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At least 1,063 records · Page 59Linked to original sources
PubMed1999

A helper virus-free packaging system for recombinant adeno-associated virus vectors.

Adeno-associated virus (AAV) is a human parvovirus that is currently receiving widespread attention for its potential use as a gene therapy vector. Construction of the recombinant AAV vector (rAAV) involves replacing most of the viral genome with a transgene of interest and then packaging this recombinant genome into an infectious virion. Most current protocols for generating rAAV entail the co-transfection of a vector plasmid and a packaging plasmid that expresses the viral replication and structural genes onto adenovirus (Ad) infected cells growing in culture. Limitations of this procedure include (1) contamination of rAAV with the Ad helper virus, (2) low yields of rAAV and (3) production of replication-competent AAV. In this report we describe new helper plasmids (pSH3 and pSH5) that eliminate the Ad co-infection requirement. The helper plasmids express the AAV rep and cap genes and the Ad E2A, VAI and E4 genes. When the helper plasmids are co-transfected onto human 293 cells with a vector plasmid in the absence of Ad infection, the rAAV vector yield is up to 80-fold greater than those obtained with the pAAV/Ad packaging plasmid. Moreover, replication competent AAV in the rAAV preparations is less than 0.00125%. The major advantages of this system are (1) the absence of infectious adenovirus and (2) the use of only two plasmids, which enhances transfection efficiencies and hence vector production. We believe that this two-plasmid transfection system will allow for more widespread use of the AAV vector system because of its simplicity and high yields. This system will be especially useful for preclinical analyses of multiple rAAV vectors.

Cell Line↗
PubMed2001

Development of disabled, replication-defective gene transfer vectors from the Jembrana disease virus, a new infectious agent of cattle.

Jembrana disease virus (JDV) is a newly isolated and characterised bovine lentivirus. It causes an acute disease in Bali cattle (Bos javanicus), which can be readily transmitted to susceptible cattle with 17% mortality. There is as yet no treatment or preventive vaccine. We have developed a gene transfer vector system based on JDV that has three components. The first of the components is a bicistronic transfer vector plasmid that was constructed to contain cis-sequences from the JDV genome, including 5'- and 3'-long terminal repeats (LTRs), 0.4kb of truncated gag and 1.1kb of 3'-env, a multiple cloning site to accommodate the gene(s) of interest for transfer, and an internal ribosome entry site plus the neomycin phosphotransferase (Neo) gene cassette for antibiotic selection. The second element is a packaging plasmid that contains trans-sequences, including gag, pol, vif, tat and rev, but without the env and packaging signals. The third is a plasmid encoding the G glycoprotein of vesicular stomatitis virus (VSV-G) to supply the vector an envelope for pseudotyping. Cotransfection of 293T cells with these three plasmid components produced VSV-G pseudotyped, disabled, replication defective, bicistronic JDV vectors encoding the green fluorescent protein (EGFP) and the Neo resistance selection maker simultaneously with a titre range of (0.4-1.2)x10(6)CFU/ml. Transduction of several replicating primary and transformed cells from cattle, primate and human sources and importantly growth-arrested cells with the JDV vectors showed high efficiency of EGFP gene transfer at 35-75%, which was stable and the expression of EGFP was long term. Furthermore, these JDV vectors were designed to suit the inclusion and expression of genes corresponding to JDV specific proteins, such as gag or env, for the development of vaccines for Jembrana disease. This strategy should also be applicable to other bovine diseases as well. The design and construction of the JDV vector system should facilitate the study of the lentivirology and pathogenesis of the diseases associated with JDV or other bovine virus infections. To our knowledge, this is the first such vector system developed from a cattle virus.

Animals↗
PubMed1998

Improved methods of HIV vector mediated gene transfer.

HIV vectors are capable of targeted gene transfer into CD4+ cells. However, extensive testing of HIV vectors in gene therapy applications is hampered by the low titer of current HIV vector could be concentrated by approximately 20 times by sulfonated cellulose column chromatography. No replication competent cytopathic HIV was detected in the concentrated vector preparation. When the vector preparation and the target cells were centrifuged at transduction, about a five-fold increase in the apparent titer was achieved. Accordingly, by combining these two techniques the overall titer was increased by approximately two orders of magnitude. Using this high efficiency strategy, we transduced human primary lymphocytes that are refractory to transduction with currently available viral vectors. Amplification and sequencing of the integration sites showed that HIV vectors could stably integrate into the chromosomes of CD4 enriched human peripheral blood mononuclear cells. These findings indicate that HIV vectors are useful for the development of gene therapy targeting lymphocytes.

CD4-Positive T-Lymphocytes↗
PubMed2004

Immune responses against adenoviral vectors and their transgene products: a review of strategies for evasion.

Human adenoviruses have been adopted as attractive vectors for in vivo gene therapy since they have a well-characterized genomic organization, can be grown to high titres and efficiently transduce a wide spectrum of dividing and non-dividing cells. However, the first-generation of adenoviral (Ad) vectors yielded only transient expression of the transgene in most immunocompetent mice. This constituted a major limitation of this early vector type. In contrast, persistent transgene expression can be established in immunodeficient mice. This suggests that the immunogenicity of adenoviral vectors limits the effective period of adenovirus-based gene therapy. Much effort has been put in devising strategies to circumvent the limitations imposed onto gene therapy by the immune system. Improvements in vector design have significantly improved the performance of the adenovirus vectors. Based on these results it is reasonable to anticipate that new modifications of the vectors will overcome some of the immunological barriers and will further expand the applicability of adenovirus-derived vectors.

Adenoviridae↗
PubMed2002

Production methods for gene transfer vectors based on adeno-associated virus serotypes.

Vectors derived from adeno-associated virus serotype 2 (AAV-2) represent a most promising tool for human gene transfer because these vectors are neither pathogenic nor toxic to the target cell, and allow long-term gene expression in a large variety of tissues. However, they are rather inefficient at infecting a number of clinically relevant cell types, and transduction by these vectors is likely hampered by neutralizing antibodies that are highly prevalent in the human population. Therefore, an increasing number of researchers are currently turning their attention to the five other serotypes of AAV, to try and develop these as novel vectors for human gene transfer, hoping to overcome the problems associated with AAV-2 vectors. Here I describe and discuss the methodology to produce these alternative AAV vectors in tissue culture. In detail, two strategies are compared that rely on transfection of cells in culture with either two or three plasmids, containing the AAV vector genome and encoding AAV and adenoviral helper functions. Either of these protocols can be used to package a recombinant AAV genome into capsids of its own serotype (generation of "real" serotypes) or to "cross-package" this vector DNA into capsids derived from another AAV serotype ("pseudotyping"). As these approaches are still in their early stages, the existing limitations of current technology are discussed, and possible further improvements proposed.

Adenoviridae↗
PubMed2002

Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors.

Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1 x 10(12) to 1 x 10(13)vector genomes/ml.

Adenoviridae↗
PubMed2002

Adeno-associated viral vectors as agents for gene delivery: application in disorders and trauma of the central nervous system.

The use of viral vectors as agents for gene delivery provides a direct approach to manipulate gene expression in the mammalian central nervous system (CNS). The present article describes in detail the methodology for the injection of viral vectors, in particular adeno-associated virus (AAV) vectors, into the adult rat brain and spinal cord to obtain reproducible and successful transduction of neural tissue. Surgical and injection procedures are based on the extensive experience of our laboratory to deliver viral vectors to the adult rat CNS and have been optimized over the years. First, a brief overview is presented on the use and potential of viral vectors to treat neurological disorders or trauma of the CNS. Next, methods to deliver AAV vectors to the rat brain and spinal cord are described in great detail with the intent of providing a practical guide to potential users. Finally, some data on the experimental outcomes following AAV vector-mediated gene transfer to the adult rat CNS are presented as is a brief discussion on both the advantages and limitations of AAV vectors as tools for somatic gene transfer.

Adenoviridae↗
PubMedDate not supplied

Development of a therapeutic adenoviral vector for cholangiocarcinoma combining tumor-restricted gene expression and infectivity enhancement.

Cholangiocarcinoma is an invasive malignancy that is most often unresectable upon diagnosis and unresponsive to chemotherapy and radiation. While adenoviral gene therapy has shown promise in treating many tumors, systemic toxicity and low tumor transduction efficiency have hampered its application in many gastrointestinal cancers. To overcome these difficulties, we have constructed an adenoviral vector utilizing a tumor-specific promoter (TSP) for selective transgene expression and a vector with an RGD-motif in the fiber-knob region for infectivity enhancement. In seeking a TSP for cholangiocarcinoma, Secretory Leukoprotease Inhibitor, Midkine, Gastrin Releasing Peptide, VEGF, Cox-2M, and Cox-2L promoters were configures in adenoviral vectors, and evaluated in cholangiocarcinoma cells lines (Oz and SkChA-1). Luciferase assays demonstrated that Cox-2 promoters (M and L) showed the highest promoter activity, with Cox-2M appearing slightly stronger than Cox-2L. Infectivity enhanced vectors with RGD-motif in the fiber-knob region were also constructed with the luciferase transgene driven by a CMV control and the Cox-2M and Cox-2L promoters. Subsequent luciferase assays comparing the unmodified vectors to the RGD-modified versions demonstrated higher levels of luciferase activity than the RGD-infected cells. This paradigm was then applied to a therapeutic HSV-TK/GCV model by constructing RGD-enhanced HSV-TK vectors driven by Cox-2M and Cox-2L promoters. In vitro cytocidal effect analysis confirmed that the RGD-modified, cox-2 (M and L) driven vectors showed a stronger cytocidal effect upon gancyclovir administration than the vectors with wild-type fiber. The Cox-2 promoter demonstrates a favorable selectivity profile for cholangiocarcinoma, and RGD-modification further enhances transduction efficiency. This combination has potential to overcome the obstacles to clinical application of adenoviral gene therapy in cholangiocarcinoma.

Adenoviridae↗
PubMed2003

Improvement of transduction efficiency of recombinant adenovirus vector conjugated with cationic liposome for human oral squamous cell carcinoma cell lines.

Adenovirus (Ad) vectors are commonly used in gene therapy trials because of their efficiency in gene transfer. However, their use is limited by immune responses that reduce transgene expression and decrease the efficiency of repeated vector administration. In this study, the efficacy of gene transduction and the tumor-cell killing effect on four human oral (SAS, HSC-2, HSC-3, HSC-4) and one murine squamous cell carcinoma cell (SCC-7, a kind gift of Dr. M. Hiraoka, Kyoto University) lines in vitro with Ad vector conjugated with catioic liposome (Ad/SUV) was evaluated. Ad/SUV resulted in two to five-fold over higher transduction efficiency in four human and one murine cell lines in vitro than Ad vector alone. The optimal Ad-SUV ratio was determined as 10(6) pfu of Ad vector with 1 micromol SUV. Ad/SUV showed more tumor-cell killing effect than Ad vector alone. Furthermore, the shielding effects of Ad vector with Ad/SUV from neutralizing antibody were evaluated. We also found that Ad/SUV is less susceptible to inactivation by neutralizing antibodies in vitro. The efficacy of gene transduction with Ad vector was blocked more than 70% with neutralizing serum, while Ad/SUV retained approximately 50% of the control activity in vitro. On the basis of these results, the anti-tumor effect with suicide gene therapy using Ad/SUV in vivo was evaluated. Three injections of Ad/SUV showed the inhibition of tumor growth compared with control in vivo. Our results suggested that an enhanced anti-tumor effect on human oral squamous cell carcinoma would be obtained with repeated administrations of Ad/SUV.

Adenoviridae↗
PubMed2003

Direct comparison of RD114-pseudotyped versus amphotropic-pseudotyped retroviral vectors for transduction of rhesus macaque long-term repopulating cells.

Recently, RD114 (feline endogenous retrovirus envelope protein)-pseudotyped retroviral particles have been shown to transduce human NOD/SCID repopulating cells efficiently. In this study, we compared directly transduction of repopulating cells with RD114-pseudotyped vector to that with standard amphotropic vector in the rhesus macaque model. G-CSF/SCF-mobilized CD34(+) rhesus peripheral blood cells were cultured in the presence of SCF, Flt-3 ligand, and MGDF on Retronectin-coated flasks. To assess directly the ability of the two pseudotypes to transduce primitive cells, both vectors were added simultaneously to the target cells every 24 h, for a total of four exposures in 96 h. The cells were reinfused after the animals received 1000 cGy total body irradiation. At the end of transduction, gene marking efficiency of CFU was higher with amphotropic LNL6 vector (mean 88.4%) vs RD114-G1Na vector (mean 18.5%). After long-term engraftment in three animals, total neo gene marking levels were 4-5% in PBMNCs and 1.5-4% in granulocytes. The RD114-G1Na marking levels were consistently higher in granulocytes than in mononuclear cells, while amphotropic LNL6 marking levels were higher in PBMNCs than in granulocytes. The differential gene marking patterns suggest that RD114 and amphotropic vectors may target distinct progenitor or stem cell populations. There was no clear advantage for RD114-pseudotyped vectors in this predictive preclinical model in terms of overall long-term marking levels; however, optimization of transduction conditions by increasing m.o.i. or inducing the receptor could potentially improve results with this novel vector system.

Amino Acid Transport System ASC↗
PubMed1993

The effects of vector control on the antibody response to antigens of Onchocerca volvulus.

The effects of exposure to infective larvae on the antibody response to a cocktail of specific recombinant antigens of Onchocerca volvulus and to a worm extract were evaluated by comparing the responses of individuals from a vector controlled area with those from an area of continuing transmission by ELISA. Individuals from the vector controlled areas were found to have reduced responses to both antigen preparations. A microfilerdermic (mf-) individuals from the area of vector control exhibited significantly lower total and subclass IgG responses to the worm extract. In contrast, the responses to the cocktail of specific recombinants were significantly reduced in individuals from the area of vector control who were still microfilerdermia positive (mf+). The distribution of IgG subclass specific responses was similar to both antigen preparations, both dominated by the IgG4 and IgG1 subclasses. IgG1 responses to the worm extract remained elevated in the vector controlled individuals but IgG4 was significantly reduced in the mf- individuals. Both subclasses reflected the total IgG response to the cocktail of recombinants and were significantly reduced in individuals from the vector controlled area, when compared to individuals from the hyperendemic area. IgG1 responses to the cocktail of recombinants are significantly lower than IgG4 in all individuals and virtually absent in individuals from the vector-controlled area. Measuring total IgG and IgG4 is more sensitive than IgG1 in detecting infection, 100 or 97% respectively, but they remain elevated in the individuals from the vector controlled areas even after 8-10 years interruption of transmission. These results have important implications for the serological monitoring of control programmes in individuals who have previously been infected.

Analysis of Variance↗
PubMedDate not supplied

Effect of protease inhibitors on yield of HSV-1-based viral vectors.

The ability to obtain high titer replication-defective herpes simplex virus (HSV) recombinant vectors will dramatically affect their use in gene therapy clinical trials. A variety of techniques and reagents have been employed to increase the overall yield of the vector. The effects of protease inhibitors on the yield of an HSV-1-based viral vector were examined. Experiments were conducted using a commercial protease inhibitor cocktail typically used in mammalian cell culture for protein production. Contrary to our expectation for enhanced vector yield, the results showed a dramatic reduction in vector yield. Moreover, it was found that AEBSF is the only component in the protease cocktail responsible for the low vector yield. On the basis of our hypothesis regarding the mode of action of AEBSF, we suggest that it should not be included in protease inhibitor cocktails designed for use in cultures aimed at production of viral vectors derived from HSV-1 or possibly several other vectors.

Culture Media↗
PubMedDate not supplied

Screening of novel excipients for improving the stability of retroviral and adenoviral vectors.

In the past decade there has been an increase in the application of viral vectors in the laboratory and clinical trials of human gene therapy, retroviral and adenoviral vectors among the most used. However, the limited stability of these vectors creates problems in the design of experiments, transport, and storage. Vectors stored at -80 degrees C must be quickly shipped on dry ice, which is somewhat cumbersome. Alternatively, viral vectors can be preserved in a lyophilized form. However, loss of viral activity during lyophilization can also be a serious problem. In this report we identify novel candidate formulations containing new compatible solutes, ectoin, hydroxyectoin, and firoin, that allow better stability of retroviral and adenoviral vectors during storage. For retroviral vectors, the maximum stabilization for long-term storage was achieved through lyophilization followed by storage at -20 degrees C using a formulation of Tris buffer pH 7.2 containing firoin (0.5 M), a half-life of 340 days being obtained. Adenoviral vectors storage at -80 degrees C in solution using Tris buffer pH 8.0 with firoin was the best method for long-term storage, with a half-life exceeding 1 year.

Adenoviridae↗
PubMed1999

A mini binary vector series for plant transformation.

A streamlined mini binary vector was constructed that is less than 1/2 the size of the pBIN19 backbone (3.5 kb). This was accomplished by eliminating over 5 kb of non-T-DNA sequences from the pBIN19 vector. The vector still retains all the essential elements required for a binary vector. These include a RK2 replication origin, the nptIII gene conferring kanamycin resistance in bacteria, both the right and left T-DNA borders, and a multiple cloning site (MCS) in between the T-DNA borders to facilitate cloning. Due to the reduced size, more unique restriction sites are available in the MCS, thus allowing more versatile cloning. Since the traF region was not included, it is not possible to mobilize this binary vector into Agrobacterium by triparental mating. This problem can be easily resolved by direct transformation. The mini binary vector has been demonstrated to successfully transform Arabidopsis plants. Based on this mini binary vector, a series of binary vectors were constructed for plant transformation.

Agrobacterium tumefaciens↗
PubMed2002

Expression vectors for studying cytoskeletal proteins in Dictyostelium discoideum.

We generated and tested a set of cloning vectors designed to facilitate the production, purification and visualization of proteins in Dictyostelium discoideum. The vectors are derived from the Dictyostelium-E. coli shuttle vector pDXA-3H (6.1 kb), which carries the origin of replication of the Dd high-copy-number plasmid, Ddp2, a high-copy-number E. coli plasmid origin of replication, an act6 promoter driven G418 resistance cassette, the bacterial ampicillin resistance gene and an expression cassette. The new cloning vectors carry expression cassettes consisting of the strong constitutive actin-15 promoter, a translation start followed by a multiple cloning site, sequences for the addition of purification or visualization tags, and Dictyostelium polyadenylation and termination signals. Vectors designed to facilitate protein visualization in living Dictyostelium cells contain either coding sequences for the cyan (CFP) or yellow (YFP) variants of green fluorescent protein (GFP). Versions of the vectors for the production of N- and C-terminal fusions with the fluorescent proteins were generated. To facilitate protein purification, vectors for the production of glutathione-S-transferase (GST) fusion proteins and Strep- or FLAG-affinity-tagged proteins were generated. Additionally, a vector for the production of His8-tagged proteins was generated, which has the G418-resistance cassette replaced by a hygromycin resistance cassette.

Amino Acid Sequence↗
PubMed2004

Expression of a transgene encoded on a non-viral episomal vector is not subject to epigenetic silencing by cytosine methylation.

Currently available vectors for mammalian cells suffer from a number of limitations which make them only partially useful for genetic modification of eukaryotic cells and organisms and for gene therapy. While integration of a vector can lead to unpredictable interactions with the host genome and silencing of the integrated transgene, most non-integrating vectors mediate only transient expression of a transgene. All available vector types can lead to transformation of the recipient cell and many of them can cause serious immunological side effects in the organism. The ideal vector has to be free of these side effects and should allow long-term expression of a transgene in the absence of selection. In this report we describe a novel non-viral episomal expression system fulfilling these criteria. The gene encoding the truncated rat NGF-receptor gene under the control of the CMV-promoter was inserted into a vector construct containing a scaffold/matrix attached region (S/MAR). This vector was then transfected into CHO cells and human HaCat cells. We show that this vector replicates episomally in these cells and is mitotically stable in the abscence of selection over more than 100 generations. Moreover, we provide the first experimental data that the CMV-promoter in an episome is not subject to silencing by cytosine methylation, thus allowing long-term expression of the transgene in the absence of selection.

Animals↗
PubMed2001

The HIV-1 DNA flap stimulates HIV vector-mediated cell transduction in the brain.

During HIV-1 reverse transcription, central initiation of the plus-strand DNA at the central polypurine tract (cPPT) and central termination at the central termination sequence (CTS) lead to the formation of a three-stranded DNA structure: the HIV-1 central DNA flap. We recently reported that the DNA flap acts as a cis-active determinant of HIV-1 genome nuclear import. Commonly employed HIV-1-derived vectors (HR vectors) lack the central DNA flap. Here we report that the insertion of this DNA flap sequence into HR vectors (TRIP vectors) improves gene transduction in neural cells, ex vivo and in vivo, in rat brain. When neural cells are exposed to increasing concentrations of TRIP vector particles, transgene expression correlates with the dose of vector. This effect contrasts with the plateau observed when using an HR vector. We further demonstrate that the increase of in vivo transduction efficiency obtained with TRIP vectors is due to the stimulation of their genome nuclear import.

Adult↗
PubMed2005

Cardiac expression profiles of the naked DNA vectors encoding vascular endothelial growth factor and basic fibroblast growth factor.

We investigated expression profiles and biological effects of the naked DNA vectors in the heart. To this end, naked DNA vector was injected into the apex of the beating rat heart after thorocotomy. When the expression of LacZ reporter was examined by reverse transcription-PCR and histochemical staining for beta-galactosidase, LacZ expression was detected only in the heart, suggesting limited dissemination of the injected vector in vivo. Even within the heart, LacZ expression was limited to the injection area (apex). Similar observations were made with other transgenes such as VEGF and basic fibroblast growth factor (bFGF), where 77% and 69% of the total transgene exprssion were detected in the heart segments containing the apex. Although VEGF and bFGF expressions were detected until 2 weeks after DNA injection, the highest levels of VEGF and bFGF were observed on day 5 and day 1, respectively. The optimal doses of the vectors were 10 microg and 25 microg for the VEGF and bFGF vectors, respectively. Interestingly, injection of bFGF vector led to 50% increase in the level of endogenous murine VEGF expression. Consistent with this finding, the number of vessels that stained positive for alpha-smooth muscle actin was increased in the bFGF vector-injected heart. These results suggest that simple injection of naked DNA vector may be sufficient to induce significant angiogenesis in the myocardium and that naked DNA gene therapy may be a feasible approach for the treatment of ischemic heart disease.

Animals↗