The Experts below are selected from a list of 8247 Experts worldwide ranked by ideXlab platform
Jeffrey M Isner - One of the best experts on this subject based on the ideXlab platform.
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Intramyocardial gene therapy with Naked DNA encoding vascular endothelial growth factor improves collateral flow to ischemic myocardium.
Human gene therapy, 1999Co-Authors: Rene A. Tio, Jeffrey M Isner, Tengis Tkebuchava, Thomas H. Scheuermann, Corinna Lebherz, Meredith Magner, Marianne Kearny, Daryll D. Esakof, James F SymesAbstract:Both VEGF protein and VEGF DNA in combination with an adenoviral vector have been shown to enhance collateral formation in a porcine model of chronic myocardial ischemia. We sought to determine whether direct intramyocardial injection of Naked DNA encoding for VEGF could similarly improve myocardial perfusion. Initially, 23 nonischemic pigs received either 200 mu g of plasmid DNA encoding beta-galactosidase (pCMV beta, n = 11) or 500 mu g of phVEGF165 (n = 12) into four separate sites in the myocardium via a small anterolateral thoracotomy incision in the fourth intercostal space. Two additional groups of pigs received an intramyocardial injection of either phVEGF165 (n = 6) or pCMV beta (n = 7) 3 to 4 weeks after implantation of an ameroid constrictor around the left circumflex coronary artery. The injections caused no change in heart rate or blood pressure, and no ventricular arrhythmias or histologic evidence of inflammation. VEGF protein was detected by Western blot in VEGF-treated animals, with the s...
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arterial gene transfer of acidic fibroblast growth factor for therapeutic angiogenesis in vivo critical role of secretion signal in use of Naked DNA
Cardiovascular Research, 1997Co-Authors: Hirotsugu Tabata, Marcy Silver, Jeffrey M IsnerAbstract:Objective: Previous studies have demonstrated that arterial gene transfer of Naked DNA encoding for a secreted protein may permit modulation of the host phenotype despite a low transfection efficiency. Acidic fibroblast growth factor (aFGF) is an angiogenic growth factor, but is not secreted by intact cells. In the current study, we investigated the hypothesis that addition of a hydrophobic leader sequence to achieve active secretion of the gene product would permit therapeutic angiogenesis following arterial gene transfer of Naked DNA encoding for aFGF. Methods: Ten days following surgical induction of unilateral hindlimb ischemia, New Zealand white rabbits were randomized to intra-arterial gene transfer with one of three plasmids: p267 (encoding non-secreted aFGF, n = 10), pMJ35 (encoding secreted aFGF) ( n = 10), or 500 μ g of pGSVLacZ (control, n = 10) (500 μ g each). All animals were studied at 30 days post-gene transfer for evidence of therapeutic angiogenesis. Results: pMJ35 transfectants had more angiographically visible collaterals (angiographic score=0.76±0.02) than either p267 (0.55±0.02, p <0.01) or LacZ (0.47±0.02, p <0.001). Limb blood pressure ratio for pMJ35 was 0.88±0.02 vs. 0.68±0.04 for p267 ( p <0.01) and 0.57±0.04 for LacZ ( p <0.001). Vascular resistance was significantly lower in the pMJ35 group, compared with that in pGSVLacZ group, both in resting state (3.2±0.4 vs. 7.4±1.4 respectively, p <0.05) and after the administration of nitroprusside. Capillary density (per mm2) was also superior in pMJ35 group (274±10) vs. p267 (204±9, p <0.01) and LacZ (177±6, p <0.001). Conclusion: The paracrine effects of a secreted gene product may obviate the need for adjunctive vectors in strategies of arterial gene therapy.
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Arterial gene transfer of acidic fibroblast growth factor for therapeutic angiogenesis in vivo: critical role of secretion signal in use of Naked DNA
Cardiovascular research, 1997Co-Authors: Hirotsugu Tabata, Marcy Silver, Jeffrey M IsnerAbstract:Objective: Previous studies have demonstrated that arterial gene transfer of Naked DNA encoding for a secreted protein may permit modulation of the host phenotype despite a low transfection efficiency. Acidic fibroblast growth factor (aFGF) is an angiogenic growth factor, but is not secreted by intact cells. In the current study, we investigated the hypothesis that addition of a hydrophobic leader sequence to achieve active secretion of the gene product would permit therapeutic angiogenesis following arterial gene transfer of Naked DNA encoding for aFGF. Methods: Ten days following surgical induction of unilateral hindlimb ischemia, New Zealand white rabbits were randomized to intra-arterial gene transfer with one of three plasmids: p267 (encoding non-secreted aFGF, n = 10), pMJ35 (encoding secreted aFGF) ( n = 10), or 500 μ g of pGSVLacZ (control, n = 10) (500 μ g each). All animals were studied at 30 days post-gene transfer for evidence of therapeutic angiogenesis. Results: pMJ35 transfectants had more angiographically visible collaterals (angiographic score=0.76±0.02) than either p267 (0.55±0.02, p
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gene transfer of Naked DNA encoding for three isoforms of vascular endothelial growth factor stimulates collateral development in vivo
Laboratory Investigation, 1996Co-Authors: Satoshi Takeshita, Yukio Tsurumi, T Couffinahl, Takayuki Asahara, Christophe Bauters, James F Symes, Napoleone Ferrara, Jeffrey M IsnerAbstract:Vascular endothelial growth factor (VEGF) is a naturally secreted endothelial cell-specific mitogen. We investigated the hypothesis that Naked DNA encoding for VEGF could be used in a strategy of arterial gene therapy to stimulate collateral artery development. Plasmid DNA encoding each of the three principal human VEGF isoforms (phVEGF 121 , phVEGF 165 , or phVEGF 189 ) was applied to the hydrogel polymer coating of an angioplasty balloon and delivered percutaneously to one iliac artery of rabbits with operatively induced hindlimb ischemia. Compared with control animals transfected with LacZ, site-specific transfection of phVEGF resulted in augmented collateral vessel development documented by serial angiography, and improvement in calf blood pressure ratio (ischemic to normal limb), resting and maximum blood flow, and capillary to myocyte ratio. Similar results were obtained with phVEGF 121 , phVEGF 165 , and phVEGF 189 , which suggests that these isoforms are biologically equivalent with respect to in vivo angiogenesis. The fact that viral or other adjunctive vectors were not required further suggests that secreted gene products may have potential therapeutic utility even when the number of successfully transfected cells remains low. Arterial gene transfer of Naked DNA encoding for a secreted angiogenic cytokine, thus, represents a potential alternative to recombinant protein administration for stimulating collateral vessel development.
Leaf Huang - One of the best experts on this subject based on the ideXlab platform.
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mechanism of Naked DNA clearance after intravenous injection
Journal of Gene Medicine, 2006Co-Authors: Christine C Conwell, Lisa M Shollenberger, Xing Yuan, Leaf HuangAbstract:Naked plasmid DNA as a non-viral vector has been extensively studied in gene therapy because of its ease of preparation, stability, biochemical simplicity, and safety compared to other nonviral and viral vectors. However, one of major hurdles of using Naked DNA in gene therapy is that injected DNA will be quickly cleared out from the circulation. Many studies have previously reported that pDNA is degraded quickly by nucleases in the blood and other compartments and is rapidly removed from the circulation and taken up by the liver, predominantly by the liver nonparenchymal cells (NPCs), after intravenous administration into mice. In the present study, we have examined the role of liver uptake and serum degradation on Naked DNA injected intravenously. The data in this study provides detailed information of Naked DNA localization and activity after intravenous injection, including 1) the rate at which the liver can take up DNA; 2) information regarding clearance of Naked DNA by the liver and serum; 3) demonstration that Naked DNA is primarily taken up by the liver endothelial cells, not Kupfer cells; and 4) effect of the enlargement of the fenestrae on transgene expression and DNA uptake by hepatocytes. First, DNA uptake was examined using liver perfusion system. Our data show that the liver was able to take up over 50% of the DNA with a single pass perfusion of 5 |[mu]|g DNA. The absolute amount of pDNA taken up by the liver increased with an increase in the amount of perfused pDNA. The liver, even just one single pass, could take up as much as approximately 25 |[mu]|g of pDNA. Second, in vivo degradation of DNA by blood was examined by transient occlusion of blood flow through the liver followed by injection of plasmid DNA. The occlusion of blood flow was performed by clipping both the portal vein and the hepatic artery. The blood was collected 1, 3, and 5 minutes after the injection. The DNA degradation was examined by loading the blood samples on a 1% agarose gel for electrophoresis, which showed a lower clearance rate compared to that of liver uptake. In addition, using confocal microscopy and labeling both the DNA and endothelial cells, it was demonstrated that Naked DNA is primarily taken up by the liver endothelial cells. The endothelial barrier could be overcome by manually massaging the liver (MML). Our TEM results clearly show that MML could enlarge the fenestrae, which increased uptake of DNA by hepatocytes and gene expression in the liver.
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Restoration of Dystrophin Expression in MDX Mice by Intravascular Injection of Naked DNA Containing Full-Length Dystrophin cDNA
Gene therapy, 2004Co-Authors: K W Liang, Feng Liu, M Nishikawa, Bin Sun, Leaf HuangAbstract:Duchenne muscular dystrophy (DMD) is a lethal, X-linked, recessive disease caused by a defect in the dystrophin gene. No effective therapy is available. Dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. However, successful treatment for DMD requires restoration of dystrophin in the affected muscle fibers to at least 20% of the normal level. Current gene transfer methods such as intramuscular injection of viral vector or Naked DNA can only transfect a small area of muscle, and therefore is of little clinical utility. We have developed a semisystemic method for gene transfer into skeletal muscle of mdx mice, an animal model for DMD. Naked DNA was injected through the tail artery or vein of mice, in which the aorta and the vena cava were clamped at the location just below the kidneys. The DNA solution was thus forced into the blood vessels of both legs. Luciferase gene expression was detected in all muscle groups in both legs. The effects of injection speed, injection volume, and ischemia time on gene expression were also optimized. LacZ staining was used to check the spread of gene expression in muscle. Although the percentage of transfected fibers was modest (approximately 10%), beta-galactosidase was found in all muscle groups of both legs. Finally, plasmid DNA encoding full-length dystrophin gene was injected into mdx mice and widespread restoration of dystrophin protein was observed in all muscles of both hind limbs. In conclusion, these results demonstrate that the semisystemic delivery of Naked DNA is a potential approach towards the long-term goal of gene therapy for DMD.
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enhanced cutaneous gene delivery following intradermal injection of Naked DNA in a high ionic strength solution
Molecular Therapy, 2002Co-Authors: Sophie Chesnoy, Leaf HuangAbstract:Intradermal injection of Naked DNA results in gene transfer to skin cells, but the efficiency of this gene transfer method is relatively low and variable. We have systematically optimized several parameters to obtain reproducible, high-level gene transfer to the mouse skin. Older mice (7 weeks) showed a significant decrease in gene expression compared with younger mice (4–5 weeks old). The composition of the solvent vehicle (electrolyte versus nonelectrolyte) strongly affected gene expression in the skin. A higher level of gene expression was achieved when Naked DNA was dissolved in isotonic phosphate buffered saline solution compared with isotonic dextrose solution. Finally, transfection efficiency in older mice was greatly improved by increasing the ionic strength of the solvent vehicle. The improved transfection efficiency was due to an enhanced DNA uptake by the skin cells. Gene transfer was most evident in the subdermal smooth muscle cells and epidermal cells. With the optimized conditions, gene transfer mediated by intradermal injection of Naked DNA was comparable in efficiency to electroporation. However, cellular distributions of the gene transfer of the two methods were different.
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systemic production of il 12 by Naked DNA mediated gene transfer toxicity and attenuation of transgene expression in vivo
Journal of Gene Medicine, 2001Co-Authors: Vivian Wai Yan Lui, Louis D. Falo, Leaf HuangAbstract:Background IL-12 is a potent antitumor cytokine for cancer gene therapy. Previously, we demonstrated that single systemic administration of Naked DNA (encoding IL-12) could serve as a good model for in vivo evaluation of the antitumor effect of a candidate gene (unpublished data). In the present study, we propose that this gene delivery method could be a very useful model for in vivo evaluation of the toxicity of a given therapeutic gene (using IL-12 as an example). By comparing the toxicities and the effects of initial IL-12 administration on subsequent transgene expression, both IL-12 gene delivery and recombinant murine IL-12 protein (rmIL-12) administration showed similar toxicity profiles. Methods Naked DNA encoding murine IL-12 (mIL-12) was delivered into mice by systemic administration. Toxicity profiles of mice treated with DNA or rmIL-12 were compared. Results Systemic administration of Naked DNA encoding mIL-12 resulted in very similar toxicity as rmIL-12 with respect to liver enzyme, hematological and immunological profiles. Repeated injection of mIL-12 gene did not recover a high level of mIL-12 production as the first injection. Moreover, initial mIL-12 administration resulted in inhibition of subsequent reporter gene expression with both viral and non-viral promoters (CMV, human α-antitrypsin or chicken β-actin promoter). This transgene inhibition effect was entirely mediated by IFN-γ as the transgene expression was fully recovered in IFN-γ knockout mice. Conclusions Systemic IL-12 therapy, with either a protein or gene therapy approach, resulted in comparable liver and systemic toxicities. Refractoriness of mIL-12 production by subsequent administration of mIL-12 gene was observed. The transgene attenuation effect of IL-12 pre-dosing (either by IL-12 or rmIL-12), mediated by IFN-γ, provided important insights for the design of IL-12 combination gene therapy and the improvement of gene vectors for IL-12 therapy. The present results show that simple injection of Naked DNA could serve as a good model for in vivo evaluation of the toxicity of a candidate therapeutic gene. Copyright © 2001 John Wiley & Sons, Ltd.
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Systemic production of IL‐12 by Naked DNA mediated gene transfer: toxicity and attenuation of transgene expression in vivo
The journal of gene medicine, 2001Co-Authors: Vivian Wai Yan Lui, Louis D. Falo, Leaf HuangAbstract:Background IL-12 is a potent antitumor cytokine for cancer gene therapy. Previously, we demonstrated that single systemic administration of Naked DNA (encoding IL-12) could serve as a good model for in vivo evaluation of the antitumor effect of a candidate gene (unpublished data). In the present study, we propose that this gene delivery method could be a very useful model for in vivo evaluation of the toxicity of a given therapeutic gene (using IL-12 as an example). By comparing the toxicities and the effects of initial IL-12 administration on subsequent transgene expression, both IL-12 gene delivery and recombinant murine IL-12 protein (rmIL-12) administration showed similar toxicity profiles. Methods Naked DNA encoding murine IL-12 (mIL-12) was delivered into mice by systemic administration. Toxicity profiles of mice treated with DNA or rmIL-12 were compared. Results Systemic administration of Naked DNA encoding mIL-12 resulted in very similar toxicity as rmIL-12 with respect to liver enzyme, hematological and immunological profiles. Repeated injection of mIL-12 gene did not recover a high level of mIL-12 production as the first injection. Moreover, initial mIL-12 administration resulted in inhibition of subsequent reporter gene expression with both viral and non-viral promoters (CMV, human α-antitrypsin or chicken β-actin promoter). This transgene inhibition effect was entirely mediated by IFN-γ as the transgene expression was fully recovered in IFN-γ knockout mice. Conclusions Systemic IL-12 therapy, with either a protein or gene therapy approach, resulted in comparable liver and systemic toxicities. Refractoriness of mIL-12 production by subsequent administration of mIL-12 gene was observed. The transgene attenuation effect of IL-12 pre-dosing (either by IL-12 or rmIL-12), mediated by IFN-γ, provided important insights for the design of IL-12 combination gene therapy and the improvement of gene vectors for IL-12 therapy. The present results show that simple injection of Naked DNA could serve as a good model for in vivo evaluation of the toxicity of a candidate therapeutic gene. Copyright © 2001 John Wiley & Sons, Ltd.
D H Du Plessis - One of the best experts on this subject based on the ideXlab platform.
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Eliciting antigen-specific egg-yolk IgY with Naked DNA.
BioTechniques, 2001Co-Authors: M Romito, G J Viljoen, D H Du PlessisAbstract:Immunization with Naked DNA was used to elicit chicken egg yolk antibodies (IgY). Layer hens were inoculated with plasmid DNA encoding the enhanced green fluorescent protein, the fusion protein of Newcastle disease virus, and VP2 of African horse sickness virus. IgY was extracted from egg yolks by polyethylene glycol precipitation. Specific antibodies were present in the yolks of eggs from hens immunized with each of the three different plasmids. This approach to raising polyclonal antibodies obviates the need to produce and purify large quantities of proteins for immunization and can potentially yield large amounts of diagnostically or therapeutically useful reagents.
M Romito - One of the best experts on this subject based on the ideXlab platform.
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Eliciting antigen-specific egg-yolk IgY with Naked DNA.
BioTechniques, 2001Co-Authors: M Romito, G J Viljoen, D H Du PlessisAbstract:Immunization with Naked DNA was used to elicit chicken egg yolk antibodies (IgY). Layer hens were inoculated with plasmid DNA encoding the enhanced green fluorescent protein, the fusion protein of Newcastle disease virus, and VP2 of African horse sickness virus. IgY was extracted from egg yolks by polyethylene glycol precipitation. Specific antibodies were present in the yolks of eggs from hens immunized with each of the three different plasmids. This approach to raising polyclonal antibodies obviates the need to produce and purify large quantities of proteins for immunization and can potentially yield large amounts of diagnostically or therapeutically useful reagents.
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Short Technical Repor t Eliciting Antigen-Specific Egg-Yolk IgY with Naked DNA
2001Co-Authors: M Romito, Dion H. Du PlessiAbstract:Immunization with Naked DNA was use d to elicit chicken egg yolk antibodies (IgY) . Layer hens were inoculated with plasmi d DNA encoding the enhanced green fluore s cent protein, the fusion protein of Newcastl e disease virus, and VP2 of African hors e sickness virus. IgY was extracted from eg g yolks by polyethylene glycol precipitation . Specific antibodies were present in the yolk s of eggs from hens immunized with each o f the three different plasmids. This approac h to raising polyclonal antibodies obviate s the need to produce and purify large qua n tities of proteins for immunization and ca n potentially yield large amounts of diagno s tically or therapeutically useful reagents .
Ming Derg Lai - One of the best experts on this subject based on the ideXlab platform.
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Delivery of noncarrier Naked DNA vaccine into the skin by supersonic flow induces a polarized T helper type 1 immune response to cancer
The journal of gene medicine, 2008Co-Authors: Chi-chen Lin, Meng-chi Yen, Chiu Mei Lin, Shih Shien Huang, Huei Jiun Yang, Nan Haw Chow, Ming Derg LaiAbstract:Background DNA vaccine is a new and powerful approach to generate immunological responses against infectious disease and cancer. The T helper type (Th)1 immune response is usually required for generating effective anti-tumor responses. A microparticulate bombardment system can induce an immune response using very low amounts of DNA. Using nozzle aerodynamics, a low pressure gene gun has been developed to decrease the noise associated with high pressure gene guns. Particles are propelled by supersonic flow through this novel nozzle. To test whether this gun could inoculate a DNA vaccine that stimulates an anti-tumor Th1 immune response, we examined the effect of direct delivery of Naked DNA (i.e. without any carrier) on the anti-tumor immune response of mice. Methods The luciferase reporter plasmid DNA was delivered using a low-pressure biolistic device and expressed in C3H/HeN, BALB/c, and C57BL/6 mice. Results Plasmid DNA expression was mainly in the epidermis. Noncarrier Naked neu DNA vaccine and gold particle-coated neu DNA vaccine (at 1 µg per mouse) had similar anti-tumor effects in C3H mice. However, cytokine profile examination showed the Th1-bias of the response induced by Naked DNA vaccine and the Th2-bias of the response induced by coated DNA vaccine. Conclusions A shift in the immune response to favour enhanced tumor rejection can be achieved by skin delivery of Naked DNA vaccine. Copyright © 2008 John Wiley & Sons, Ltd.
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752. Delivery of Non-Microparticle Naked DNA Vaccine Using Supersonic Flow by a Low-Pressure Gene Gun
Molecular Therapy, 2006Co-Authors: Chi-chen Lin, Ying-chang Wang, Men-chi Yen, Ming Derg LaiAbstract:DNA vaccines are a new and powerful approach to generation of immunological responses against infectious disease and cancer. DNA can be delivered either into muscle by simple injection or into epidermal by gene gun. Intramuscular injection requires large amount of DNA (100microgram/per mouse) to elicit the immune response; in contrast, microparticlulate bombardment system can induce immune response using very low amount of DNA (1 microgram/per mouse). One disadvantage of gene gun bombardment is that non-biodegradable gold or tungsten may skew the immune response or cause adverse side effect when accumulated. In this report, we have demonstrated the direct delivery of Naked DNA without any microparticle using a modified gene gun. The modified gene gun is based on the aerodynamic theory that a supersonic flow is generated when the pressure difference between the inside and the outside pressure of the nozzle is greater than 1.9atm. This high speed airflow can carry the particle from stationary state to accelerate to an extreme high speed (200m/sec). In this way, the Naked DNA may be directly transformed into the mammalian cells. Previous study indicated that this modified gene gun achieve similar deliver efficacy as the gene gun commercially available using gold-coated DNA. In this report, we showed that the modified gene gun can achieve 10|[ndash]|40% delivery efficacy as the gold particle coated with DNA in Balb/C mice, C57BL/6 mice, and C3H mice using luciferase gene reporter driven by CMV promoter. Then, we examined the immune responses elicited by inoculation of DNA encoding HBV large surface protein expressed by its own promoter. The non-microparticle DNA and gold-coated DNA elicited similar humoral immunity as shown by antibody titer. Similar overall cytokine profile was induced by either type of DNA vaccine. Furthermore, we examined the therapeutic responses with the DNA vaccine against the extracellular domain of neu on the mouse tumor (MBT-2) naturally overexpressing neu in C3H mice. The results indicated that non-microparticle Naked neu DNA vaccine can achieve similar anti-tumor effect as the gold-coated neu DNA vaccine in C3H mice at the dose of 1 microgram/per mouse.
