Biomedical subjects

Bradley S Fletcher

Publications and source records attributed to Bradley S Fletcher.

13 recordsLinked to original sources

PubMed2006

Sleeping Beauty-mediated eNOS gene therapy attenuates monocrotaline-induced pulmonary hypertension in rats.

Pulmonary hypertension (PH) is a life-threatening disorder with high mortality rates and limited treatment options. Gene therapy is an alternative treatment strategy, yet viral vectors have inherent disadvantages including immune activation. The Sleeping Beauty (SB) transposon is a nonviral method of gene delivery that overcomes some of these drawbacks. A SB-based transposon harboring a constitutively active endothelial nitric oxide synthase (eNOS) gene was administered to Sprague-Dawley rats via tail vein injection using the carrier polyethylenimine. Two days after transposon delivery, monocrotaline (MCT) was administered to induce PH. Hemodynamic, histological, and molecular measurements were performed four weeks later. Animals coinjected with transposase showed a significant reduction in pulmonary arterial pressure (PABP, 31.67+/-6.03 mmHg, P<0.01), an attenuation of right ventricle (RV) to whole heart (WH) wt ratios (0.227+/-0.0252, P<0.05) and a decrease in the pulmonary vessel wall thickness index (36.87%, P<0.001), compared with those animals receiving the eNOS transposon and a nonfunctional transposase (PABP 44.33+/-4.04 mmHg; RV/WH ratio 0.280+/-0.01; wall thickness index 62.14%) or control animals receiving MCT injection alone (PABP 49.67+/-3.22 mmHg; RV/WH ratio 0.290+/-0.0265; wall thickness index 71.99%). The physiological improvements correlated with therapeutic gene expression, suggesting that transposon-based genetic approaches have utility in the treatment of PH.

Animals↗

PubMed2006

Sleeping Beauty-based gene therapy with indoleamine 2,3-dioxygenase inhibits lung allograft fibrosis.

Sleeping Beauty (SB) transposon is a natural nonviral gene transfer system that can mediate long-term transgene expression. Its potential utility in treating organ transplantation-associated long-term complications has not yet been explored. In the present study we generated an improved SB transposon encoding the human gene indoleamine-2,3-dioxygenase (hIDO), an enzyme that possesses both T cell-suppressive and antioxidant properties and selectively delivered the SB transposon in combination with a hyperactive transposase plasmid to donor lung using the cationic polymer polyethylenimine (PEI) as transfection reagent. This nonviral gene therapeutic approach led to persistent and uniform transgene expression in the rat lung tissue without noticeable toxicity and inflammation. Importantly, IDO activity produced by hIDO transgene showed a remarkable therapeutic response, as evident by near normal pulmonary function (peak airway pressure and oxygenation), histological appearance, and reduced collagen content in lung allografts. In addition, we established a hIDO-overexpressing type II cell line using the SB-based gene transfer system and found that hIDO-overexpressing lung cells effectively inhibited transforming growth factor-beta-stimulated fibroblast proliferation in vitro. In summary, the SB-based gene therapy with hIDO represents a new strategy for treating lung transplantation-associated chronic complications, e.g., obliterative bronchiolitis.

Animals↗

PubMed2006

Sustained FVIII expression and phenotypic correction of hemophilia A in neonatal mice using an endothelial-targeted sleeping beauty transposon.

Hemophilia A, deficiency of coagulation factor VIII (FVIII), is an attractive candidate for gene therapy as expression of modest amounts of FVIII can provide therapeutic benefit. Most gene transfer approaches for hemophilia have focused on the liver, as this is the major source of endogenous FVIII; however, increasing evidence suggests that endothelial cells are capable of synthesis and release of FVIII. Here the Sleeping Beauty (SB) transposon is employed to target long-term expression of the human B-domain-depleted FVIII gene (approved gene symbol F8) within lung endothelia of hemophilic mice. As the formation of inhibitory antibodies to FVIII has been a significant impediment toward achieving therapeutic plasma levels after gene or protein therapy, we chose to perform gene transfer in neonatal mice, which are more likely to be immune tolerant. Using this approach, low therapeutic levels of FVIII ( approximately 10%), as well as phenotypic correction of the bleeding disorder, were achieved in all animals that received the FVIII transposon and functional transposase throughout the duration of the study (24 weeks). Rechallenge of these animals with additional gene transfer did not result in significant increases in FVIII levels, due mainly to increases in inhibitory antibodies. These studies demonstrate the feasibility of using endothelial-targeted SB transposons for the treatment of hemophilia A.

Animals↗

PubMed2006

Sleeping beauty transposon-mediated nonviral gene therapy.

Safe and effective delivery of genetic material to mammalian tissues would significantly expand the therapeutic possibilities for a large number of medical conditions. Unfortunately, the promise of gene therapy has been hampered by technical challenges, the induction of immune responses, and inadequate expression over time. Despite these setbacks, progress continues to be made and the anticipated benefits may come to fruition for certain disorders. In terms of delivery, nonviral vector systems are particularly attractive as they are simple to produce, can be stored for long periods of time, and induce no specific immune responses. A significant drawback to nonviral systems has been the lack of persistent expression, as plasmids are lost or degraded when delivered to living tissues. The recent application of integrating transposons to nonviral gene delivery has significantly helped to overcome this obstacle, because it allows for genomic integration and long-term expression. Recent advances in transposon-based vector systems hold promise as new technologies that may unlock the potential of gene therapy; however, technical and safety issues still need refinement.

Animals↗

PubMed2005

Novel action of indoleamine 2,3-dioxygenase attenuating acute lung allograft injury.

RATIONALE: Lung allografts are prone to reperfusion injury and acute rejection, which, in addition to infiltrating lymphocytes, are accompanied by neutrophil infiltration and neutrophil-associated oxidative stress. Indoleamine 2,3-dioxygenase (IDO) is a unique cytosolic enzyme that possesses T-cell-suppressive and antioxidant properties. OBJECTIVES: The purpose of this study was to determine if genetic up-regulation of IDO could ameliorate acute lung allograft injury. METHODS: Lung orthotopic transplants were performed using Lewis donors and Sprague-Dawley rat recipients (allografts) or the same strain (isografts). Plasmid-encoding human IDO was delivered to donor lungs in vivo using a nonviral gene-transfer vector, polyethylenimine. Transplanted lungs were evaluated at 6 d post-transplantation based on pulmonary function, histology, inflammatory responses, and their associated oxidative stress. Basic biology of the IDO-overexpressing lung cells was evaluated in vitro in response to external oxidant. MEASUREMENTS AND MAIN RESULTS: This gene delivery method led to uniform transgene expression in lung tissue distributed in airway, alveolar epithelial, and endothelial cells. IDO overexpression in lung allografts resulted in a significant protective effect with improvement in functional properties (peak airway pressure and oxygenation) and histologic appearance. Although IDO was able to block local T-cell responses, it failed to abrogate neutrophilic infiltration and the inflammation-associated oxidative stress. IDO-enhanced lung cells were resistance to oxidant-induced necrosis and apoptosis by limiting intracellular reactive oxygen species formation. CONCLUSIONS: These results demonstrate that IDO prevents acute lung allograft injury through augmenting the local antioxidant defense system and inhibiting alloreactive T-cell responses.

Acute Disease↗

PubMed2005

Hyperactive transposase mutants of the Sleeping Beauty transposon.

Transposable elements have enormous potential to overcome one of the major hurdles in nonviral gene delivery, namely the lack of long-term gene expression. The Sleeping Beauty (SB) transposon is a promising vector system for nonviral gene therapy as it has the highest transposition activity of all known DNA transposons within mammalian cells. In an effort to generate a more efficient delivery vehicle, we conducted a systematic evaluation of several novel and previously identified SB transposase mutants. The results indicate that certain combinations of mutants do not enhance transposition, whereas others give a synergistic response. The most active mutant, designated HSB17, shows nearly 17-fold higher transposition activity compared to the original transposase SB10 when tested within the same expression cassette. In addition, synergistic activity is observed when this hyperactive mutant is combined with an improved transposon. Animal studies utilizing the hyperactive transposase show enhanced long-term reporter gene expression. These modifications further expand the utility of this transposon-based gene transfer system.

Amino Acid Sequence↗

PubMed2005

Modulation of epithelial morphology, monolayer permeability, and cell migration by growth arrest specific 3/peripheral myelin protein 22.

Peripheral myelin protein 22 (PMP22) is associated with a subset of hereditary peripheral neuropathies. Although predominantly recognized as a transmembrane constituent of peripheral nerve myelin, PMP22 is localized to epithelial and endothelial cell-cell junctions, where its function remains unknown. In this report, we investigated the role of PMP22 in epithelial biology. Expression of human PMP22 (hPMP22) slows cell growth and induces a flattened morphology in Madin-Darby canine kidney (MDCK) cells. The transepithelial electrical resistance (TER) and paracellular flux of MDCK monolayers are elevated by hPMP22 expression. After calcium switch, peptides corresponding to the second, but not the first, extracellular loop of PMP22 perturb the recovery of TER and paracellular flux. Finally, subsequent to wounding, epithelial monolayers expressing hPMP22 fail to migrate normally. These results indicate that PMP22 is capable of modulating several aspects of epithelial cell biology, including junctional permeability and wound closure.

Animals↗

PubMed2005

The WD-repeat protein GRWD1: potential roles in myeloid differentiation and ribosome biogenesis.

A cDNA fragment originally identified in U-937 cells as a vitamin D(3)-regulated gene is here designated the glutamate-rich WD-repeat (GRWD1) gene. WD-repeat proteins are a class of functionally divergent molecules that cooperate with other proteins to regulate cellular processes. GRWD1 encodes a 446-amino-acid protein containing a glutamate-rich region followed by four WD repeats. The yeast homologue of GRWD1, Rrb1, has been shown to be an essential protein involved in ribosome biogenesis. Northern analysis of GRWD1 message levels in the myeloid cell line HL-60 undergoing differentiation induced by vitamin D(3) or retinoic acid demonstrate downregulation coincident with slowing of cellular proliferation. A siRNA designed to downregulate GRWD1 similarly results in a decrease in cellular proliferation within 293 cells. Metabolic labeling of cells expressing the siRNA to GRWD1 shows a decrease in global protein synthesis. Finally, nuclear fractionation studies show cosedimentation of GRWD1 with preribosomal complexes, as well as the WD-repeat-containing protein Bop1, which has previously been implicated in ribosome biogenesis. These studies suggest that within mammalian cells GRWD1 plays a role in ribosome biogenesis and during myeloid differentiation its levels are regulated.

Base Sequence↗

PubMed2005

Impaired proteasome activity and accumulation of ubiquitinated substrates in a hereditary neuropathy model.

Accumulation of misfolded proteins and alterations in the ubiquitin-proteasome pathway are associated with various neurodegenerative conditions of the CNS and PNS. Aggregates containing ubiquitin and peripheral myelin protein 22 (PMP22) have been observed in the Trembler J mouse model of Charcot-Marie-Tooth disease type 1A demyelinating neuropathy. In these nerves, the turnover rate of the newly synthesized PMP22 is reduced, suggesting proteasome impairment. Here we show evidence of proteasome impairment in Trembler J neuropathy samples compared with wild-type, as measured by reduced degradation of substrate reporters. Proteasome impairment correlates with increased levels of polyubiquitinated proteins, including PMP22, and the recruitment of E1, 20S and 11S to aggresomes formed either spontaneously due to the Trembler J mutation or upon proteasome inhibition. Furthermore, myelin basic protein, an endogenous Schwann cell proteasome substrate, associates with PMP22 aggregates in affected nerves. Together, our data show that in neuropathy nerves, reduced proteasome activity is coupled with the accumulation of ubiquitinated substrates, and the recruitment of proteasomal pathway constituents to aggregates. These results provide novel insights into the mechanism by which altered degradation of Schwann cell proteins may contribute to the pathogenesis of certain PMP22 neuropathies.

Animals↗

PubMed2004

Transposon-based RNAi delivery system for generating knockdown cell lines.

RNA interference is rapidly becoming a powerful tool for genetic analyses in mammalian systems. A potential drawback to transient small inhibitory RNA silencing is the short duration of downregulation it confers, usually only 24-72h. Viral-based vector systems for the long-term delivery of RNA hairpins have been developed, yet they require expertise in viral production and transduction. Here we describe a simple plasmid-based system for the generation of long-term gene knockdown utilizing RNA interference combined with the gene delivery capabilities of the mammalian Tc1-like transposon Sleeping Beauty. Designated Maleficent, this system is shown to downregulate exogenous expression of GFP in a constitutively positive cell line. In addition, targeting of the endogenously expressed lamin A gene results in long-term silencing with significant reduction in protein levels (> 95%). Maleficent therefore provides a relatively easy, efficient, and stable means of delivering RNAi hairpins to generate long-term gene-specific knockdown cell lines.

Animals↗

PubMed2004

Endothelial targeting of the Sleeping Beauty transposon within lung.

Endothelial cells have complex roles in the pathophysiology of vascular and heart disease and are increasingly being recognized as targets for gene therapy. The intravenous administration of plasmid DNA complexed to lipid tends to target transfection of endothelial cells within the lung; however, expression from the transgene remains transient. Here we utilize the integrating capability of the Sleeping Beauty (SB) transposon for durable gene transfer within lung endothelia. To restrict expression of the transgene, an endothelial cell-specific promoter, endothelin-1, was placed within the transposon. Further refinements to the transposon increased in vitro transposition efficiency by 3.6-fold. Utilizing this optimized transposon we evaluated the expression of two reporter molecules, secreted alkaline phosphatase (SEAP) and intracellular GFP, following administration of DNA-polyethylenimine complexes to mice. Long-term expression (>2 months) of SEAP occurred only with cotransfection of adequate amounts of transposase. Localization studies using the GFP reporter, at 3 days and 6 weeks postinjection, demonstrated that the majority of transgene-expressing cells were of endothelial origin, while the second most abundant cell type was type II pneumocyte. These results suggest that the SB transposon can be adapted to target particular cell types, in this case, endothelial cells. Such an approach may be useful for gene therapy paradigms involving the long-term modulation of vascular and endothelial cell biology.

Alkaline Phosphatase↗

PubMed2003

Glucose deprivation enhances targeting of GLUT1 to lipid rafts in 3T3-L1 adipocytes.

Glucose deprivation dramatically increases glucose transport activity in 3T3-L1 adipocytes without changing the concentration of GLUT1 in the plasma membrane (PM). Recent data suggest that subcompartments within the PM, specifically lipid rafts, may sequester selected proteins and alter their activity. To evaluate this possibility, we examined the distribution of GLUT1 in Triton X-100-soluble and -insoluble fractions. Our data show that 77% of the GLUT1 pool in PMs isolated from control 3T3-L1 adipocytes was extracted by 0.2% Triton X-100. After glucose deprivation for 12 h, only 56% of GLUT1 was extracted by detergent. In contrast, there was a twofold increase in the GLUT1 content of the detergent-resistant fraction. To evaluate whether GLUT1 interacts with a specific protein within lipid rafts, we focused on stomatin, recently shown to interact with and inhibit GLUT1 activity. Stomatin is distributed about equally between the PM and the biosynthetic compartments, and its expression is not affected by glucose deprivation. Nearly 90% of the PM pool of stomatin is in detergent-resistant lipid rafts. In normal 3T3-L1 adipocytes, we were unable to demonstrate an interaction between GLUT1 and stomatin in coimmunoprecipitation experiments. However, in stomatin-overexpressing cells, there was clear coprecipitation of stomatin with GLUT1 antibodies. Glucose deprivation increased this interaction threefold, which may reflect the increase of GLUT1 in lipid rafts. Despite this, there was little change in transport activity in glucose-deprived, stomatin-overexpressing cells vs. that in control cells. Thus GLUT1 interacts with stomatin in lipid rafts, but this interaction per se does not alter transport activity. Rather, stomatin may serve as an anchor for GLUT1 in lipid rafts, the environment of which favors activation.

3T3 Cells↗

PubMed2002

Glycogen phosphorylase is activated in response to glucose deprivation but is not responsible for enhanced glucose transport activity in 3T3-L1 adipocytes.

We have previously shown that glucose deprivation activates glucose transport in a time- and protein synthesis-dependent fashion in 3T3-L1 adipocytes, a mouse cell line. Coincident with this is loss of glycogen. Because glycogen phosphorylase (GP) is responsible for glycogen degradation, we have examined its regulation to determine the relationship between transport activation and glycogen turnover. We first cloned the adipose GP cDNA and found sequence similarity to rat and human liver GP. Because the mouse liver GP cDNA sequence was unavailable, we cloned this cDNA as well and showed 100% identity between mouse adipose and liver sequences. A 3.1 kb transcript was readily observed in total RNA isolated from mouse liver or adipose by Northern blot analysis but, surprisingly, not in either total or poly(A) selected RNA from 3T3-L1 adipocytes. To evaluate regulation in 3T3-L1 adipocytes, we amplified GP mRNA from total RNA using multiplex, semi-quantitative PCR but found that expression did not change in response to deprivation. GP protein levels did not change either. However, endogenous GP activity from glucose-deprived cells was significantly elevated relative to controls, due to an increase in the phosphorylated form of GP (GPa). Finally, we overexpressed GP to determine its direct influence on the glucose transport system. These results were negative, which suggests that the nutrient control of glucose transport and GP occurs independently despite kinetic similarities in transport activation and glycogen turnover.

3T3 Cells↗