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At least 73 records · Page 4Linked to original sources
PubMed1997

Prolonged reduction of high blood pressure with an in vivo, nonpathogenic, adeno-associated viral vector delivery of AT1-R mRNA antisense.

To produce a prolonged decrease in blood pressure, we have developed a nonpathogenic adeno-associated viral vector (AAV) with the antisense DNA for AT1-R. AAV has many advantages over other viral vectors. AAV does not stimulate inflammation or immune reaction. AAV enters nondividing cells and does not replicate. Therefore, it is an appropriate choice for gene therapy. Recombinant AAV was prepared with a cassette containing a cytomegalovirus promoter and the cDNA for the AT1 receptor inserted in the antisense direction. The cassette was packaged in the virion. Stable transfection of NG108-15 cells with the PAAV-AS (plasmid AAV) antisense to AT1-R produced a significant reduction in AT1 receptors. A single injection of the rAAV-AS (viral vector) was made in adult spontaneously hypertensive rats, either directly in the hypothalamus (1 microL) or in the lateral ventricles (5 microL). The result shows that there is a significant decrease of blood pressure (approximately 23 +/- 2 mm Hg) for up to 9 weeks after injection. Control injections of mock vector produced no change in blood pressure during the same time period in age-matched controls. In young spontaneously hypertensive rats (3 weeks), a single intracardiac injection of recombinant rAAV-AS reduced blood pressure and slowed the development of hypertension compared with controls (P < .01). The results suggest that a prolonged reduction in high blood pressure can be achieved with AAV vectors delivering antisense to inhibit AT1 receptors with a single administration.

Animals↗
PubMed2003

Plasmid chemokines and colony-stimulating factors enhance the immunogenicity of DNA priming-viral vector boosting human immunodeficiency virus type 1 vaccines.

Heterologous "prime-boost" regimens that involve priming with plasmid DNA vaccines and boosting with recombinant viral vectors have been shown to elicit potent virus-specific cytotoxic T-lymphocyte responses. Increasing evidence, however, suggests that the utility of recombinant viral vectors in human populations will be significantly limited by preexisting antivector immunity. Here we demonstrate that the coadministration of plasmid chemokines and colony-stimulating factors with plasmid DNA vaccines markedly increases the immunogenicity of DNA prime-recombinant adenovirus serotype 5 (rAd5) boost and DNA prime-recombinant vaccinia virus (rVac) boost vaccine regimens in BALB/c mice. In mice with preexisting anti-Ad5 immunity, priming with the DNA vaccine alone followed by rAd5 boosting elicited only marginal immune responses. In contrast, cytokine-augmented DNA vaccine priming followed by rAd5 vector boosting was able to generate potent immune responses in mice with preexisting anti-Ad5 immunity. These data demonstrate that plasmid cytokines can markedly improve the immunogenicity of DNA prime-viral vector boost vaccine strategies and can partially compensate for antivector immunity.

AIDS Vaccines↗
PubMed2002

In vivo antitumor activity of Sindbis viral vectors.

BACKGROUND: Sindbis virus, a blood-borne virus transmitted by mosquitoes, has been used as a vector to efficiently express exogenous genes in vitro and in vivo and to induce apoptosis. Because Sindbis virus infects mammalian cells by interacting with the high-affinity laminin receptors, which are expressed at higher levels in several human cancers than in normal cells, we determined whether a Sindbis viral vector could be used to target cancers in vivo. METHODS: C.B-17-SCID mice with established xenografts were given daily intraperitoneal injections of the Sindbis viral vector SinRep/LacZ containing the bacterial beta-galactosidase gene. Control mice were untreated or received injections with phosphate-buffered saline. Tumor size was measured daily. Expression of beta-galactosidase and Factor VIII (a marker for endothelial cells) was determined by immunohistochemical staining of tumor sections. Apoptosis was analyzed by TUNEL (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labeling) staining. C.B-17-SCID beige mice, which lack natural killer (NK) cells, were used to assess the importance of NK cells in antitumor efficacy of Sindbis viral vectors. RESULTS: Tumors from mice treated with SinRep/LacZ were statistically significantly smaller than tumors from control mice. This effect was observed for tumor xenografts derived from BHK (kidney, hamster), LS174T (colon, human), HT29 (colon, human), and CFPAC (pancreas, human) cells. Expression of beta-galactosidase co-localized with that of Factor VIII in tumor sections. Tumors from SinRep/LacZ-treated mice contained more apoptotic cells than tumors from control mice. Complete tumor regression was observed in three of five C.B-17-SCID mice but in none of five C.B-17-SCID beige mice treated with SinRep/LacZ. CONCLUSION: Sindbis viral vectors efficiently targeted tumors in vivo, were apparently delivered through the circulation, and were more effective in the presence of NK cells.

Animals↗
PubMed1999

Immune modulation by IL-10 gene transfer via viral vector and plasmid DNA: implication for gene therapy.

In the present report, we have evaluated and compared the modulatory effect of the cytokine interleukin (IL)-10 expressed via viral vector or plasmid DNA on viral antigen-induced cutaneous inflammatory lesions. Our data demonstrate the superior potency of both recombinant vaccinia virus and herpes simplex virus IL-10 expression vectors after single intramuscular administration, but the effects were only short term and only functioned in animals lacking immunity to the viral vectors used for modulation. In contrast, modulatory effects achieved by plasmid DNA expressing IL-10 were delayed in onset and milder in effect but were far more persistent than those achieved by viral vectors. Moreover, plasmid DNA expressing IL-10 provided effective modulation when given repeatedly to animals. Our data also showed that IL-10 gene delivery resulted in a systemic and durable modulatory effect while the effect caused by a single IL-10 protein treatment was transient and confined to the injected site. Our results imply that the viral vector system is superior for obtaining short-term effects, whereas the plasmid DNA approach represents a better strategy to achieve gene therapy to modulate chronic inflammatory lesions.

Animals↗
PubMed2003

Gene delivery by viral vectors in primary cultures of lacrimal gland tissue.

PURPOSE: To test the feasibility of gene transfer into lacrimal gland tissue in primary culture, using different viral vectors. METHODS: Lacrimal glands were dissected from adult Sabra rats and divided by pincers to 0.3-0.4 mm fragments. Tissue was maintained under primary organ culture conditions using the "raft" technique. The ability of three different viral vectors to conduct beta-galactosidase (beta-gal) gene delivery was examined: adenovirus (Ad5CMVLacZ), vaccinia (VSC9), and herpesvirus (tkLTRZ(1)). Tissue fragments were incubated for 60 minutes with one of the viral vectors and transferred to fresh medium. After 3 and 7 days, beta-gal expression was examined by X-gal staining in gross preparations and in histologic sections. RESULTS: At 3 days, beta-gal expression was observed in 33% of tissue fragments exposed to the vaccinia vector and in 18% and 14% of fragments exposed to the adenoviral and herpes vectors, respectively. After 7 days in culture, successful gene delivery occurred in 77% of vaccinia, 41% of adenovirus, and only 13% of herpesvirus applications. Vector-specific reporter gene expression patterns were observed: With the vaccinia vector, lacrimal duct cells were predominantly stained; in contrast, the adenoviral vector tended to transduce the interacinar areas, with beta-gal expression mainly occurring within the myoepithelial cells. CONCLUSIONS: Vaccinia and adenovirus are efficient vectors for gene transfer into lacrimal gland tissue in primary culture. The specific expression pattern obtained by the vaccinia vector probably reflects its characteristic tissue tropism to lacrimal duct cells. The results presented in this ex vivo system may be a first step toward expressing genes with products that could be continuously delivered to the eye through the tears. Such proteins could include anti-inflammatory, anti-angiogenic, anti-herpetic, anti-bacterial, or anti-glaucomatous agents, among others.

Adenoviridae↗
PubMed2006

Evolving gene therapy approaches for osteosarcoma using viral vectors: review.

This review begins with an introduction to the malignant bone tumor, osteosarcoma [OS] and then moves to a discussion of the commonly used vectors for gene transfer. We first briefly highlight non-viral vectors including polymeric and liposomal delivery systems but concentrate predominantly on the 5 leading viral vectors used in cancer gene therapy, specifically retroviruses, adeno-associated viruses, herpes viruses and lentiviruses with the most detailed analysis reserved for adenoviruses. The 3 main strategies for gene therapy in osteosarcoma are next summarized. As part of this review, the several prodrug-converting enzymes utilized in OS suicide gene therapy are examined. The text then turns to a discussion of adenovirus-mediated gene transfer and the need for tumor targeting via transductional or transcriptional approaches. Because of practical problems with use of replication-incompetent viruses in achieving complete tumor kill in vivo, virotherapy utilizing replication competent viruses has come to the fore. This topic is, thus, next reviewed which allows for a natural transition to a discussion of armed therapeutic viruses many of which are conditionally replicating adenoviruses carrying transgenes with established anti-tumor efficacy. We recognize that several other issues have arisen which hamper progress in the field of cancer gene therapy. We, therefore, review viral-induced toxicity in the host and vector delivery issues which have been found to potentially influence safety. We end with a brief perspective including commenting on animal models used in examining delivery strategies for osteosarcoma gene therapy. The challenges remaining are touched upon most especially the need to deal with pulmonary metastatic disease from OS.

Bone Neoplasms↗
PubMed2003

Systemic dissemination of viral vectors during intratumoral injection.

Intratumoral injection is a routine method for local viral gene delivery that may improve interstitial transport of viral vectors in tumor tissues and reduce systemic toxicity. However, the concentration of transgene products in normal organs, such as in the liver, may still exceed normal tissue tolerance if the products are highly toxic. The elevated concentration in normal tissues is likely to be caused by the dissemination of viral vectors from the tumor. Therefore, we investigated transgene expression in the liver, the serum, and a mouse mammary carcinoma (4T1) in mice after intratumoral injection of adenoviral vectors for mouse interleukin-12, luciferase, enhanced green fluorescence protein, or beta-galactosidase. We also performed numerical simulations of virus transport in tumors after intratumoral injection, based on the Krogh cylinder model. Our experimental data and numerical simulations demonstrated that virus dissemination was significant in mice and it occurred mainly during the intratumoral injection. To reduce virus dissemination, we mixed these vectors with a viscous alginate solution and injected the mixture into the tumors. Our data showed that the alginate solution could significantly reduce virus dissemination while having minimal effects on transgene expression in tumors and on interleukin-12-induced tumor growth delay. These data suggest that virus dissemination is a potential problem in local viral gene therapy of cancer and that the dissemination could be significantly reduced by the alginate solution without compromising the efficacy of gene therapy.

Adenoviridae↗
PubMed2002

Humoral immune response to recombinant adenovirus and adeno-associated virus after in utero administration of viral vectors in mice.

Adenovirus and adeno-associated virus vector-mediated gene delivery is limited by the induction of a humoral immune response that prevents readministration. To determine whether viral delivery in the "preimmune" fetus would produce dose- or time-dependent tolerance, we evaluated the humoral immune response after sequential pre- and postnatal virus administration. We evaluated six injection route and viral dose combinations of adenovirus (intra-amniotic, intrahepatic, and intramuscular injection at 4 x 10(8) and 2 x 10(9) particles/fetus) at d 15 postconception (p.c.); three route and dose combinations at d 13 p.c. (intramuscular injection at 1 x 10(8), 3 x 10(8), and 5 x 10(8) particles/fetus); and one route and dose combination of adeno-associated virus (intramuscular at 2.5 x 10(10) genome copies/fetus) at d 15 p.c. In utero injection of either viral vector at any route and dose combination resulted in the production of low titers of neutralizing antivirus and antitransgene (beta-galactosidase) antibodies. This primary immune response only partially blocked transgene expression after the readministration of viral vectors postnatally. However, delivery of the virus postnatally triggered an immune response that completely blocked transgene expression after a third viral injection. Together, these results suggest that, for B6/129 F1 mice, in utero injection of recombinant adenovirus or adeno-associated virus between d 13 and 15 p.c. does not induce tolerance to the viral vector or transgene product.

Adenoviridae↗
PubMed2005

Modulation of human dendritic-cell function following transduction with viral vectors: implications for gene therapy.

Genetic modification of dendritic-cell (DC) function is an attractive approach to treat disease, either using mature DCs (mDCs) to immunize patients, or immature DCs (iDCs) to induce tolerance. Viral vectors are efficient at transducing DCs, and we have investigated the effect of transduction with a variety of viral vectors on the phenotype and function of DCs. Adenovirus (Ad), human immunodeficiency virus (HIV), equine anemia virus (EIAV), and Moloney murine leukemia virus (MMLV) all up-regulate costimulatory molecules and major histocompatibility complex (MHC) class II expression on DCs, as well as, in the case of Ad and lentiviral vectors, inducing production of Th1 and proinflammatory cytokines. Following transduction there is activation of double-stranded (ds) RNA-triggered pathways resulting in interferon (IFN) alpha/beta production. In addition, the function of virally infected DCs is altered; iDCs have an increased, and mDCs a decreased, ability to stimulate a mixed lymphocyte reaction (MLR). Viral transduction of mDCs results in up-regulation of the indoleamine 2,3-dioxygenase (IDO) enzyme, which down-regulates T-cell responsiveness. Inhibition of IDO restores the ability of mDCs to stimulate an MLR, indicating that IDO is responsible for the modulation of mDC function. These data have important implications for the use of viral vectors in the transduction of DCs.

Adenoviridae↗
PubMed1995

Viral vectors in gene therapy.

The use of DNA as a drug is both appealing and simple in concept. Indeed in many instances the feasibility of such an approach has been established using model systems. In practical terms, however, the delivery of DNA to human tissues presents a wide variety of problems that differ with each potential therapeutic application. In this review, the design, production, and application of viral vectors for human gene therapy are considered. Although viral vectors are an obvious starting point because viruses have evolved efficient mechanisms to introduce and express their nucleic acid into recipient cells, by the same token the viral hosts have evolved sophisticated mechanisms to rid themselves of such pathogens. The challenge for the therapeutic use of viral vectors is to achieve efficient and often extended expression of the exogenous gene while evading the host defenses. Methodology used and progress towards that goal are reviewed.

Adenoviridae↗
PubMed1999

Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein.

We are interested in using recombinant adeno-associated viral vectors in the treatment of hemophilia A. Because of the size constraints of recombinant adeno-associated viral vectors, we delivered the heavy and light chains of the human factor 8 (hFVIII) cDNA independently by using two separate vectors. Recombinant AAV vectors were constructed that utilized the human elongation factor 1alpha promoter, a human growth factor polyadenylation signal, and the cDNA sequences encoding either the heavy or light chain of hFVIII. Portal vein injections of each vector alone, a combination of both vectors, or a hFIX control vector were performed in C57BL/6 mice. An ELISA specific for the light chain of hFVIII demonstrated very high levels (2-10 microgram/ml) of protein expression in animals injected with the light chain vector alone or with both vectors. We utilized a chromogenic assay in combination with an antibody specific to hFVIII to determine the amount of biologically active hFVIII in mouse plasma. In animals injected with both the heavy and light chain vectors, greater than physiological levels (200-400 ng/ml) of biologically active hFVIII were produced. This suggests that coexpression of the heavy and light chains of hFVIII may be a feasible approach for treatment of hemophilia A.

Animals↗
PubMed2006

Immunogenicity of cytopathic and noncytopathic viral vectors.

The impact of cytolytic versus noncytolytic viral infections on host responses is not well understood, due to limitations of the systems that have been used to address this issue. Using paired cytopathic and noncytopathic rabies viruses that differ by only two amino acids, we investigated several fundamental aspects of the immune response to these viral vectors. Greater cytopathic capacity translated into a greater degree of cross-priming to CD8(+) T cells (T(CD8)(+)) and more-robust short-term humoral and cellular responses. However, long-term responses to the two viruses were similar, suggesting that direct priming drives the bulk of the T(CD8)(+) antirabies response and that enhanced acute responses associated with greater virally mediated cellular destruction were balanced by other factors, such as prolonged antigen expression associated with noncytopathic virus. Such compensatory mechanisms may be in place to ensure comparable immunologic memories to various pathogens.

Animals↗
PubMed1995

Testing the concept of virally vectored immunosterilisation for the control of wild rabbit and fox populations in Australia.

Virally vectored immunosterilisation is a concept whereby a gene encoding an antigen from an animal's reproductive system is inserted into a virus and, during infection, stimulates the formation of antibodies to that antigen such that the animal is rendered infertile. There is good evidence that certain proteins from sperm or egg when introduced parenterally will induce infertility. This paper summarises the work of the Cooperative Research Centre for the Biological Control of Vertebrate Pest Populations and reviews progress toward the isolation of the genes for gamete antigens from rabbits and foxes and their introduction into suitable viral vectors as a means of control of these pests in Australia.

Animals↗
PubMed2002

Gene transfer mediated by different viral vectors following direct cannulation of mouse submandibular salivary glands.

The salivary gland has been suggested as an accessible organ for gene transfer to express recombinant proteins locally in the saliva, as well as for secretion to the blood circulation. The aim of this study was to evaluate the efficiency of gene transfer to salivary glands using different viral vectors: adenovirus, vaccinia, herpes simplex type 1 (HSV), and two retroviral vectors (murine leukemia virus (MuLV) and lentivirus). We show, by in situ staining and beta-galactosidase reporter activity assay, that the adenoviral and vaccinia vectors were able to deliver the reporter gene efficiently to acinar and duct cells. The HSV vector was less efficient and infected only the acinar cells. The lentiviral vector infected acinar and duct cells, but at a relatively low efficiency. The MuLV vector did not infect the salivary gland unless cell proliferation was induced. Host immune responses to viral infection, inflammation, apoptosis and lymphocyte infiltration, in the transduced glands, were assessed. The DNA viral vectors induced local lymphocyte infiltration and apoptosis. In contrast, the retroviral vectors did not induce an immune response. Our results describe the outcome of salivary gland infection with each of the five different viral vectors and indicate their advantages and limitations for transferring genes to the salivary glands.

Adenoviridae↗
PubMed2006

Nuclear-targeted minicircle to enhance gene transfer with non-viral vectors in vitro and in vivo.

BACKGROUND: To develop more efficient non-viral vectors, we have previously described a novel approach to attach a nuclear localisation signal (NLS) to plasmid DNA, by generating a fusion protein between the tetracycline repressor protein TetR and an SV40 NLS peptide (TetR-NLS). The high affinity of TetR for the DNA sequence tetO is used to bind the NLS to DNA. We have now investigated the ability of this system displaying the SV40 NLS or HIV-1 TAT peptide to enhance nuclear import of a minimised DNA construct more suitable for in vivo gene delivery: a minicircle. METHODS: We have produced a new LacZ minicircle compatible with the TetR system. After transfection of the minicircle in combination with TetR-NLS or TetR-TAT using different transfection agents, we first measured beta-galactosidase activity in vitro. We then used a special delivery technique, in which DOTAP/cholesterol liposomes and DNA/protein complexes are sequentially injected intravenously, to evaluate the activity of this system in vivo. RESULTS: In vitro results showed a 30-fold increase in transfection efficiency of the nuclear-targeted minicircle compared to normal plasmid lipofection. Results on cell cycle arrested cells seem to indicate a different mechanism between the TetR-NLS and TetR-TAT. Finally, we demonstrate a more than 6-fold increase in beta-galactosidase expression in the mouse lung using the minicircle and the TetR-TAT protein. This increase is specific for the peptide sequence and is not observed with the control protein TetR. CONCLUSIONS: Our results indicate that the combination of a minicircle DNA construct with a TetR nuclear-targeting system is able to potentiate gene expression of non-viral vectors.

Animals↗
PubMed2026

Recent advances for the pharmaceutical production of highly attenuated poxviruses as viral vector platforms.

INTRODUCTION: Highly attenuated poxviruses serve as potent viral vectors, oncolytic agents, and therapeutic vaccines. They can accommodate and stably maintain a large genomic payload of foreign inserts. Their limited replication in human cells provides an excellent safety profile, but it concomitantly necessitates higher doses of infectious particles for full therapeutic efficacy. AREAS COVERED: We review recent advances in bioprocesses for the pharmaceutical production of poxvirus-based vectors, focusing mainly on the vaccinia virus and the Orf virus. These include upstream processing using highly permissive cell substrates, optimized feeding strategies, and a virus phenotype that facilitates downstream processing. The study explores ongoing challenges and identifies strategies to adapt the downstream process to intensified upstream processes in order to achieve an economic end-to-end production. EXPERT OPINION: For notably increased virus yields of up to 2 log after amplification, we propose to replace classic adsorption chromatography by a collective and continuous purification platform for separating the virus from process-related impurities. Filtration operations facilitate process scalability while reducing volumes, which is beneficial for a flow-through polishing to meet pharmaceutical quality attributes. Combined with artificial intelligence modeling, these advancements alleviate financial pressures on healthcare systems and accelerate the production of novel vaccine candidates for clinical use.

Humans↗
PubMed2006

Production of adeno-associated viral vectors in insect cells using triple infection: optimization of baculovirus concentration ratios.

The production of viral vectors or virus-like particles for gene therapy or vaccinations using the baculovirus expression system is gaining in popularity. Recently, reports of a viral vector based on adeno-associated virus (AAV) produced in insect cells using the baculovirus expression vector system have been published. This system requires the triple infection of cells with baculovirus vectors containing the AAV gene for replication proteins (BacRep), the AAV gene for structural proteins (BacCap), and the AAV vector genome (BacITR). A statistical approach was used to investigate the multiplicities of infection of the three baculoviruses and the results were extended to the production of AAVs containing various transgenes. Highest AAV yields were obtained when BacRep and BacCap, the baculovirus vectors containing genes that code for proteins necessary for the formation of the AAV vector, were added in equal amounts at high multiplicities of infection. These combinations also resulted in the closest ratios of infectious to total AAV particles produced. Overexpression of the AAV structural proteins led to the production of empty AAV capsids, which is believed to overload the cellular machinery, preventing proper encapsidation of the AAV vector transgene, and decreased the viability of the insect cells. Delaying the input of BacCap, to reduce the amount of capsids produced, resulted in lower infectious AAV titers then when all three baculoviruses were put into the system at the same time. The amount of BacITR added to the system can be less than the other two without loss of AAV yield.

Animals↗