Use Of Vectors Research Articles

Virus Vectors for use in the Central Nervous System: Problems in the Use of Herpes Simplex Virus as a Vector

Publisher Summary This chapter describes problems in the use of herpes simplex virus (HSV) as a vector for expression of foreign gene. The use of HSV1 as a vector has been investigated by a number of groups and these studies have included both replicating and nonreplicating viruses. The use of HSV1 as a vector became a practical goal with the discovery that the ICP4 gene could be deleted and the virus grown as a host range mutant. It is found that functional promoter expression during latency may require two elements, and that the upstream latency associated transcript (LAT) promoter and the long terminal repeats (LTR) together provide these functions. It is observed that as specific regions of the upstream part of the LAT promoter were removed, particularly between −330 and −250, expression in neurons decreased. It is suggested that targeted expression is nothing more than making a good match between transcription factor binding sites on the promoter and high levels of the same transcription factor in a given cell type. A reasonable explanation for the lack of expression of 8117-43 in the hypoglossal nucleus may have been a lack of transcription factors that could bind to either the LTR or the LAT enhancer. It is suggested that in neurons of the dorsal root ganglia, where the factors can bind to the LAT enhancer, expression is high, and in other neurons it is lower or not observed.

Virus Vectors for use in the Central Nervous System: Nonneurotropic Adenovirus: A Vector for Gene Transfer to the Brain and Gene Therapy of Neurological Disorders

Virus Vectors for use in the Central Nervous System: Nonneurotropic Adenovirus: A Vector for Gene Transfer to the Brain and Gene Therapy of Neurological Disorders

Virus Vectors for use in the Central Nervous System: adeno-associated viral vectors

Virus Vectors for use in the Central Nervous System: adeno-associated viral vectors

Virus Vectors for use in the Central Nervous System: Retroviral Vectors for Gene Delivery to Neural Precursor Cells

Virus Vectors for use in the Central Nervous System: Retroviral Vectors for Gene Delivery to Neural Precursor Cells

Targeting Retroviral Integration?

Can retroviral integration be targeted to preselected locations in the human genome? If so, targeting might improve the safety of retroviral vectors for use in gene therapy. Directing integration of vectors to benign locations in the human genome, for example, might reduce the risk of transformation by insertional activation of oncogenes. This issue has become a lot less theoretical with the recent report from Li and coworkers describing transformation by a gene therapy vector [1]. Interpreting this experiment is not straightforward, however, in part because the marker gene transduced by the vector probably contributed to oncogenesis.

Development and use of gene transfer for treatment of cardiovascular disease.

Gene therapy holds promise for the treatment of cardiovascular diseases for which effective pharmacological therapies are insufficient or unavailable. Recent studies have suggested that modification of current gene delivery systems combined with the use of efficacious therapeutic genes may ultimately be successful for clinical vascular gene therapy. Although certain applications such as vein-graft failure may be best suited for short-term transient overexpression of therapeutic genes, other disorders including human essential hypertension and atherosclerosis require sustained overexpression of genes. Hence, design and use of vector systems for delivery of genes to the required site in vivo requires careful consideration. Both viral and nonviral gene therapy vectors show low efficiency for gene transfer into vascular cells and demonstrate a lack of selectivity, as vectors have natural tropism for other cells and tissues. Recent work has focused on the design, development, and utility of vascular cell-selective gene therapy vectors for use in distinct and diverse vascular gene therapy scenarios. Using phage display technology we have isolated small peptide ligands that mediate selective binding to either vascular endothelial cells or vascular smooth muscle cells. When engineered into either adenoviral (Ad) or adeno-associated viral (AAV) vectors, candidate peptides enabled the virus to selectively bind to the desired cell type thus generating novel vascular cell-selective gene transfer. As preclinical studies have highlighted both the potential for vascular gene therapy as well as defining the potential pitfalls, the development of disease-selective gene therapeutics will increase safety and efficiency of gene therapy for future clinical use.

Induction of a strong HIV-specific CD8+ T cell response in mice using a fowlpox virus vector expressing an HIV-1 multi-CTL-epitope polypeptide.

Recombinant avipoxvirus vectors are attractive candidates for use in vaccination strategies for infections such as human immunodeficiency virus type 1 (HIV-1), where induction of a CD8+ T cell response is thought to be an important component of protective immunity. Here, we report the expression of a multiepitope polypeptide (TAB9) composed of the central 15 amino acids of the V3 loop from six different isolates of HIV-1 in a fowlpox virus (FWPV) vector, and the use of this vector (FPTAB9LZ) to induce strong HIV-specific CD8+ T cell responses in mice. In animals immunized twice intravenously with FPTAB9LZ, almost 2% of the CD8+ T cells in the spleen were shown to produce IFN-gamma in response to stimulation with HIV-1 peptides 1 week after the second immunization. The most dominant response was to the HIV-1 IIIB peptide. A strong HIV-specific response was also induced by intraperitoneal immunization of mice with FPTAB9LZ, whilst subcutaneous immunization elicited a weaker response. Intraperitoneal immunization with FPTAB9LZ was also shown to provide protection against challenge with a recombinant vaccinia virus expressing antigens, including those in TAB9. These results confirm the potential of FWPV vectors for use in HIV vaccination strategies.

Separating fact from fiction: assessing the potential of modified adenovirus vectors for use in human gene therapy.

One of the major hurdles to successful gene therapy of genetic and/or acquired disease is the ability to efficiently introduce a foreign gene into the tissue of interest and, in the case of some genetic diseases, achieve long-term expression of the transgene. Due to their ability to transduce a wide variety of cell types in a cell-cycle independent fashion, adenovirus (Ad)-based vectors have received considerable attention in recent years as delivery vehicles for multiple gene therapy applications. Effective use of early "first-generation" versions of these vectors was hampered by not only the induction of strong immune responses in the host to the Ad vector and transduced cells, but also to direct acute and chronic toxicity caused by the vector itself. Furthermore, transgene expression was typically transient, lasting only a few weeks. Despite these limitations, these vectors have been used in a number of human clinical trials, eliciting both interesting as well as controversial results, some of which are summarized herein. Because of these limitations, a number of advances in adenovirus "vectorology", manifested primarily as the development of multiply attenuated Ads and vectors deleted of all viral protein coding sequences, has resulted in vectors which retain all of the advantages of Ad vectors and, in addition, do not exhibit the deleterious characteristics associated with [E1-]deleted Ads. This review focuses on the current state of the art regarding the potential for human use of Ad-based vectors, and how the use of this vector continues to offer the potential for successful use as a gene delivery tool for the treatment of a great number of human genetic and non-genetic diseases.

1302. Development of B Lineage Specific Lentiviral Vectors for Use in Gene Therapy of Human B Lymphocyte Disorders

1302. Development of B Lineage Specific Lentiviral Vectors for Use in Gene Therapy of Human B Lymphocyte Disorders

Expression of the S. aureus hysA gene in S. carnosus from a modified E. coli–staphylococcal shuttle vector

We have modified an E. coli–staphylococcal shuttle vector for use in the general cloning and expression of genes from pathogenic staphylococci in Staphylococcus carnosus. As S. carnosus is non-pathogenic, this expression system will facilitate the study of the roles of individual gene products in the disease process. To evaluate the use of this expression system, a DNA fragment containing the Staphylococcus aureus hyaluronate lyase ( hysA) gene was cloned into the modified vector, pNW21, and introduced into S. carnosus. Hyaluronate lyase was both produced and secreted by S. carnosus. In addition, the secreted HysA protein was enzymatically active, as determined using a zymographic assay.

Flow regime identification in a two-phase flow using wavelet transform

This study addresses the problem of the automatic flow regime identification in two-phase flows in pipes. A novel wavelet transform-based approach is proposed and validated using time series of differential pressure fluctuations. The experimental data on the differential pressure measured in a vertically installed Venturi meter for air–water flow were analyzed and found to be appropriate for flow regime identification. The wavelet spectrum of the measured signal is shown to characterize the flow patterns completely, and the vector of the wavelet variances is proposed as the characteristic vector for use in an on-line flow regime identification system.

Vaccination of cats with an attenuated recombinant myxoma virus expressing feline calicivirus capsid protein

Myxoma virus, a member of the Poxviridae family (genus Leporipoxvirus) is the agent responsible for myxomatosis in the European rabbit. Recombinant myxoma viruses expressing the capsid gene of an F9 strain of feline calicivirus (FCV) were constructed from an apathogenic, laboratory attenuated, isolate of myxoma virus. The FCV capsid genes were recombined into the myxoma growth factor (MGF) locus of the myxoma genome and expressed from synthetic poxvirus promoters. Myxoma virus is unable to replicate productively in feline cells in vitro, however, cells infected with recombinant viruses do express the heterologous antigens from both late and early/late synthetic promoters. Cats immunised with myxoma–FCV recombinant virus generated high levels of serum neutralising antibody and were protected from disease on subsequent challenge with virulent FCV. Furthermore, there was no evidence of transmission of myxoma–FCV recombinant virus from vaccinated to non-vaccinated cats. These results demonstrate the potential of myxoma virus as a safe vaccine vector for use in non-lepori species and in particular the cat.

Comparison of two schemes for derivation of atmospheric motion vectors

[1] This paper presents the operational scheme of the National Satellite Meteorological Center (NSMC) of the China Meteorological Administration (CMA) to derive atmospheric motion vectors. The NSMC scheme is compared with a method developed at the European Organization for the Exploitation of Meteorological Satellites (EUMETSAT) in preparation for Meteosat Second Generation. Both schemes employ similar basic principles in terms of feature tracking and height assignment, however there are also some important differences. Furthermore, the EUMETSAT scheme assigns quality indicators to each wind vector at the end of the processing chain, whereas the NMSC scheme has inbuilt quality checking at different processing steps allowing for reinstatement of winds rejected by a first quality check. The evaluation of the performance is gained from two periods: a week in January and a week in July 1999. European Centre for Medium-Range Weather Forecast analyses and radiosonde data are used as independent data for evaluation of the two schemes. It is shown that correlating infrared image data with water vapor data before height adjustment, as performed in the NSMC scheme, has a great potential to better distinguish high and low cloud and to provide high-density wind fields. The utilization of radiative transfer calculations for the estimation of the height of thin clouds in the EUMETSAT scheme is shown to be imperative for good quality wind fields. Finally, the feature of the EUMETSAT scheme to assign quality indicators improves the utility of the wind vectors for use in numerical weather prediction models. It is suggested that a combination of the different features of both schemes potentially provide highly increased spatial density in the wind field with improved quality.

In Vivo Application of Non-viral Vectors to the Liver

The liver plays a central role in many inherited and acquired genetic disorders, and thus is a potential target for nucleic acid therapies. Despite the great strides made in basic molecular biology over the last two decades successful gene therapy remains elusive. Most recently, there has been considerable effort to develop non-viral gene therapy approaches, in part, to overcome the potential complications associated with viral delivery systems. This review outlines the different non-viral approaches available to the liver, and includes a detailed review of recent advances in delivery vectors for use in techniques such as gene augmentation, with particular emphasis on useful applications of antisense and ribozyme technology.

Plasmid-only rescue of influenza A virus vaccine candidates.

The potential threat of another influenza virus pandemic stimulates discussion on how to prepare for such an event. The most reasonable prophylactic approach appears to be the use of effective vaccines. Since influenza and other negative-stranded RNA viruses are amenable to genetic manipulation using transfection by plasmids, it is possible to outline new reverse genetics-based approaches for vaccination against influenza viruses. We suggest three approaches. First, we use a plasmid-only rescue system that allows the rapid generation of high-yield recombinant vaccine strains. Second, we propose developing second-generation live influenza virus vaccines by constructing an attenuated master strain with deletions in the NS1 protein, which acts as an interferon antagonist. Third, we suggest the use of Newcastle disease virus recombinants expressing influenza virus haemagglutinin proteins of pandemic (epizootic) strains as novel vaccine vectors for use in animals and possibly humans.

Endocrine-related resources from the National Institutes of Health.

Endocrine-related resources from the National Institutes of Health.

Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria.

Full exploitation of the information available in bacterial genome sequences requires the availability of facile tools for rapid genetic manipulation. One bacterium for which new genetic tools are needed is the methylotroph Methylobacterium extorquens AM1. IncQ and small IncP vectors were shown to be unsuitable for use in this bacterium, but a spontaneous mutant of a small IncP plasmid was isolated that functioned efficiently in M. extorquens AM1. This plasmid was sequenced and used as a base for developing improved broad-host-range cloning vectors. These vectors were found to replicate in a wide variety of bacterial species and have the following advantages: (1) high copy number in Escherichia coli; (2) small size (7.2 and 8.0 kb); (3) complete sequences; (4) variety of unique restriction sites; (5) blue-white screening via lacZalpha; (6) conjugative mobilization between bacterial species; and (7) readily adaptable into species-specific promoter-probe and expression vectors. Two low-background promoter-probe vectors were constructed based on these cloning vectors with either lacZ or xylE as reporter genes; these were shown to report gene expression effectively in M. extorquens AM1. Specific expression vectors were developed for use in M. extorquens AM1, which were shown to express foreign genes at significant levels, and a simple strategy is outlined to develop specific expression vectors for other bacteria. The strong mxaF promoter was used for expression, since E. coli lac-derived promoters were expressed at very low levels. This suite of genetic tools will enable a more sophisticated analysis of the physiology of M. extorquens AM1, and these vectors should also be valuable tools in the study of a variety of bacterial species.

Inducible and constitutive expression using new plasmid and integrative expression vectors for Thermus sp.

To develop molecular tools and examine inducible and constitutive gene expression in Thermus thermophilus. Two plasmid promoter probe vectors and an integrative promoter probe vector were constructed using a promoterless thermostable kanamycin nucleotidyltransferase (KmR) cassette. Three expression vectors were constructed based on a constitutive promoter J17, that functions in both Thermus and Escherichia coli. An inducible expression vector was constructed using the heat-shock inducible promoter (70 to 85 degrees C) from the dnaK gene of T. flavus, and the malate dehydrogenase gene (mdh) from T. flavus was cloned and expressed in both E. coli and T. thermophilus HB27. This report describes the construction and use of improved promoter probe and expression vectors for use in Thermus species. The mdh gene can be used as a high temperature (85 degrees C) reporter gene for Thermus sp. The dnaK promoter is thermo-inducible. The expression vectors and molecular tools described here are significant improvements over previously reported vectors for Thermus sp. The mdh gene and the thermo-inducible dnaK promoter will facilitate high temperature studies employing Thermus species.

Factors Affecting Long-Term Expression of a Secreted Transgene Product after Intravenous Administration of a Retroviral Vector

We have studied parameters affecting in vivo expression of human growth hormone (hGH) in mice after intravenous administration of a retroviral vector encoding the protein as a model system for clotting factor VIII gene therapy. Such treatment results in a brief burst of high-level expression followed by lower level sustained expression of the hGH in the circulation. The major targets for transduction in the mouse are liver and spleen. Such direct transduction (i.e., without surgical or chemical induction of cell division) requires vector at high titer (≥108 cfu/ml) and is dose dependent. Transduction efficiency decreases with increasing age of the recipient. Nevertheless, long-term expression in adults is observed after administration of vector as a split dose on 2 consecutive days. We also show that anti-vector immune responses may enhance long-term expression and that both anti-vector and anti-transgene immunity can be modulated. This work provides a framework for the rational development of means to enhance the efficiency of retroviral vectors for use in clinical gene replacement therapy.

Systemic immunity and mucosal immunity are induced against human immunodeficiency virus Gag protein in mice by a new hyperattenuated strain of Listeria monocytogenes.

Vaccines designed to control chronic infections by intracellular agents such as human immunodeficiency virus type 1 (HIV-1) require the induction of cell-mediated immune responses to rid the host of pathogen-infected cells. Listeria monocytogenes has characteristics that make it an attractive vaccine vector for use against such infections. Here we show that parenteral immunization with a new highly attenuated strain of this organism provided complete protection against systemic and mucosal challenges with a recombinant vaccinia virus expressing HIV-1 gag. Immunization also generated a strong, long-term memory cytotoxic-T-lymphocyte (CTL) response in spleen, mesenteric lymph nodes, and Peyer's patches directed against the gag protein. Oral immunization with this attenuated strain also produced complete, long-lasting protection against the recombinant virus but only against mucosal virus challenge. Curiously, oral immunization was associated with a transient CTL response in the three lymphoid tissues examined.