular matrix




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