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At least 1,081 records · Page 60Linked to original sources
PubMed2006

HIV vaccines: new frontiers in vaccine development.

A human immunodeficiency virus (HIV) vaccine is the most promising and feasible strategy to prevent the events during acute infection that simultaneously set the course of the epidemic in the community and the course of the disease for the individual. Because safety concerns limit the use of live, attenuated HIV and inactivated HIV, a variety of alternate approaches is being investigated. Traditional antibody-mediated approaches using recombinant HIV envelope proteins have shown no efficacy in 2 phase III trials. Current HIV vaccine trials are focusing primarily on cytotoxic T lymphocyte-mediated products that use viral vectors, either alone or as boosts to DNA plasmids that contain viral genes. The most immunogenic of these products appear to be the recombinant adenovirus vector vaccines, 2 of which are now in advanced clinical development.

AIDS Vaccines↗
PubMed2006

Canine adenovirus vectors for lung-directed gene transfer: efficacy, immune response, and duration of transgene expression using helper-dependent vectors.

A major hurdle to the successful clinical use of some viral vectors relates to the innate, adaptive, and memory immune responses that limit the efficiency and duration of transgene expression. Some of these drawbacks may be circumvented by using vectors derived from nonhuman viruses such as canine adenovirus type 2 (CAV-2). Here, we evaluated the potential of CAV-2 vectors for gene transfer to the respiratory tract. We found that CAV-2 transduction was efficient in vivo in the mouse respiratory tract, and ex vivo in well-differentiated human pulmonary epithelia. Notably, the in vivo and ex vivo efficiency was poorly inhibited by sera from mice immunized with a human adenovirus type 5 (HAd5, a ubiquitous human pathogen) vector or by human sera containing HAd5 neutralizing antibodies. Following intranasal instillation in mice, CAV-2 vectors also led to a lower level of inflammatory cytokine secretion and cellular infiltration compared to HAd5 vectors. Moreover, CAV-2 transduction efficiency was increased in vitro in human pulmonary cells and in vivo in the mouse respiratory tract by FK228, a histone deacetylase inhibitor. Finally, by using a helper-dependent CAV-2 vector, we increased the in vivo duration of transgene expression to at least 3 months in immunocompetent mice without immunosuppression. Our data suggest that CAV-2 vectors may be efficient and safe tools for long-term clinical gene transfer to the respiratory tract.

Adenoviruses, Canine↗
PubMed2006

Optimizing cancer immunotherapy trials: back to basics.

Attempts to raise effective immunity against cancer are benefiting from information on the nature of the immunity involved and its regulation and, perhaps, now it is time to step back and define our approach in molecular terms prior to clinical testing. Although there are immunological differences between mice and patients, results from murine studies are encouraging early 'translation' of concepts to the clinic and it is vital to take immunological principles emerging from mice into clinical vaccine design. One is the requirement to break tolerance against over-expressed self-antigens, a potentially risky procedure but necessary for several cancer targets. A study in this issue of the European Journal of Immunology attempts to do this by using xenogeneic antigens, albeit with variable outcome. The unstated goal is to activate T-cell help but this can be achieved more effectively by harnessing a predictable anti-microbial repertoire. The second issue lies in the delivery of antigen. One strategy is "prime/boost" using DNA priming and boosting with a viral vector; however, this induces blocking immunity against viral proteins, and must be used judiciously. There are other physical methods to increase immunity such as electroporation, which can itself be used in 'prime/boost' sequence. These twin problems of engagement of T-cell help and delivery of adequate antigen can now be addressed by applying immunological logic to cancer vaccines.

Antigens, Neoplasm↗
PubMed2005

Comparison between recombinant baculo- and adenoviral-vectors as transfer system in cardiovascular cells.

The development of effective gene-therapeutic applications for cardiovascular disorders is in part limited by the lack of appropriate delivery systems. In an attempt to overcome this deficiency, we investigated the ability of baculoviral vectors to transduce human cardiovascular cells, for which data are missing in literature. Additionally, baculovirus ability to transduce target cells was compared to that of an adenoviral vector, a well characterized and widely used viral vector. Transduction experiments, performed using baculo/adenoviral vectors expressing the enhanced green fluorescence protein, revealed that, under the experimental condition considered, baculoviruses but not adenoviruses efficiently transduce human coronary smooth muscle cells (hCSMC); an opposite behavior was noticed for human coronary endothelial cells (hCEC). Thus, baculoviral vectors are potentially indicated as transfer system in the treatment of coronary restenosis, where growth inhibitory genes should reach hCSMC but not hCEC. When used to transduce human cardiomyocytes and fibroblasts, both vectors behaved similarly. Finally, studies on cellular DNA replication revealed a more prolonged and pronounced negative effect on cells transduced by adenoviral compared to baculoviral vectors. Our data indicate that baculoviruses represent an attractive alternative to adenoviruses as transfer vectors in cardiovascular cells and that baculovirus have the potential to be used as gene transfer system in cardiovascular diseases such as restenosis.

Adenoviridae↗
PubMed2007

Controlled delivery of glial cell line-derived neurotrophic factor by a single tetracycline-inducible AAV vector.

An autoregulated tetracycline-inducible recombinant adeno-associated viral vector (rAAV-pTet(bidi)ON) utilizing the rtTAM2 reverse tetracycline transactivator (rAAV-rtTAM2) was used to conditionally express the human GDNF cDNA. Doxycycline, a tetracycline analog, induced a time- and dose-dependent release of GDNF in vitro in human glioma cells infected with rAAV-rtTAM2 serotype 2 virus. Introducing the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) downstream to the rtTAM2 coding sequence, resulted in a more rapid induction and a higher basal expression level. In vivo, 8 weeks after a single injection of the rAAV-rtTAM2-GDNF vector encapsidated into AAV serotype 1 capsids in the rat striatum, the GDNF protein level was 60 pg/mg tissue in doxycycline-treated animals whereas in untreated animals, it was undistinguishable from the endogenous level ( approximately 4 pg/mg tissue). However, a residual GDNF expression in the uninduced animals was evidenced by a sensitive immunohistochemical staining. As compared to rAAV1-rtTAM2-GDNF, the rAAV1-rtTAM2-WPRE-GDNF vector expressed a similar concentration of GDNF in the induced state (with doxycycline) but a basal level (without doxycycline) approximately 2.5-fold higher than the endogenous striatal level. As a proof for biological activity, for both vectors, downregulation of tyrosine hydroxylase was evidenced in dopaminergic terminals of doxycycline-treated but not untreated animals. In conclusion, the rAAV1-rtTAM2 vector which expressed biologically relevant doses of GDNF in the striatum in response to doxycycline with a basal level undistinguishable from the endogenous striatal level, as measured by quantitative ELISA assay, constitutes an interesting tool for local conditional transgenesis.

Animals↗
PubMed2004

Xenograft models for liver metastasis: Relationship between tumor morphology and adenovirus vector transduction.

The improvement of initial tumor cell transduction with viral vectors is a major task in tumor gene therapy. We have developed mouse tumor models with hepatic metastases to study transduction of tumor cells after systemic adenovirus vector application. The tumor models were established by intraportal transplantation of human tumor cell lines into immunodeficient mice. Liver metastases derived from cervix, colon, breast, and liver cancer lines were analyzed for distribution of extracellular matrix, vascularization, and transgene expression after tail vein injection of adenovirus vectors. Overall, xenografts resembled the morphology of corresponding tumors in cancer patients. Adenovirus-mediated gene delivery depended on tumor vascularization and direct contact between blood vessels and tumor cells. These models represent important tools for studying and improving tumor gene therapy approaches.

Adenoviridae↗
PubMed2003

HVJ-envelope vector for gene transfer into central nervous system.

To overcome some problems of virus vectors, we developed a novel non-viral vector system, the HVJ-envelope vector (HVJ-E). In this study, we investigated the feasibility of gene transfer into the CNS using the HVJ-E both in vitro and in vivo. Using the Venus reporter gene, fluorescence could be detected in cultured rat cerebral cortex neurons and glial cells. In vivo, the reporter gene (Venus) was successfully transfected into the rat brain by direct injection into the thalamus, intraventricular injection, or intrathecal injection, without inducing immunological change. When the vector was injected after transient occlusion of the middle cerebral artery, fluorescence due to EGFP gene or luciferase activity could be detected only in the injured hemisphere. Finally, luciferase activity was markedly enhanced by the addition of 50 U/ml heparin (P<0.01). Development of efficient HVJ-E for gene transfer into the CNS will be useful for research and clinical gene therapy.

Animals↗
PubMed2003

Oncolytic viruses: clinical applications as vectors for the treatment of malignant gliomas.

Gene therapy using viral vectors for the treatment of primary brain tumors has proven to be a promising novel treatment modality. Much effort in the past has been placed in utilizing replication-defective viruses to this end but they have shown many disadvantages. Much recent attention has been focused on the potential of replication-competent viruses to discriminatingly target, replicate within, and destroy tumor cells via oncolysis, leaving adjacent post-mitotic neurons unharmed. The engineered tumor-selective herpes simplex-1 virus (HSV-1) mutants G207 and HSV1716 have completed Phase I investigations in the treatment of recurrent high-grade glioma. The results of these clinical trials are reviewed here. This review also aims to examine the manipulation and development of other viruses for the treatment of malignant glioma, including Newcastle disease virus, reovirus, poliovirus, vaccinia virus, and adenoviruses, in particular the adenovirus mutant ONYX-015.

Adenoviridae↗
PubMed2005

Detection of plasmid DNA vectors following gene transfer to the murine airways.

Non-viral gene therapy is being considered as a treatment for cystic fibrosis. In clinical studies and in studies using the mouse airways as a model, current formulations result in only transient transgene expression. A number of reasons for this have been proposed including the loss of plasmid DNA from cells. The aim of these studies was to investigate why transgene expression from non-viral vectors is transient in the mouse lung. Plasmid DNA encoding the luciferase reporter gene was complexed with the cationic lipid GL67 and delivered to the mouse airways. The persistence of plasmid DNA in the mouse lungs was investigated using quantitative PCR and Southern hybridization. Results showed that intact plasmid DNA persisted in the mouse lung in the absence of any detectable luciferase activity. The de novo methylation of plasmid DNA in vivo was investigated as a potential cause of this transient gene expression but results suggested that plasmid DNA does not become de novo methylated in the mouse lung. Therefore processes other than the loss of plasmid DNA from the lung or the de novo methylation of plasmid DNA vectors must be responsible for the transient transgene expression.

Adenoviridae↗
PubMed2004

Plant sterol biosynthesis: identification of two distinct families of sterol 4alpha-methyl oxidases.

In plants, the conversion of cycloartenol into functional phytosterols requires the removal of the two methyl groups at C-4 by an enzymic complex including a sterol 4alpha-methyl oxidase (SMO). We report the cloning of candidate genes for SMOs in Arabidopsis thaliana, belonging to two distinct families termed SMO1 and SMO2 and containing three and two isoforms respectively. SMO1 and SMO2 shared low sequence identity with each other and were orthologous to the ERG25 gene from Saccharomyces cerevisiae which encodes the SMO. The plant SMO amino acid sequences possess all the three histidine-rich motifs (HX3H, HX2HH and HX2HH), characteristic of the small family of membrane-bound non-haem iron oxygenases that are involved in lipid oxidation. To elucidate the precise functions of SMO1 and SMO2 gene families, we have reduced their expression by using a VIGS (virus-induced gene silencing) approach in Nicotiana benthamiana. SMO1 and SMO2 cDNA fragments were inserted into a viral vector and N. benthamiana inoculated with the viral transcripts. After silencing with SMO1, a substantial accumulation of 4,4-dimethyl-9beta,19-cyclopropylsterols (i.e. 24-methylenecycloartanol) was obtained, whereas qualitative and quantitative levels of 4alpha-methylsterols were not affected. In the case of silencing with SMO2, a large accumulation of 4alpha-methyl-Delta7-sterols (i.e. 24-ethylidenelophenol and 24-ethyllophenol) was found, with no change in the levels of 4,4-dimethylsterols. These clear and distinct biochemical phenotypes demonstrate that, in contrast with animals and fungi, in photosynthetic eukaryotes, these two novel families of cDNAs are coding two distinct types of C-4-methylsterol oxidases controlling the level of 4,4-dimethylsterol and 4alpha-methylsterol precursors respectively.

Arabidopsis↗
PubMed1997

Gene transfer by guanidinium-cholesterol cationic lipids into airway epithelial cells in vitro and in vivo.

Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis. Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo. The results obtained indicate that the lipid bis(guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture. Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo. Transfected cells were detected both in the surface epithelium and in submucosal glands. In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system.

Adenoviridae↗
PubMed2003

Nucleic acid-matrix attachment recognition regions--as facilitators in plasmid transfer.

Non-viral gene transfer is an alternative to viral vectors for gene transfer. However, non-viral transgene expression remains undesirably low and transient. Matrix attachment regions (MARs) are DNA elements that are defined by their high affinity for the nuclear matrix. MARs may also be related to long-term transgene expression in vitro. The purpose of this research is to evaluate human interferon-beta MARs element in various cell types. This was accomplished by constructing MARs-containing plasmid DNA (pDNA) and comparing their transgene expression with non-MARs-containing pDNA. We found that MARs-containing pDNA increased and prolonged the expression in Chinese hamster ovary (CHO) cells, but not in human neuroblastoma cells (SKnSH) and neuronal cells (primary neuron, astroglia and microglia). From the cotransfection experiment, MARs-containing pDNA had a trans effect on another pDNA expression. A PCR method was used to monitor the intracellular distribution of pDNA after cellular fractionalization. We found that non-MAR containing pDNA demonstrates similar intracellular distribution as non-MARs-containing pDNA.

Animals↗
PubMed2002

Stability of a recombinant adenoviral vector: optimization of conditions for storage, transport and delivery.

Recombinant viral vectors have been developed for use as therapeutic agents and for the introduction of exogenous genes into living cells. However, little is known about the viability and stability of such recombinant viruses during storage, transport and delivery under various conditions. We describe here an analysis of the stability of an adenoviral vector in crude solutions of cell lysates during freezing and thawing and during storage at various temperatures in the presence and in the absence of glycerol. For example, the titer of adenoviruses in crude lysates of infected cells was reduced only ten-fold or three-fold after two hundred rounds of freezing and thawing or after incubation at 28 degrees C for 14 days, respectively. Our observations indicate that recombinant adenoviral vector was more stable than expected both during freezing and thawing and during storage at low temperatures. Our results confirm the importance of appropriate conditions for the delivery and transport of recombinant adenoviral vectors.

Adenoviridae↗
PubMed2002

Retroviral-mediated IL-12 gene transduction into human CD34+ cell-derived dendritic cells.

Genetically modified dendritic cells (DCs) with Th1 type cytokine genes are useful for activating anti-tumor immune response. We made human interleukin (IL)-12 p70 gene-transduced DCs generated from CD34+ progenitor cells using a retrovirus system and investigated the function of IL-12-producing DCs. We used the pMX retroviral vector and made cytokine gene-containing viral vectors referred to as GFP pMX and hIL-12 pMX. Supernatants from BOSC23 cells transfected with GFP pMX and hIL-12 pMX were harvested and used for transfection of DC. Cord blood CD34+ cells were incubated with supernatants containing retrovirus for 48 h with cytokines such as IL-3, IL-6, SCF, Flt3 ligand (FL), bFGF and IGF-I. The cells were cultured for 12 days in the presence of GM-CSF, SCF, FL, IL-4 and TNF-alpha to get mature DC-enriched population. Analysis of surface marker on DCs and allogeneic MLR assay were also performed. After a 14-day culture, 60-70% of cultured CD34+ cells were DC marker (CD1a, DEC205) positive. The IL-12 p70 protein levels in supernatant of DC-GFP and DC-hIL-12 were 0.2 ng/ml and 53 ng/ml/5 x 10(5) DCs for 72 h, respectively. The addition of CH296 fibronectin fragment (FN) increased 3-fold IL-12 gene transduction efficiency into DCs. MLR assay showed that IL-12-producing DC exhibited more potent T cell growth-stimulating activity compared with GFP-DC. These results suggested that genetically modified CD34+ cell-derived DCs with human IL-12 gene are fully efficient in T cell priming, and could be a good tool for effective cancer immunotherapy.

Antigens, CD1↗
PubMed1997

Lipidic vector systems for gene transfer.

Clinical application of gene therapy depends on the development of suitable gene transfer vehicles (vectors). Although generally not as efficient as viral vectors, nonviral systems such as lipidic vectors have the potential advantages of being less toxic, nonrestrictive in cargo DNA size, potentially targetable, and easy to produce in relatively large amounts. More important, lipidic vectors generally lack immunogenicity, allowing repeated in vivo transfection using the same vector. In this paper, we will attempt to summarize some of the recent advances in lipidic gene delivery vectors. Three types of lipidic gene transfer vectors are described: 1) DNA/cationic liposome complexes, 2) DNA encapsulated in neutral or anionic liposomes, and 3) liposome-entrapped, polycation-condensed DNA (LPDI and LPDII). We review the various factors affecting vector structure and gene delivery efficiency, and we discuss the possible mechanisms of gene transfer and their implications in vector design.

Animals↗
PubMedDate not supplied

Defective herpes simplex virus vectors expressing thymidine kinase for the treatment of malignant glioma.

Viral vectors used for cancer gene therapy have usually been either replication-incompetent vectors expressing a gene product that leads to the destruction of the tumor or replication-competent vectors that are inherently cytotoxic to the tumor cells. We have sought to combine the attributes of these different approaches using a defective herpes simplex virus (HSV) vector that consists of a defective particle, containing tandem repeats of the HSV thymidine kinase (TK) gene, and a replication-competent, non-neurovirulent HSV mutant as a helper virus. HSV-TK activity in defective vector-infected cells was significantly greater than that in helper virus-infected cells which contained a single copy of HSV-TK. Infection of cells with this defective vector renders them, as well as surrounding uninfected cells, sensitive to killing by ganciclovir. Ganciclovir treatment of C57BL/6 mice bearing TK-defective vector/helper virus-infected subcutaneous GL261 gliomas resulted in significantly decreased tumor size.

Animals↗
PubMed2006

Verification of functional AAV-mediated neurotrophic and anti-apoptotic factor expression.

The use of viral vectors for gene delivery offer many advantages for both basic research and therapeutic application through the continuous expression of a gene product within a target region. It is vital however that any gene product is correctly expressed in a biologically active form, and this should be confirmed prior to large scale in vivo studies. Using adeno-associated viral (AAV) vectors to direct the expression of either a neurotrophic factor or an anti-apoptotic protein, we have developed a range of in vitro assays to verify functional transgenic protein expression. Brain-derived neurotropic factor (BDNF) activity was confirmed by demonstrating enhanced generation of GABAergic neurons in embryonic (E15) striatal cultures and AAV-mediated glial-derived neurotrophic factor (GDNF) function using an assay for dopaminergic differentiation of embryonic (E14) ventral mesencephalic cultures. To assess functional anti-apoptotic factor expression we designed cell-survival assays, using embryonic cortical cultures to confirm Bcl-x(L) activity and the HT1080 cell-line for X-linked inhibitor of apoptosis protein (XIAP) activity following AAV-mediated expression. This study demonstrates that the use of functional assays provides valuable confirmation of desired biotherapeutic expression prior to extensive investigation with new gene delivery vectors.

Adenoviridae↗
PubMed2003

Development and characterization of novel empty adenovirus capsids and their impact on cellular gene expression.

Adenovirus (Ad) has been extensively studied as a eukaryotic viral vector. As these vectors have evolved from first-generation vectors to vectors that contain either very few or no viral genes ("gutless" Ad), significant reductions in the host innate immune response upon infection have been observed. Regardless of these vector improvements an unknown amount of toxicity has been associated with the virion structural proteins. Here we demonstrate the ability to generate high particle numbers (10(11) to 10(12)) of Ad empty virions based on a modification of Cre/lox gutless Ad vectors. Using a battery of analyses (electron microscopy, atomic force microscopy, confocal images, and competition assays) we characterized this reagent and determined that it (i) makes intact virion particles, (ii) competes for receptor binding with wild-type Ad, and (iii) enters the cell proficiently, demonstrating an ability to carry out essential steps of viral entry. To further study the biological impact of these Ad empty virions on infected cells, we carried out DNA microarray analysis. Compared to that for recombinant Ad, the number of mRNAs modulated upon infection was significantly reduced but the expression signatures were similar. This reagent provides a valuable tool for studies of Ad in that researchers can examine the effect of infection in the presence of the virion capsid alone.

Adenoviridae↗