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At least 1,333 records · Page 74Linked to original sources
PubMed2001

Heparan sulfate: anchor for viral intruders?

Heparan sulfates (HS) are ubiquitous, polyanionic carbohydrate chains linked to core proteins in cell membranes and extracellular matrices of all eukaryotes. Due to the complex nature of the HS-biosynthesis, a wealth of different structures are produced. These seem to have a well defined distribution in different tissues and cells throughout development. Binding of endogenous proteins with different functional properties such as growth factors, adhesion molecules or enzymes, is one of the functions of HS. Besides interaction with endogenous factors, glycosaminoglycans (GAG) and especially HS have also been demonstrated to function as receptors for a number of different pathogens. What roles may HS play in the pathogenesis and tropism of different intruders like parasites or viruses? What implications does binding of viruses to HS have for the development of drugs or the application of viral vectors for gene targeting? In this review an attempt is made to collect our present knowledge on viral usage of HS and the implications that follow.

Adaptation, Physiological↗
PubMedDate not supplied

Production of retrovirus and adenovirus vectors for gene therapy: a comparative study using microcarrier and stationary cell culture.

In gene therapy, retrovirus and adenovirus vectors are extensively used as gene-delivery vehicles and further large-scale processing of these viral vectors will be increasingly important. This study examined stationary and microcarrier cell culture systems with respect to the production of a retrovirus vector (encoding a monounit hammerhead ribozyme gene with an intron) and an adenovirus vector (encoding a reporter lacZ gene). Cytodex 1 and Cytodex 3 solid microcarriers were found to be able to provide good cell growth and high-titer vector production in suspension cultures. Porous microcarriers such as Cytopore 2 gave slightly lower but still efficient growth but produced significantly lower titers of retrovirus and adenovirus vector from the producer cells. The specific retrovirus production was not proportionally related to the specific growth rate of the producer cells. High MOI infection was essential for high-titer production of adenovirus vector in 293 cells. Hydrodynamic shear forces on microcarrier-grown cells increased the production yield for retrovirus vector but decreased for adenovirus vector. The cellular productivity was much more efficient for adenovirus vector produced in 293 cells as compared to the retrovirus vector produced in PA317-RCM1 cells. These findings can provide further insight into the feasibility of applying microcarrier cell culture technology to produce gene-therapy virus vectors.

3T3 Cells↗
PubMed1999

Angiogenesis induced in muscle by a recombinant adenovirus expressing functional isoforms of basic fibroblast growth factor.

The present work studies the effects of a replication-deficient adenovirus (Ad), Ad-RSVbFGF, bearing the human basic fibroblast growth factor (bFGF) cDNA, as a potential vector for therapeutic angiogenesis of ischemic diseases. The different isoforms of the protein were expressed from the viral vector in various cell types and, although the cytoplasmic isoform does not possess a signal peptide, we observed its release from a muscle cell line. The proteins were fully functional when tested in a long-term survival assay of quiescent fibroblasts. After endothelial cell infection with Ad-RSVbFGF, we observed an 80&percnt increase in the mean length of the capillary-like tubes that differentiated in a three-dimensional model of angiogenesis. We evaluated angiogenesis directly in mice 14 days after subcutaneous injection of Matrigel plugs containing Ad-RSVbFGF. A marked neovascularization was observed in the Matrigel plugs and in the surrounding tissues. Finally, the recombinant virus was injected into the hindlimb muscles of mdx mice. A 2.5-fold increase in bFGF content of the muscle was observed 6 days after injection, without any significant variations detected in the animal sera. Immunohistological detection showed an increased number of large-caliber vessels in the treated muscles as compared with control muscles. These results demonstrate that Ad-mediated transfer of the human bFGF gene can induce angiogenesis in muscle, making this tissue a potential target for the treatment of ischemic diseases.

Adenoviridae↗
PubMed1999

Intrauterine gene transfer: gestational stage-specific gene delivery in mice.

Intrauterine gene transfer in mice by intraplacental microinjection of recombinant adenoviral or retroviral vectors carrying beta-galactosidase (beta-gal) reporter gene was analyzed in relation to gestational stage, viral titer and promoters. After injections of viral vectors on days 9.5, 11.5 or 14.5 post coitum (p.c.), embryos, fetuses and adult animals were analyzed for beta-gal expression on days 13.5 p.c., 18.5 p.c. and at 2 months of age, respectively. Injection of adenoviral vectors on day 9.5 or day 11.5 p.c. resulted in high beta-gal expression in the heart or liver, respectively. Injection on either day also gave expression in other tissues including vasculature and hindbrain. This temporal pattern of adenoviral transduction correlated with the expression level of integrin beta3 subunit, which is known to be a component involved in adenoviral transduction. Adenovirus-mediated intrauterine gene transfer resulted in persistent beta-gal expression in multiple cell foci in the liver and hearts of 2-month-old adult animals treated in utero, indicating stable integration of the transgene into the host cell genome at a low frequency. Although at low efficiency, injection of retroviral vector on day 9.5 and 11.5 p.c. resulted in beta-gal expression in embryonic liver, while day 9.5 p.c. injection resulted in persistent beta-gal expression in 2-month-old adult heart. This is the first study to show gestational stage-specific gene transfer via intraplacental microinjection of adenoviral vectors.

Adenoviridae↗
PubMed2005

[Gene therapy and molecular therapeutic for head and neck cancer].

Despite recent progress in radiotherapy, chemotherapy and surgical techniques, the survival of patients with head and neck squamous cell carcinomas has not improved significantly over the past three decades. To conquer these malignant tumors, various new therapies have been under development, including gene therapies. In this paper, we have reviewed recent advances in gene therapies and molecular target therapy for head and neck cancers, with a special focus on the replication-selective adenovirus vector. At present, replication-defective adenovirus-based vectors have been widely used as convenient and safe vectors for transferring therapeutic genes into target cancer cells. However, these vectors have limited the efficacy of treatment by restricting the number of tumor cells to which the therapeutic gene can be delivered. Replication-selective adenoviral vectors (RSAV) are adenoviruses designed to have a limited ability to replicate themselves in the targeted tumor cells but not in other normal tissues. Tumor cell killing is achieved not by the genes delivered by the vectors but by the oncolysis induced by the replicated viruses by their original nature as adenovirus. Amplified viral vectors also spread to the adjacent tumor cells and kill these cells in the same manner. Recently, increased evidences of antitumor activity of RSAVs have been demonstrated preclinically, and several clinical trials have demonstrated the safety and clinical activity of replication-selective viruses.

Adenoviridae↗
PubMed1990

[Transgenic chickens].

Transgenic mice are produced by retroviral insertion, micro-injection in the early embryo, and recently by transfection of embryonic stem cells. Transgenic chickens were only made by retroviral vectors based on avian leukemia virus (ALV) and reticuloendotheliosis (REV) genomes. A replication-defective retroviral vector is preferentially used because these do not induce infectious virus. Since chickens are lacking endogenous REV sequences, a replication defective REV vector is most useful for practical application. Transgenic disease resistance is most likely obtained by blocking of viral receptors. By this approach recently transgenics with resistance against ALV infection were made at the Regional Poultry Disease Laboratory in East Lansing. Inhibition of virus replication by antisense DNA, which is complementary to viral mRNA, is promising for the future. Considerable research efforts still have to be made, however. Production of biomedical proteins will most likely be the first practical use of transgenic chickens. For the time being, vaccines will be used for the control of infectious diseases. The current live-virus vaccines will be replaced by inactivated (sub-unit) vaccines and thereafter by recombinant DNA vaccines based on viral vectors.

Animals↗
PubMed2006

Bioluminescence imaging after HSV amplicon vector delivery into brain.

BACKGROUND: Firefly luciferase (Fluc) has routinely been used to quantitate and analyze gene expression in vitro by measuring the photons emitted after the addition of ATP and luciferin to a test sample. It is now possible to replace luminometer-based analysis of luciferase activity and measure luciferase activity delivered by viral vectors directly in live animals over time using digital imaging techniques. METHODS: An HSV amplicon vector expressing Fluc cDNA from an inducible promoter was delivered to cells in culture and into the mouse brain. In culture, expression of Fluc was measured after induction in a dose-dependent manner by a biochemical assay, and then confirmed by Western blot analysis and digital imaging. The vectors were then stereotactically injected into the mouse brain and Fluc expression measured non-invasively using bioluminescence imaging. RESULTS: Rapamycin-mediated induction of Fluc from an HSV amplicon vector in culture resulted in dose-dependent expression of Fluc when measured using a luminometer and by digital analysis. In mouse cortex, a single injection of an HSV amplicon vector (2 microl, 1x10(8) transducing units (t.u.)/ml) expressing Fluc from a viral promoter (CMV) was sufficient to detect robust luciferase activity for at least 1 week. Similarly, an HSV amplicon vector expressing Fluc under an inducible promoter was also detectable in the mouse cortex after a single dose (2 microl, 1x10(8) t.u./ml) for up to 5 days, with no detectable signal in the uninduced state. CONCLUSIONS: This HSV amplicon vector-based system allows for fast, non-invasive, semi-quantitative analysis of gene expression in the brain.

Animals↗
PubMed1993

Heterologous protection against influenza by injection of DNA encoding a viral protein.

Cytotoxic T lymphocytes (CTLs) specific for conserved viral antigens can respond to different strains of virus, in contrast to antibodies, which are generally strain-specific. The generation of such CTLs in vivo usually requires endogenous expression of the antigen, as occurs in the case of virus infection. To generate a viral antigen for presentation to the immune system without the limitations of direct peptide delivery or viral vectors, plasmid DNA encoding influenza A nucleoprotein was injected into the quadriceps of BALB/c mice. This resulted in the generation of nucleoprotein-specific CTLs and protection from a subsequent challenge with a heterologous strain of influenza A virus, as measured by decreased viral lung titers, inhibition of mass loss, and increased survival.

Animals↗
PubMed1997

Expression of transgenes in normal and neoplastic anterior pituitary cells using recombinant adenoviruses: long term expression, cell cycle dependency, and effects on hormone secretion.

Adenovirus vectors have recently been used to transfer genes into a variety of cell types, including neurons, glial cells, Schwann cells, and epithelial cells. To evaluate the efficiency of gene transfer into pituitary cells using viral vectors, we used replication-deficient recombinant adenovirus vectors (RAds) encoding beta-galactosidase driven by various viral promoters. We tested the ability of RAds to infect and express beta-galactosidase within the different identified cell populations of the anterior pituitary anterior pituitary gland and also in tumor cells of anterior pituitary origin, i.e. GH3 and AtT20 cells. Our results demonstrate that transgenes encoded by RAds are expressed within all cell types of the adenohypophysis in vitro and also within AtT20 and GH3 endocrine tumor cells. Our long term expression studies indicate that long term expression with low cytotoxicity can be achieved, but that the longevity of transgene expression from RAds depends on the proliferative status of the target cells. Slowly dividing cells (endocrine population) express transgenes for longer than actively dividing cells (tumor cells and nonendocrine anterior pituitary cells). The ability of anterior pituitary cells to secrete ACTH or LH through the regulated secretory pathway decreased after infection with RAds at high multiplicity of infection (> or = 20 plaque-forming units/target cell), whereas cell viability was not affected. We also demonstrate that a higher percentage of cells expressed the transgene beta-galactosidase when we infected actively dividing GH3 cells compared with the infection of growth-arrested GH3 cells. This could reflect differential virus entry or differential activity of the individual promoters during different stages of the cell cycle. This work demonstrates that high efficiency gene transfer into all pituitary cell types can be achieved with RAds, and that this system can be exploited to characterize and experimentally manipulate pituitary-specific gene expression. The higher efficiency of infection and transgene expression in actively dividing cells compared to that in their growth-arrested counterparts could also be exploited for the treatment of pituitary adenomas that do not respond to classical treatment strategies, using suicide or cytotoxic gene therapy.

Adenoviridae↗
PubMed2006

Effect of osmotic pressure on the production of retroviral vectors: Enhancement in vector stability.

The use of Moloney murine leukaemia virus (MoMLV) derived retroviral vectors in gene therapy requires the production of high titer preparations. However, obtaining high titers of infective MoMLV retroviral vectors is difficult due to the vector inherent instability. In this work the effect of the cell culture medium osmotic pressure upon the virus stability was studied. The osmolality of standard medium was raised from 335 up to 500 mOsm/kg using either ionic (sodium chloride) or non-ionic osmotic agents (sorbitol and fructose). It was observed that, independently of the osmotic agent used, the infectious vector inactivation rate was inversely correlated with the osmolality used in the production media; therefore, the use of high medium osmolalities enhanced vector stability. For production purposes a balance must be struck between cell yield, cell productivity and retroviral stability. From the conditions tested herein sorbitol addition, ensuring osmolalities between 410 and 450 mOsm/kg, yields the best production conditions; NaCl hampered the viral infectious production while fructose originates lower cell yields. Lipid extractions were performed for cholesterol and phospholipid analyses showing that more stable viral vectors had a 10% reduction in the cholesterol content. A similar reduction in cholesterol was observed in the producer cells. A detailed analysis of the major phospholipids composition, type and fatty acid content, by mass spectrometry did not show significant changes, confirming the decrease in the cholesterol to phospholipids ratio in the viral membrane as the major reason for the increased vector stability.

Cell Line↗
PubMed1999

Efficient generation of recombinant adenoviral vectors by Cre-lox recombination in vitro.

BACKGROUND: Although recombinant adenovirus vectors are attractive for use in gene expression studies and therapeutic applications, the construction of these vectors remains relatively time-consuming. We report here a strategy that simplifies the production of adenoviruses using the Cre-loxP system. MATERIALS AND METHODS: Full-length recombinant adenovirus DNA was generated in vitro by Cre-mediated recombination between loxP sites in a linearized shuttle plasmid containing a transgene and adenovirus genomic DNA. RESULTS: After transfection of Cre-treated DNA into 293 cells, replication-defective viral vectors were rapidly obtained without detectable wild-type virus. CONCLUSION: This system facilitates the development of recombinant adenoviral vectors for basic and clinical research.

Adenoviridae↗
PubMed2006

Use of herpes simplex virus type 1-based amplicon vector for delivery of small interfering RNA.

Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis of mammalian cells. The use of DNA-based plasmid vectors to achieve transient and stable expression of siRNA has been developed to avoid the problems of double-stranded oligonucleotides transfection. These vectors direct the transcription of small hairpin RNAs (shRNAs) from a polymerase-III (H1 or U6)-RNA gene promoter. However, numerous disadvantages remain, including low transfection efficiency and difficulty in transfecting primary cells. To overcome some of these problems, the use of viral vectors for siRNA delivery has been described. Retroviral, adenoviral, adeno-associated and herpes viral shRNAs delivery systems have been successfully used to silence genes, in vitro and in vivo. The use of a herpes simplex virus type 1 (HSV-1)-based amplicon vector for siRNA delivery into mammalian cells, using human polyomavirus BK (BKV)-transformed cells as a model system is described. The results demonstrate the ability of amplicon vectors to inhibit the expression of BKV T-Ag and tumorigenicity of BKV-transformed cells. We show that the use of the amplicon vector is highly efficient for the delivery of siRNA molecules. The unique ability of these vectors to deliver multiple copies of siRNA may provide a useful tool in the development of novel anticancer therapy.

Animals↗
PubMedDate not supplied

Improving glucose and glutamine metabolism of human HEK 293 and Trichoplusia ni insect cells engineered to express a cytosolic pyruvate carboxylase enzyme.

Metabolic engineering has been defined as a directed improvement of product formation or cellular properties by modification of specific biochemical pathways or introduction of new enzymatic reactions by recombinant DNA technology. The use of metabolic flux analysis (MFA) has helped in the understanding of the key limitation in the metabolic pathways of cultured animal cells. The MFA of the major nutrients glucose and glutamine showed that the flux of glucose to the TCA cycle and its subsequent utilization is limited as a result of the lack of certain key enzymes in the pathway. One of the key enzymes controlling this flux is pyruvate carboxylase. Introduction of this enzyme into mammalian cells has been shown to improve the utilization of glucose and limit the production of lactate and ammonia, which are deleterious to cell growth. In the present work a yeast pyruvate carboxylase gene has been introduced into mammalian (HEK 293) and insect (Trichoplusia ni High-Five) cells, resulting in the cytosolic expression of the enzyme. In both cases the resulting transfected cells were able to utilize glucose and glutamine more efficiently and produce lower amounts of lactate and ammonia. Differences in the amino acid utilization pattern were also observed, indicating changes in the basic metabolism of the cells. The performance of the transfected cells as expression systems for adenovirus and baculovirus vectors, respectively, has also been examined. The results obtained and their impact on the process development for protein and viral vector production are discussed.

Animals↗
PubMed2002

Cationic lipids in gene delivery: principles, vector design and therapeutical applications.

Gene therapy will change medicine by treating the diseases at their core levels revolutionizing the way to deliver functional proteins. The development of this technology relies in designing optimal systems for DNA transfer and expression (transfection), cationic lipids being a promising alternative. Being safer than viral vectors, they also allow the delivery of larger plasmids and can be easily GMP-manufactured and stored. The main problem associated with the use of these vectors is their transfection efficiency, which is still inferior to viral methods. In this paper we present an overview of the correlations between the chemical structure and biological activity for the principal classes of cationic lipids. Key issues in the design of this class of transfection agents are presented, as well as the future trends.

Animals↗
PubMed2003

Lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha.

The practical application of gene therapy as a treatment for cystic fibrosis is limited by poor gene transfer efficiency with vectors applied to the apical surface of airway epithelia. Recently, folate receptor alpha (FR alpha), a glycosylphosphatidylinositol-linked surface protein, was reported to be a cellular receptor for the filoviruses. We found that polarized human airway epithelia expressed abundant FR alpha on their apical surface. In an attempt to target these apical receptors, we pseudotyped feline immunodeficiency virus (FIV)-based vectors by using envelope glycoproteins (GPs) from the filoviruses Marburg virus and Ebola virus. Importantly, primary cultures of well-differentiated human airway epithelia were transduced when filovirus GP-pseudotyped FIV was applied to the apical surface. Furthermore, by deleting a heavily O-glycosylated extracellular domain of the Ebola GP, we improved the titer of concentrated vector severalfold. To investigate the folate receptor dependence of gene transfer with the filovirus pseudotypes, we compared gene transfer efficiency in immortalized airway epithelium cell lines and primary cultures. By utilizing phosphatidylinositol-specific phospholipase C (PI-PLC) treatment and FR alpha-blocking antibodies, we demonstrated FR alpha-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FR alpha was observed in primary cultures of human airway epithelia. Understanding viral vector binding and entry pathways is fundamental for developing cystic fibrosis gene therapy applications.

Animals↗
PubMedDate not supplied

Gene therapy in Duchenne muscular dystrophy.

Experiments in dystrophin gene transgenic mice have supported the concept of treating Duchenne muscular dystrophy (DMD) by demonstrating that regional expression of recombinant dystrophin in dystrophic muscle leads to regional restoration of normal muscle morphology and that dystrophin mini-genes driven by muscle specific regulatory elements are probably more effective than the full-length dystrophin gene. As a gene therapy trial for DMD, dystrophin cDNAs were introduced into skeletal muscle fibers of dystrophin-deficient mice (mdx) through direct DNA injection into plasmid expression vectors, and by replication-defective recombinant retrovirus or adenovirus vectors. With direct injection of dystrophin cDNA into a plasmid expression vector or retrovirus vectors, less than 10% of adult mdx fibers of the injected muscle expressed dystrophin. On the other hand, greater efficiency has been reported for recombinant adenovirus injection into young mdx muscle. However, it is necessary to develop vectors, viral or plasmid DNA, which can be injected intravenously and directed to muscle tissues. This will involve designing vectors possessing appropriate cell-type specific tropism and/or gene transcriptional activity for DMD treatment. This is anticipated to be a vital component in the second stage of experiments aimed at DMD treatment.

Animals↗
PubMed1999

Solvoplex: a new type of synthetic vector for intrapulmonary gene delivery.

A novel type of synthetic vector, termed solvoplex, is described that can greatly enhance gene expression in lung after intrapulmonary delivery. Solvoplexes consist of plasmid DNA and organic solvents. Several organic solvents were analyzed, and luciferase reporter gene expression was observed after intrapulmonary delivery of solvoplexes containing DPSO (di-n-propylsulfoxide), TMU (tetramethylurea), or BMSO (butylmethylsulfoxide). Expression levels correlated with the amount of solvent used at constant DNA amounts. Highest expression was obtained in the lung after intratracheal injection with 15% DPSO resulting in an increase up to 440-fold compared with DNA alone. DPSO-solvoplexes (15%) gave higher reporter gene expression than polyplexes (ExGen 500) or lipoplexes (DOTAP-cholesterol or DOTAP-DOPE). Solvoplex-mediated gene expression did not depend on the delivery mode, and was observed in both mice and rats. Readministration of DPSO-solvoplexes was possible. A second injection after 4 weeks resulted in expression levels similar to the first administration. Histological analyses using lacZ and GFP reporter genes demonstrated gene expression in the lung airway epithelium after intratracheal and microspray delivery. When luciferase expression levels in lung homogenates were compared with adenovirus vectors, DPSO-solvoplexes were 4- or 100-fold less efficient, depending on the promoter used in the viral vector. A quantitative histological comparison between solvoplexes and adenovirus vectors in the best expressing regions revealed that solvoplexes yielded about 2% LacZ-positive cells in the lung airway epithelium, and adenovirus vectors about 20%. Using the microsprayer system, we demonstrated that DNA remained intact in solvoplexes on spraying and that reporter gene expression was observed in mice after intrapulmonary delivery of a solvoplex spray. DNA in DPSO-solvoplexes remained stable and functional after prolonged storage at room temperature.

Animals↗
PubMed1999

The 5' noncoding region of grapevine chrome mosaic nepovirus RNA-2 triggers a necrotic response on three Nicotiana spp.

The 5' noncoding region (NCR) of grapevine chrome mosaic nepovirus (GCMV) was cloned in a viral vector derived from potato virus X (PVX). The recombinant virus obtained was inoculated to Nicotiana benthamiana, N. clevelandii, and N. tabacum plants. Infected plants developed necrotic symptoms in place of the vein clearing and mosaic typically observed after inoculation with PVX. Northern (RNA) blot analysis showed that the replication of PVX was not specifically altered by the presence of the GCMV 5' NCR. Inoculation of recombinant PVX harboring deleted forms of the GCMV 5' NCR showed that the three stem-loop structures at the 3' end of the 5' NCR (nucleotides 153 to 206) are dispensable for the induction of necrosis. Further deletion analysis indicated that neither the 5'-most 70 nucleotides of the 5' NCR nor the downstream region (nucleotides 71 to 217) alone is able to induce the necrotic symptoms. In the presence of both the sequence encoding the GCMV coat protein and the GCMV 3' NCR, the GCMV 5' NCR failed to induce necrosis in the PVX background. The mechanisms by which the expression of the 5' NCR might modify PVX symptoms are discussed.

Base Sequence↗