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At least 649 records · Page 36Linked to original sources
PubMed2004

Targeting specific neuronal populations using adeno- and lentiviral vectors: applications for imaging and studies of cell function.

We employ viral vectors to address questions related to the function of specific types of neurones in the central control of blood pressure. Adenoviral vectors (AVVs) or lentiviral vectors (LVVs) can be used to visualize specifically living GABAergic or noradrenergic (NAergic) neurones or to interfere with intracellular signalling within these cell types. Here, we review recent in vitro, in situ and in vivo applications of these vectors in the rat brainstem as performed in our laboratories. In organotypic slice cultures prepared from defined cardiovascular brainstem areas, viral vectors were used to study the electrophysiological properties, intracellular signalling and gene expression in selected neuronal phenotypes. In vivo, vectors were microinjected into brainstem nuclei to inhibit specific aspects of cell signalling by expression of dominant negative proteins, for example. Outcomes for cardiovascular control were measured either acutely in situ or chronically in vivo with radio telemetry in freely moving rats. We showed that AVVs and LVVs have distinct properties that need to be considered prior to their application. For example, LVVs can be manufactured very quickly, have no immunogenicity and can be pseudotyped to display higher tropism for neurones than glia. However, comparatively lower production yields of LVVs may limit their use for some types of applications. In contrast, AVVs require a lengthy construction period, are easy to amplify to high yields at moderate cost but may trigger an immune response when used at high titres in vivo. These features make AVVs particularly suitable for in vitro applications. As the two vector types complement each other in several ways we generated a shuttle system that simplifies transfer of transgene cassettes between the backbones of AVVs and LVVs. Thus, AVVs and LVVs are powerful experimental tools that can be used in a variety of experimental designs in vivo, in situ and in vitro.

Adenoviridae↗
PubMed2001

Gene therapy for muscular dystrophies: current status and future prospects.

Since the identification in 1987 of the gene for Duchenne muscular dystrophy (DMD), research on the molecular pathogenesis of muscular dystrophy has progressed extensively. In particular, discovery of the DMD gene product, dystrophin, led to the identification of dystrophin-associated proteins and, subsequently, the recognition of other types of muscular dystrophy caused by the defects in each of the sarcoglycan genes. On the other hand, effective therapy for DMD has not yet been established. Some of the viral vectors, such as adeno-associated virus vectors or lentiviral vector, have been proven to enable the long-term expression of the exogenous gene without overt host immune reactions. However, dystrophin cDNAs are too large (14kb) to be accommodated in these viral vectors. To solve this problem, we and other research groups succeeded in truncating full-length dystrophin cDNA to small dystrophin cDNA (4 to 5kb), the products of which protect dystrophin-deficient mdx muscle from contraction-induced membrane damage when introduced by viral vectors or as a transgene into mdx mice. The usefulness of these truncated dystrophin cDNAs should be confirmed using other animal models such as dystrophic dogs. To develop successful treatment of DMD, the authors believe that several different approaches should be used, such as cell transfer therapy, drug design to up-regulate utrophin, or a strategy to repair the mutation in vivo.

Animals↗
PubMed2001

Therapeutic effects of viral vector-mediated antiangiogenic gene transfer in malignant ascites.

Malignant ascites is a common complication of advanced intraabdominal neoplasms for which standard treatments are suboptimal. Evidence suggests that tumor-mediated angiogenesis and enhanced vascular permeability in the peritoneal wall due to high levels of vascular endothelial growth factor play a fundamental role in the pathogenesis of malignant ascites. To explore the advantage of viral vector-mediated "targeted antiangiogenic therapy" in ascites formation, we constructed and administered adenoviral vectors encoding several different antiangiogenic proteins (angiostatin, endostatin, platelet factor 4, and a fusion protein between angiostatin and endostatin) alone or in combination intraperitoneally in mice with peritoneal carcinomatosis from breast cancer (TA3 cells) and ovarian cancer (SKOV-3 i.p. and ES-2 cell lines) to explore the potential of additive or synergistic activity. Our data demonstrated statistically significant downregulation of ascites formation, tumor growth, vascularity, and prolongation of animal survival after intraperitoneal treatment with antiangiogenic adenoviral vectors in three different ascites tumor models. Combined treatment proved to be more effective than treatment with one vector alone. Reduced ascites formation was accompanied by decreased microvascular density in the peritoneal wall and increased apoptosis of tumor cells after administration of antiangiogenic vectors in vivo. Of interest was the observation that AdPF4 caused a significant decrease in the level of VEGF secreted by tumor cells both in vitro and in TA3 ascites tumor-bearing animals in vivo. These data suggest that adenoviral vector-mediated delivery of genes encoding antiangiogenic proteins may represent a potentially new treatment modality for malignant ascites.

Adenoviridae↗
PubMed2003

Suppression of tumor growth by viral vector-mediated gene transfer of N-terminal truncated platelet factor 4.

Platelet factor four (PF4), an inhibitor of endothelial cell proliferation in vitro, inhibits angiogenesis and tumor growth in vivo in experimental animals. The present study was designed to determine whether gene therapy-mediated expression of a form of PF4 lacking 16 amino acids of N-terminus from tumor cells could inhibit angiogenesis and tumor growth in vivo. Two replication-defective recombinant retroviral vectors were constructed. One encodes human PF4 (rRV-PF4) and the other encodes the N-truncated peptide (rRVp17-70). These vectors were then used to transduce KB cells, a human head and neck squamous carcinoma cell line. Expression of PF4 and p17-70 transgenes was confirmed by Western blot analysis. In vitro, both rRV-PF4 and rRVp17-70 were able to inhibit selectively the proliferation of human umbilical vascular endothelial cells (HUVEC) but not KB cells. In vivo activity was assessed by injecting 10(7) KB cells subcutaneously into nude mice and by monitoring subsequent tumor growth, xenograft vascular histochemistry, and animal survival. Viral vector-mediated cDNA transfer of PF4 and p17-70 resulted in inhibiting solid tumors through an anti-angiogenic action in vivo. Our data indicate that targeting tumor angiogenesis using viral-mediated gene transfer of full-length and N-terminal truncated PF4 represents a promising strategy for cancer gene therapy.

Animals↗
PubMed2000

Adeno-associated viral vector-mediated vascular endothelial growth factor gene transfer induces neovascular formation in ischemic heart.

Vascular endothelial growth factor (VEGF) plays important roles in physiological and pathological angiogenesis. Recent studies have demonstrated that direct injection of VEGF protein, plasmid DNA, or an adenoviral vector encoding the VEGF gene into ischemic myocardium or limb can induce collateral blood vessel formation and improve perfusion of the ischemic areas. However, these approaches have limitations ranging from a short-lasting effect to angioma formation. In this study, we investigated the feasibility of using adeno-associated viral (AAV) vectors to deliver VEGF genes to mouse myocardium. A cytomegalovirus promoter was used to drive genes for a human VEGF isoform, VEGF(165), and LacZ. A mouse myocardial ischemic model was generated by ligation of the anterior descending coronary artery. Approximately 10(11) copies of the AAV-VEGF vector mixed with 10(10) copies of AAV-LacZ were injected to one site of normal myocardium and a total of 10(11) copies of AAV-VEGF were injected to multiple sites of myocardium around the ischemic region. LacZ gene expression was observed up to 3 months after the vector inoculation. After AAV-VEGF inoculation, neoangiogenesis was observed in the ischemic heart model but not in normal heart tissue. An inflammatory-cell infiltration was not observed in the AAV-VEGF- and AAV-LacZ-inoculated hearts, and angioma-like structure was not observed. These results indicated that injection of the AAV vector directly to myocardium could mediate efficient gene transfer and transgene expression and that VEGF gene delivered by AAV vector can induce angiogenesis in ischemic myocardium.

Animals↗
PubMed2005

Toxicogenomics of cationic lipid-based vectors for gene therapy: impact of microarray technology.

Implementation of the high-throughput microarray gene expression profiling technology towards "toxicogenomics" has advanced identification process for safer drugs in the century of 'omics' technology. Applying such technology, in fact, to identify mechanisms for cellular toxicity can provide a means to clarify safety liabilities early in the drug discovery and developments process. The underlying principle in gene therapy is primarily targeting a specific gene (e.g., for silencing). Hence, massive efforts have been devoted to validate the gene-based therapeutics, regardless of toxicogenomics potential of delivery systems. Of the gene delivery systems, viral and non-viral vectors, as two main paradigms, have so far been widely used for delivering of the genome-based therapeutics such as oligonucleotide, small interfering RNA and DNA. However, the use of viral vectors was narrowed due to the safety concerns. Non-viral vectors were utilized as safer alternatives for gene delivery in vitro and ex-vivo; though their success for in vivo gene therapy has been limited due to low efficiency and safety issues. Fundamental principle for gene therapy is to deliver gene-based therapeutics into target cells for specific gene targeting ideally with minimal cellular toxicity. Until now, few works have been conducted about geno-compatibility of delivery systems itself, including cationic lipid-based nanosystems. Inadvertent toxicogenomic impact of gene delivery systems (e.g., cationic lipids) may intrinsically affect the outcome of gene therapy, where often only a single desired genetic change is sought. Further, there exists a possibility that gene changes induced by the lipid delivery system itself could exacerbate, attenuate or even mask the desired effects of the gene-based therapeutics. This review will focus on toxicogenomics impact of the cationic lipid-based formulations for gene therapy.

Animals↗
PubMed2003

Coupling of importin beta binding peptide on plasmid DNA: transfection efficiency is increased by modification of lipoplex's physico-chemical properties.

BACKGROUND: Non-viral vectors for gene transfer are less immunogenic than viral vectors but also less efficient. Significant effort has focused on enhancing non-viral gene transfer efficiency by increasing nuclear import of plasmid DNA, particularly by coupling nuclear localization peptidic sequences to plasmid DNA. RESULTS: We have coupled a 62-aminoacid peptide derived from hSRP1alpha importin beta binding domain, called the IBB peptide to plasmid DNA by using the heterobifunctional linker N-(4-azido-2,3,5,6 tetrafluorobenzyl)-6-maleimidyl hexanamide (TFPAM-6). When covalently coupled to plasmid DNA, IBB peptide did not increase the efficiency of cationic lipid mediated transfection. The IBB peptide was still able to interact with its nuclear import receptor, importin beta, but non-specifically. However, we observed a 20-fold increase in reporter gene expression with plasmid DNA / IBB peptide complexes under conditions of inefficient transfection. In which case, IBB was associated with plasmid DNA through self assembling ionic interaction. CONCLUSIONS: The improvement of transfection activity was not due to an improved nuclear import of DNA, but rather by the modification of physicochemical properties of IBB peptide / plasmid complexes. IBB peptide increased lipoplex size and these larger complexes were more efficient for gene transfer.

Active Transport, Cell Nucleus↗
PubMed2002

Adenoassociated virus vectors for genetic immunization.

Genetic immunization has initiated a new era of vaccine research, which provides a stable and long-lived source of the protein antigen. Such a vaccine is a simple, robust, and effective means of eliciting both antibody- and cell-mediated immune responses compared with protein or peptide vaccines. Although naked DNA vaccines are relatively simple to produce and handle without significant toxicity or host immunity, those using viral vectors have shown greater efficacy in gene transfer and in inducing both protective and therapeutic immunity in preclinical models. However, clinical translation of results obtained in animal studies with viral vectors has not met with anticipated success so far because of inherent limitations of the vector-associated immunity and antigen specificity. Thus, understanding the requirements for the elicitation of target-specific immunity in host requires that a major cellular arm be unraveled, and modifications of the existing viral vectors and testing of newer ones encompass the technological arm of vaccine research. In this article, I give a comprehensive account of the potential of adenoassociated virus, a nonpathogenic human parvovirus in vaccine development.

Animals↗
PubMed2002

Prospects for cationic polymers in gene and oligonucleotide therapy against cancer.

Gene and antisense/ribozyme therapy possesses tremendous potential for the successful treatment of genetically based diseases, such as cancer. Several cancer gene therapy strategies have already been realized in vitro, as well as in vivo. A few have even reached the stage of clinical trials, most of them phase I, while some antisense strategies have advanced to phase II and III studies. Despite this progress, a major problem in exploiting the full potential of cancer gene therapy is the lack of a safe and efficient delivery system for nucleic acids. As viral vectors possess toxicity and immunogenicity, non-viral strategies are becoming more and more attractive. They demonstrate adequate safety profiles, but their rather low transfection efficiency remains a major drawback. This review will introduce the most important cationic polymers used as non-viral vectors for gene and oligonucleotide delivery and will summarize strategies for the targeting of these agents to cancer tissues. Since the low efficiency of this group of vectors can be attributed to specific systemic and subcellular obstacles, these hurdles, as well as strategies to circumvent them, will be discussed. Local delivery approaches of vector/DNA complexes will be summarized and an overview of the principles of anticancer gene and antisense/ribozyme therapy as well as an outline of ongoing clinical trials will be presented.

Animals↗
PubMed2001

Preexisting immunity to poliovirus does not impair the efficacy of recombinant poliovirus vaccine vectors.

Recombinant viruses are attractive candidates for the development of novel vaccines. A number of viruses have been engineered as vaccine vectors to express antigens from other pathogens or tumors. Inoculation of susceptible animals with this type of recombinant virus results in the induction of both humoral and cellular immune responses directed against the foreign antigens. A general problem to this approach is that existing immunity to the vector can diminish or completely abolish the efficacy of the viral vector. In this study, we investigated whether poliovirus recombinants are capable of inducing effective immunity to the foreign antigen in previously vaccinated animals. Antipoliovirus immunity was induced in susceptible mice by intraperitoneal immunization with live poliovirus. Immunized mice developed antibodies directed against capsid proteins that effectively neutralized poliovirus in vitro and protected animals from a lethal challenge with a high dose of pathogenic poliovirus. To test whether preexisting immunity reduces the efficacy of vaccination with recombinant poliovirus, immunized mice were inoculated with a recombinant poliovirus expressing the C-terminal half of chicken ovalbumin (Polio-Ova). Animals developed ovalbumin-specific antibodies and cytotoxic T lymphocytes (CTL). While the antibody titers observed in preimmune and naive mice were similar, the overall CTL response appeared to be reduced in preimmune mice. Importantly, vaccination with Polio-Ova was able to effectively protect preimmune mice against lethal challenge with a tumor expressing the antigen. Thus, preexisting immunity to poliovirus does not compromise seriously the efficacy of replication-competent poliovirus vaccine vectors. These results contrast with those observed for other viral vaccine vectors and suggest that preexisting immunity does not equally affect the vaccine potential of individual viral vectors.

Animals↗
PubMed1997

Improved adenoviral vectors for gene therapy of Duchenne muscular dystrophy.

We have been exploring the feasibility of gene therapy for Duchenne muscular dystrophy by characterizing parameters important for the design of therapeutic protocols. These studies have used transgenic mice to analyze expression patterns of multiple dystrophin vectors, and have been accompanied by the development of viral vectors for gene transfer to dystrophic mdx mouse muscle. Analysis of transgenic mdx mice indicates that greater than 50% of the fibers in a muscle group must express dystrophin to prevent development of a significant dystrophy, and that low-level expression of truncated dystrophins can function very well. These results suggest that gene therapy of DMD will require methods to transduce the majority of fibers in critical muscle groups with vectors that express moderate levels of dystrophin proteins. Strategies for the development of viral vectors able to deliver dystrophin genes to muscle include the use of muscle specific regulatory sequences coupled with deletion of viral gene sequences to limit virus-induced immune rejection of transduced tissues. These strategies should enable production of adenoviral vectors expressing full-length dystrophin proteins in muscle.

Adenoviridae↗
PubMed2004

Viral vector-mediated transduction of a modified thrombospondin-2 cDNA inhibits tumor growth and angiogenesis.

Gene therapy represents a possible alternative to the chronic delivery of recombinant antiangiogenic proteins to cancer patients. We have constructed retroviral and adenoviral vectors that express murine N-terminal fragments of thrombospondin-2 (NfTSP2), a potent endogenous inhibitor of tumor growth and angiogenesis. To test the possibility of anticancer gene therapy using NfTSP2, we tested whether an ex vivo retrovirus-mediated procedure could be used for the treatment of tumors. The treatment of tumor-bearing mice with syngenic immortalized cell lines expressing NfTSP2 led to a tumor volume reduction up to 70% as compared with the controls (P<0.005). In addition, the established tumors were eradicated in 40% of the mice treated with NfTSP2-expressing cells. Furthermore, the intratumoral injection of the NfTSP2-expressing adenoviral vector to the human squamous cell carcinoma in nude mice resulted in a significant reduction of the growth rates and the volumes of the carcinoma (P<0.05). Immunohistochemical staining of the tumors indicated that the total area and the average size of tumor vessels were significantly reduced in the treatment group versus the controls (P<0.05). In conclusion, the present study clearly demonstrates that the viral vector-mediated transfer of the NfTSP2 gene could inhibit the growth of tumors by perturbing tumor-associated angiogenesis.

Adenoviridae↗
PubMed2005

12th annual congress of the European Society of Gene Therapy.

The 2004 European Society of Gene Therapy (ESGT) meeting took place at Tampere Hall in Finland and highlighted advances in a variety of topics, including cancer, zinc-fingers, stem cells, small interfering RNA (siRNA), microRNA, and recent developments of non-viral and viral vectors. This meeting was attended by 513 participants from 32 countries, and included 106 oral and 224 poster presentations. One of the aims of this meeting was to take a critical look at gene therapy and the prospects for the future. Se-veral presentations reported on RNA-based technologies, such as siRNA, as potential new classes of therapeutics against a wide range of diseases and for use in expression libraries to identify functional genes involved in biological phenotypes. Critical assessments were made of other aspects of gene therapy, such as genome editing and the use of protein transduction domains (PTDs) in gene- and protein-based therapies, where many researchers have failed to reproduce initial findings reported in the literature. Safety issues related to viral vectors were also important areas of discussion, especially following details released by the UK Gene Therapy Advisory Committee of perhaps the first known case of lentiviral vector-associated oncogenesis. Finally, updates were presented on the clinical development of viral vectors in anticancer therapies with evidence of significant improvements in the mean survival of patients.

Clinical Trials as Topic↗
PubMed2005

Characterisation of systemic dissemination of nonreplicating adenoviral vectors from tumours in local gene delivery.

Systemic virus dissemination is a potential problem during local gene delivery in solid tumours. However, the kinetics and pathways of the dissemination have not been well characterised during the first 24 h after the infusion is started. To this end, we infused adenoviral vectors for luciferase or enhanced green fluorescence protein into three different tumour models in mice. During and/or after the infusion, we determined the amount of adenoviruses in the tumour, blood, and liver, and examined the transgene expression in the liver, lung, blood, and tumour. In addition, we intravenously injected tumour cells expressing luciferase and examined the biodistribution of these cells in the body. We observed transgene expression in the liver and tumour at 24 h after the infusion, but could not detect transgene expression in the blood and lung. The peak concentration of viral vectors in the plasma occurred during the intratumoral infusion. At 10 min after the infusion, few viral vectors remained in the blood and the ratio of copy numbers of adenoviruses between liver and tumour was > 2 in 80% and > or = 10 in 40% of the mice. Most tumour cells injected intravenously accumulated in the lung within the first 24 h. Taken together, these data indicated that systemic virus dissemination occurred mainly during the first 10 min after the intratumoral infusion was started, and that the dissemination was due to infusion-induced convective transport of viral vectors into leaky tumour microvessels.

Adenoviridae↗
PubMed2000

A therapeutic HIV vaccine using coxsackie-HIV recombinants: a possible new strategy.

The ultimate goal in the treatment of HIV-infected persons is to prevent disease progression. A strategy to accomplish this goal is to use chemotherapy to reduce viral load followed by immunotherapy to stimulate HIV-specific immune responses that are observed in long-term asymptomatic individuals. An effective, live, recombinant virus, expressing HIV sequences, would be capable of inducing both CTL and CD4(+) helper T cell responses. To accomplish these goals, the viral vector must be immunogenic yet retain its avirulent phenotype in a T cell-deficient host. We have identified a coxsackievirus variant, CB4-P, that can induce protective immunity against a virulent variant. In addition, the CB4-P variant remains avirulent in mice lacking CD4(+) helper T cells, suggesting that CB4-P may be uniquely suited as a viral vector for a therapeutic HIV vaccine. Two strategies designed to elicit CTL and CD4(+) helper T cell responses were used to construct CB4-P/HIV recombinants. Recombinant viruses were viable, genetically stable, and retained the avirulent phenotype of the parental virus. In designing a viral vector for vaccine development, an issue that must be addressed is whether preexisting immunity to the vector would affect subsequent administration of the recombinant virus. Using a test recombinant, we showed that prior exposure to the parental CB4-P virus did not affect the ability of the recombinant to induce a CD4(+) T cell response against the foreign sequence. The results suggest that a "cocktail" of coxsackie/HIV recombinants may be useful as a therapeutic HIV vaccine.

AIDS Vaccines↗
PubMed2006

Long-term adeno-associated viral vector-mediated expression of truncated TrkB in the adult rat facial nucleus results in motor neuron degeneration.

Adult facial motor neurons continue to express full-length TrkB tyrosine kinase receptor (TrkB.FL), the high-affinity receptor for the neurotrophins BDNF and neurotrophic factor-4/5 (NT-4/5), suggesting that they remain dependent on target-derived and locally produced neurotrophins in adulthood. Studies on the role of TrkB signaling in the adult CNS have been hampered by the early lethality of bdnf, nt-4/5, and trkB knock-out mice. We disrupted TrkB.FL signaling in adult facial motor neurons using adeno-associated viral vector-mediated overexpression of a naturally occurring dominant-negative TrkB receptor, TrkB.T1. Expression of TrkB.T1 resulted in neuronal atrophy and downregulation of NeuN (neuronal-specific nuclear protein) and ChAT expression in facial motor neurons. A subset of transduced neurons displayed signs of motor neuron degeneration that included dendritic beading and rounding of the soma at 2 months of TrkB.T1 expression. Cell counts revealed a significant reduction in motor neuron number in the facial nucleus at 4 months after onset of expression of TrkB.T1, suggesting that a proportion of TrkB.T1-expressing motor neurons became undetectable as a result of severe atrophy or was lost because of cell death. In contrast, overexpression of TrkB.FL did not result in a decrease in facial motor neuron number. Our results indicate that a subset of facial motor neurons remains dependent on TrkB ligands for the maintenance of structural and molecular characteristics in adulthood.

Animals↗
PubMed2000

Folate-targeted non-viral DNA vectors for cancer gene therapy.

The vitamin folic acid exhibits high affinity for the endocytosed, membrane-bound folate receptor. Conjugation of folic acid via its gamma-carboxyl group to various macromolecules results in binding to, and endocytosis into, cells expressing the folate receptor. The folate receptor is overexpressed on a wide range of cancers, therefore folic acid has been investigated as a targeting ligand for the specific delivery of therapeutic compounds to cancer cells. This review will introduce folate-targeting of macromolecules to cancer cells in vitro and in vivo, and discuss the accumulation of such compounds in non-target tissues in vivo. Folate-targeting of non-viral DNA vectors in vitro and in vivo will be discussed in detail, with particular emphasis on the recent advances in this field.

Animals↗
PubMed2002

Tumor antigen-specific induction of transcriptionally targeted retroviral vectors from chimeric immune receptor-modified T cells.

High-level systemic delivery of viral vectors to tumors has proved problematic as a result of immune neutralization, nonspecific adhesion, and clearance of circulating viral particles. Some cell types localize to tumors in response to particular biological properties associated with tumor growth. Their use to deliver viral vectors to tumors would allow precious viral stocks to be protected until they can be released at high local concentrations. Here, we describe a mechanism by which retroviral vector production by T cells can be regulated by a tumor-specific trigger through engagement of a chimeric immune receptor (CIR) with its target antigen. The virus that is released from the T cells can also be transcriptionally targeted. Finally, we show that it is possible to use vector-loaded, antigen-triggered human T cells as therapeutic, tumor-specific vector delivery cells in models of both local intratumoral and systemic delivery to both lung and liver metastases. This strategy incorporates multiple levels of targeting into the delivery system at the stages of surface targeting, viral production, and gene expression.

Adenoviridae↗