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Vector Encoding (vector + encoding)
Selected AbstractsGene delivery of Cu/Zn-superoxide dismutase improves graft function after transplantation of fatty livers in the ratHEPATOLOGY, Issue 6 2000Thorsten G. Lehmann Oxygen-derived free radicals play a central role in reperfusion injury after organ transplantation, and fatty livers are particularly susceptible. Endogenous radical scavengers such as superoxide dismutase (SOD) degrade these radicals; however, SOD is destroyed rapidly when given exogenously. Therefore, an adenoviral vector encoding the Cu/Zn-SOD gene (Ad.SOD1) was used here to test the hypothesis that organ injury would be reduced and survival increased in a rat model of transplantation of fatty livers. Donors received chow diet (untreated), high-fat diet, or ethanol-containing high-fat diet. Some of the ethanol-fed donors were infected either with the gene lacZ encoding bacterial ,-galactosidase (Ad.lacZ), or Ad.SOD1. After liver transplantation, SOD activity and protein expression in liver, survival, histopathology, release of transaminases, free radical adducts in bile, and activation of NF-,B, I,B kinase (IKK), Jun-N-terminal kinase (JNK), and TNF, were evaluated. Ad.SOD1 treatment increased survival dramatically, blunted transaminase release, and reduced necrosis and apoptosis significantly. Free radical adducts were increased two-fold in the ethanol group compared with untreated controls. Ad.SOD1 blunted this increase and reduced the activation of NF-,B. However, release of TNF, was not affected. Ad.SOD1 also blunted JNK activity after transplantation. This study shows that gene therapy with Ad.SOD1 protects marginal livers from failure after transplantation because of decreased oxygen radical production. Genetic modification of fatty livers using viral vectors represents a new approach to protect marginal grafts against primary nonfunction. [source] Acceleration of cartilage repair by genetically modified chondrocytes over expressing bone morphogenetic protein-7JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 4 2003Chisa Hidaka Background: Cartilage has a limited capacity to heal. Although chondrocyte transplantation is a useful therapeutic strategy, the repair process can be lengthy. Previously we have shown that over expression of bone morphogenetic protein-7 (BMP-7) in chondrocytes by adenovirus-mediated gene transfer leads to increased matrix synthesis and cartilage-like tissue formation in vitro. In this context we hypothesized that implantation of genetically modified chondrocytes expressing BMP-7 would accelerate the formation of hyaline-like repair tissue in an equine model of cartilage defect repair. Methods: Chondrocytes treated with adenovirus vector encoding BMP-7 (AdBMP-7) or as control, an adenovirus vector encoding an irrelevant gene (Escherichia coli cytosine deaminase, AdCD) were implanted into extensive (15 mm diameter) articular cartilage defects in the patellofemoral joints of 10 horses. Biopsies were performed to evaluate early healing at 4 weeks. At the terminal time point of 8 months, repairs were assessed for morphology, MRI appearance, compressive strength, biochemical composition and persistence of implanted cells. Results: Four weeks after surgery AdBMP-7-treated repairs showed an increased level of BMP-7 expression and accelerated healing, with markedly more hyaline-like morphology than control. Quantitative real-time polymerase chain reaction (PCR) analysis of the repair tissue 8 months after surgery showed that few implanted cells persisted. By this time, the controls had healed similarly to the AdBMP-7-treated defects, and no difference was detected in the morphologic, biochemical or biomechanical properties of the repair tissues from the two treatment groups. Conclusions: Implantation of genetically modified chondrocytes expressing BMP-7 accelerates the appearance of hyaline-like repair tissue in experimental cartilage defects. Clinical relevance: Rehabilitation after cell-based cartilage repair can be prolonged, leading to decreased patient productivity and quality of life. This study shows the feasibility of using genetically modified chondrocytes to accelerate cartilage healing. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Expression of ,re,y luciferase gene in Erwinia amylovoraLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 4 2003Giovanna Gentilomi Abstract In this study we describe an ef,cient stable genetic transformation of the phytopathogenic bacterium Erwinia amylovora using a recombinant expression vector encoding the ,re,y luciferase gene of Photinus pyralis, which is further controlled by IPTG-inducible promoter. Stably transformed E. amylovora cells maintain the same infectivity as the wild-type strain and, after induction with IPTG, produce luciferase. Luminescence produced by the action of luciferase on an exogenous substrate was easily detectable by a simple and rapid bioluminescent assay (BL). The transformed E. amylovora strain maintains the same high emission level, even after passage in pears, until about 15 days post-infection. Our ,ndings therefore show that the luciferase assay can be conveniently used to follow the bacterial movement in plant tissue and its dissemination in controlled environments. [source] Two and three-dimensional gene transfer from enzymatically degradable hydrogel scaffoldsMICROSCOPY RESEARCH AND TECHNIQUE, Issue 9 2010Yuguo Lei Abstract The ability to genetically modify mesenchymal stem cells (MSCs) seeded inside synthetic hydrogel scaffolds would offer an alternative approach to guide MSC differentiation. In this report, we explored gene transfer to MSCs seeded on top or inside matrix metalloproteinase (MMP) degradable hydrogels that were loaded with DNA/poly(ethylene imine) (PEI) polyplexes. DNA/PEI polyplexes were encapsulated inside poly(ethylene glycol) (PEG) hydrogels crosslinked with MMP degradable peptides via Michael Addition chemistry. Gene transfer was visualized and quantified through using a vector encoding for green fluorescent protein and luciferase. We found that gene transfer to MSCs was possible for cells seeded both in two and three dimensions. The amount of luciferase expression was similar for cells seeded in two and three dimensions even though the number of cells in three dimensions is significantly higher, indicating that gene transfer to cells seeded in two dimensions is more efficient than for cells seeded in three dimensions. The use of hydrogel scaffolds that allow cellular infiltration to deliver DNA may result in long-lasting signals in vivo, which are essential for the regeneration of functional tissues. Microsc. Res. Tech. 73:910,917, 2010. © 2010 Wiley-Liss, Inc. [source] Dendritic cells lentivirally engineered to overexpress interleukin-10 inhibit contact hypersensitivity responses, despite their partial activation induced by transduction-associated physical stressTHE JOURNAL OF GENE MEDICINE, Issue 3 2010Verena Besche Abstract Background Dendritic cells (DCs) constitute an attractive target for immunotherapeutic approaches. Because DCs are largely refractory to transfection with plasmid DNA, several viral transduction protocols were established. The potential side-effects of lentiviral transduction on the phenotype and activation state of DCs left unstimulated after transduction have not been assessed. There is a need to analyse these parameters as a result of the requirement of using DCs with a low activation state for therapeutic strategies intended to induce tolerance. Methods Lentivirally-transduced bone marrow (BM)-derived DCs (LV-DCs) in comparison with mock-transduced (Mock-DCs) and untreated DCs were analysed with regard to the induction of maturation processes on the RNA, protein and functional level. BM-DCs engineered to overexpress interleukin (IL)-10 were analysed for therapeutic potential in a mouse model of allergic contact dermatitis. Results Compared with untreated DCs, Mock-DCs and LV-DCs displayed an altered gene expression signature. Mock-DCs induced a stronger T cell proliferative response than untreated DCs. LV-DCs did not further augment the T cell proliferative response, but induced a slightly different T cell cytokine pattern compared to Mock-DCs. Accordingly, the gene promoter of the DC maturation marker fascin mediated efficient expression of the model transgene IL-10 in unstimulated-transduced BM-DCs. Nevertheless, IL-10 overexpressing BM-DCs exerted tolerogenic activity and efficiently inhibited the contact hypersensitivity response in previously hapten-sensitized mice. Conclusions Lentiviral transduction of BM-DCs results in their partial activation. Nevertheless, the transduction of these DCs with a vector encoding the immunomodulatory cytokine IL-10 rendered them tolerogenic. Thus, lentivirally-transduced DCs expressing immunomodulatory molecules represent a promising tool for induction of tolerance. Copyright © 2010 John Wiley & Sons, Ltd. [source] Development and characterization of a triple combination gene therapy vector inhibiting HIV-1 multiplicationTHE JOURNAL OF GENE MEDICINE, Issue 10 2008Maria B. Asparuhova Abstract Background RNA-based approaches are promising for long-term gene therapy against HIV-1. They can target virtually any step of the viral replication cycle. It is also possible to combine anti-HIV-1 transgenes targeting different facets of HIV replication to compensate for limitations of any individual construct, maximizing efficacy and decreasing chances of escape mutations. We have previously developed two strategies to inhibit HIV-1 multiplication. One was a short hairpin RNA targeting the host factor cyclophilin A implicated in HIV-1 replication. Additionally, an antisense derivative of U7 small nuclear RNA was designed to induce the skipping of the HIV-1 Tat and Rev internal exons. Results In the present study, we have established an additional tRNAval promoter-driven shRNA against the coding sequence of viral infectivity factor. When human T-cell lines or primary CD4+ T cells are transduced with a triple lentiviral vector encoding these three therapeutic RNAs, HIV-1 multiplication is very efficiently suppressed. Moreover, all three therapeutic RNAs exhibit antiviral effects at early stages of the viral replication cycle (i.e. prior to viral cDNA integration or gene expression). Conclusions These findings make this triple lentiviral vector an attractive candidate for a gene therapy against HIV/AIDS. Copyright © 2008 John Wiley & Sons, Ltd. [source] In vivo gene delivery of glial cell line,derived neurotrophic factor for Parkinson's diseaseANNALS OF NEUROLOGY, Issue S3 2003Jeffrey H. Kordower PhD Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects approximately 1,000,000 Americans. The cause of the disease remains unknown. The histopathological hallmarks of the disease are dopaminergic striatal insufficiency secondary to a loss of dopaminergic neurons in the substantia nigra pars compacta and intracellular inclusion called Lewy bodies. Currently, only symptomatic treatment for PD is available. Although some treatments are efficacious for many years, all have significant limitations and new therapeutic approaches are needed. Gene therapy is ideal for delivering therapeutic molecules to site-specific regions of the central nervous system. Via gene therapy, a piece or pieces of DNA placed into a carrying vector encoding for a substance of interest can be introduced into specific cells. Although there are several ways that gene therapy can be applied for PD, this review focuses on in vivo gene delivery of glial cell line,derived neurotrophic factor (GDNF) as a neuroprotective strategy for PD. Ann Neurol 2003;53 (suppl 3):S120,S134 [source] Cooperative effects of bcl-2 and AAV-mediated expression of CNTF on retinal ganglion cell survival and axonal regeneration in adult transgenic miceEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2006Simone G. Leaver Abstract We used a gene therapy approach in transgenic mice to assess the cooperative effects of combining anti-apoptotic and growth-promoting stimuli on adult retinal ganglion cell (RGC) survival and axonal regeneration following intraorbital optic nerve injury. Bi- cistronic adeno-associated viral vectors encoding a secretable form of ciliary neurotrophic factor and green fluorescent protein (AAV-CNTF-GFP) were injected into eyes of mice that had been engineered to over-express the anti-apoptotic protein bcl-2. For comparison this vector was also injected into wildtype (wt) mice, and both mouse strains were injected with control AAV encoding GFP. Five weeks after optic nerve injury we confirmed that bcl-2 over-expression by itself promoted the survival of axotomized RGCs, but in contrast to previous reports we also saw regeneration of some mature RGC axons beyond the optic nerve crush. AAV-mediated expression of CNTF in adult retinas significantly increased the survival and axonal regeneration of RGCs following axotomy in wt and bcl-2 transgenic mice; however, the effects were greatest in the transgenic strain. Compared with AAV-GFP-injected bcl-2 mice, RGC viability was increased by about 50% (mean, 36 738 RGCs per retina), and over 1000 axons per optic nerve regenerated 1,1.5 mm beyond the crush. These findings exemplify the importance of using a multifactorial therapeutic approach that enhances both neuroprotection and regeneration after central nervous system injury. [source] Evaluating the Ability of Polyhydroxyalkanoate Synthase Mutants to Produce P(3HB -co- 3HA) from Soybean OilMACROMOLECULAR BIOSCIENCE, Issue 1 2009Takeharu Tsuge Abstract Polyhydroxyalkanoate (PHA) synthase from Pseudomonas sp 61-3 (PhaC1Ps) is able to synthesize P(3HB -co- 3HA), consisting of a 3HB unit and medium-chain-length 3HA units of 6,12 carbon atoms. Expression vectors encoding 76 PhaC1Ps mutants with an amino acid replacement at position 130, 325, 477 or 481 were individually introduced into Ralstonia eutropha. The mutant enzyme genes were evaluated in terms of their abilities to synthesize P(3HB -co- 3HA) using soybean oil as a carbon source. 20 mutants showed significantly high accumulation levels of PHA exceeding 30 wt.-% and as high as 57 wt.-%. It was found that hydrophobic amino acids at the positions are more likely to enhance accumulation of PHA in R. eutropha. [source] Salivary gland delivery of pDNA-cationic lipoplexes elicits systemic immune responsesORAL DISEASES, Issue 6 2002V Sankar OBJECTIVE: To test the ability of two cationic lipoplexes, Vaxfectin and GAP-DLRIE/DOPE, to facilitate transfection and elicit immune responses from plasmid DNAs (pDNAs) after retrograde instillation into salivary glands. METHODS: Two pDNA expression vectors encoding either the influenza NP protein or human growth hormone (hGH) were complexed with the cationic lipid transfection reagents, GAP-DLRIE/DOPE or Vaxfectin, and delivered to the submandibular glands of rats. Samples from rats receiving the influenza NP protein pDNA and cationic lipoplexes were analyzed for anti-influenza NP-specific IgG1, IgG2a, and IgG2b in serum, and IgA in saliva, by an enzyme- linked immunosorbent assay (ELISA). Cytotoxic T-cell lymphocyte (CTL) assays were also performed. Transgene protein expression (hGH) was determined from extracts of submandibular glands of rats receiving hGH lipoplexes. RESULTS: Serum antibodies (IgG) against the NP protein developed and were highest in all rats vaccinated with GAP-DLRIE/DOPE or Vaxfectin. The major serum IgG subclass stimulated by this route of immunization was IgG2b, followed by IgG2a. CTL assay results showed statistically significantly higher percentage killing in the Vaxfectin group than controls (P < 0.05). No rats developed IgA antibodies to NP protein in saliva. Animals receiving plasmid encoding hGH and either lipoplex expressed significantly higher amounts of hGH compared with those receiving the hGH plasmid and water. Although hGH expression was higher in the animals receiving pDNA/Vaxfectin (, 30-fold > pDNA/water), the difference with those receiving pDNA/GAP-DLRIE/DOPE (,10-fold > pDNA/water) was not significant. CONCLUSIONS: Retrograde instillation of pDNA complexed with Vaxfectin into the salivary glands can stimulate cytotoxic and humoral responses to the influenza NP protein antigen. Optimization of the conditions required to stimulate humoral and secretory antibody formation may facilitate use of this tissue for specific clinical applications of pDNA immunization. [source] Cytokine stimulation and the choice of promoter are critical factors for the efficient transduction of mouse T cells with HIV-1 vectorsTHE JOURNAL OF GENE MEDICINE, Issue 2 2010David E. Gilham Abstract Background HIV-1 fails to successfully infect mouse T cells as a result of several blocks in the viral replication cycle. We investigated whether this also impacted on the use of HIV-1 derived lentiviral vectors for stable gene transfer into mouse T cells. Methods Freshly isolated primary mouse T cells were immediately mixed with lentiviral vectors encoding an enhanced green fluorescent protein marker gene and transduction frequency was determined after 5 days of culture. Results Optimal transduction required both mouse T cell activation and cytokine support. Furthermore, transduction was also dependent upon the promoter chosen, with the rank order of potency being PGK > EF1 > SFFV > CMV. HIV-1 lentiviral vectors also efficiently transduced cytokine-stimulated T cells (in the absence of antibody driven T cell activation), albeit with a lower level of transgene expression compared to fully-activated T cells. Conclusions The present study demonstrates that primary mouse T cells can be efficiently transduced with HIV-1 lentiviral vectors, opening up prospects for their use in mouse models of gene-modified adoptive cellular therapy. Copyright © 2009 John Wiley & Sons, Ltd. [source] Comparative analysis of antitumor activity of CD40L, RANKL, and 4-1BBL in vivo following intratumoral administration of viral vectors or transduced dendritic cellsTHE JOURNAL OF GENE MEDICINE, Issue 2 2006Zoya R. Yurkovetsky Abstract The tumor necrosis factor (TNF) family comprises a group of ligands that regulate cell proliferation, differentiation, activation, maturation and apoptosis through interaction with the corresponding TNF receptor family members. In this study, we have evaluated whether adenovirus-mediated intratumoral gene transfer of CD40L, RANKL, or 4-1BBL elicits an immune response to established murine MC38 and TS/A tumors. Intratumoral administration of the recombinant adenoviral vectors expressing CD40L, RANKL or 4-1BBL 7 days post-tumor cell inoculation resulted in significant inhibition of MC38 tumor growth for all three ligands when compared with control groups treated with either saline or control adenovirus. However, intratumoral injection of Ad-4-1BBL or Ad-CD40L resulted in a significantly stronger inhibition of TS/A tumor progression than did Ad-RANKL treatment. We also demonstrated that intratumoral administration of dendritic cells (DC) transduced with adenoviral vectors encoding the TNF-related ligands resulted in a significant inhibition of MC38 tumor growth as compared with control groups treated with Ad-LacZ-transduced DC or saline-treated DC. In addition, DC overexpressing CD40L secreted considerably more IL-12 and expressed higher levels of the co-stimulatory molecules, CD80, CD86 and CD40, than did DC overexpressing LacZ, 4-1BBL or RANKL. We have also demonstrated that DC/CD40L, DC/4-1BBL, and DC/RANKL survived significantly longer than control DC or DC infected with the LacZ vector. Taken together, these results demonstrate that adenoviral gene transfer of CD40L, RANKL or 4-1BBL elicit a significant antitumor effect in two different tumor models, with CD40L gene transfer inducing the strongest antitumor effect. Copyright © 2005 John Wiley & Sons, Ltd. [source] Cytotoxicity and antiangiogenesis by fibroblast growth factor 2,targeted Ad-TK cancer gene therapy,THE LARYNGOSCOPE, Issue 4 2009Koichiro Saito MD Abstract Objectives: Human head and neck squamous cell carcinoma (HNSCC) in addition to lung, skin, ovarian, and other cancers overexpress fibroblast growth factor (FGF) receptors on both individual tumor cells and endothelial cells within the tumor microenvironment. The purpose of this study was to investigate whether FGF2-targeted gene therapy could redirect adenoviral vectors encoding the herpes simplex virus thymidine kinase gene (Ad-TK) to FGF receptors on tumor and endothelial cells with the intent of improving both the efficiency of transgene expression and the antitumor response. Study Design and Methods: An Ad-TK vector consisting of a conjugate of FGF2 linked to a Fab, fragment against the adenoviral knob region was directly delivered to human HNSCC xenograft tumors in nude mice, which were subsequently dosed with ganciclovir. Tumor specimens were assessed for herpes simplex virus thymidine kinase (HSV- tk) transgene mRNA expression, FGF1/2 receptor expression, terminal deoxynucleotidyl transferase biotin,deoxy uridine triphosphate nick end labeling assay for apoptosis, CD31 immunohistochemistry to estimate tumor microvessel density, and tumor volume change. Results: FGF2-retargeted Ad-TK gene therapy demonstrated significant increases in both HSV- tk mRNA expression and cellular apoptosis levels, and a significant decrease in tumor volume size compared with all other groups. Furthermore, microvessel density was significantly lower in the FGF2-retargeted Ad-TK group, indicating a strong antiangiogenesis effect. Conclusions: These data suggest that FGF2-retargeted Ad-TK produces a combination of expected direct antitumor cytotoxicity and a newly reported antiangiogenesis effect that could prove promising as a novel therapeutic approach in the treatment of FGF receptor,expressing cancers. Laryngoscope, 2009 [source] The impact of retroviral suicide gene transduction procedures on T cellsBRITISH JOURNAL OF HAEMATOLOGY, Issue 4 2003Waseem Qasim Summary., Retroviral vectors encoding the herpes simplex thymidine kinase gene have been used to render T cells sensitive to the prodrug ganciclovir. Such genetically modified T cells have been used in clinical trials for their graft-versus-leukaemia effects following allogeneic haematopoietic stem cell transplantation. In the event of graft-versus-host disease (GVHD) the cells were susceptible to elimination through exposure to ganciclovir. We have investigated the impact of T-cell activation, required for successful retrovirus-mediated gene transfer, on T-cell receptor repertoire profile, subset distribution and antiviral potential. Using a combination of antibodies against CD3 and CD28, T cells were transduced at high efficiency when exposed to retrovirus between 48 and 72 h later. Lymphocytes had undergone up to seven cycles of cell division by the end of the procedure. Although the T-cell receptor V, repertoire was not altered after retroviral transduction, there were notable shifts in subset profiles with an increased proportion of CD45RO cells in transduced populations. T cells continued to proliferate for several days after transduction and were difficult to sustain under the extended culture conditions required to generate virus-specific T cells. These observations may explain the lower than expected levels of GVHD and poor antiviral immunity reported in recent trials. [source] | |||||||||||||