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Long-term Expression (long-term + expression)
Selected AbstractsGene therapy in epilepsyEPILEPSIA, Issue 1 2009Véronique Riban Summary Results from animal models suggest gene therapy is a promising new approach for the treatment of epilepsy. Several candidate genes such as neuropeptide Y and galanin have been demonstrated in preclinical studies to have a positive effect on seizure activity. For a successful gene therapy-based treatment, efficient delivery of a transgene to target neurons is also essential. To this end, advances have been made in the areas of cell transplantation and in the development of recombinant viral vectors for gene delivery. Recombinant adeno-associated viral (rAAV) vectors in particular show promise for gene therapy of neurological disorders due to their neuronal tropism, lack of toxicity, and stable persistence in neurons, which results in robust, long-term expression of the transgene. rAAV vectors have been recently used in phase I clinical trials of Parkinson's disease with an excellent safety profile. Prior to commencement of phase I trials for gene therapy of epilepsy, further preclinical studies are ongoing including evaluation of the therapeutic benefit in chronic models of epileptogenesis, as well as assessment of safety in toxicological studies. [source] An active triple-catalytic hybrid enzyme engineered by linking cyclo-oxygenase isoform-1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protectorFEBS JOURNAL, Issue 23 2008Ke-He Ruan It remains a challenge to achieve the stable and long-term expression (in human cell lines) of a previously engineered hybrid enzyme [triple-catalytic (Trip-cat) enzyme-2; Ruan KH, Deng H & So SP (2006) Biochemistry45, 14003,14011], which links cyclo-oxygenase isoform-2 (COX-2) to prostacyclin (PGI2) synthase (PGIS) for the direct conversion of arachidonic acid into PGI2 through the enzyme's Trip-cat functions. The stable upregulation of the biosynthesis of the vascular protector, PGI2, in cells is an ideal model for the prevention and treatment of thromboxane A2 (TXA2)-mediated thrombosis and vasoconstriction, both of which cause stroke, myocardial infarction, and hypertension. Here, we report another case of engineering of the Trip-cat enzyme, in which human cyclo-oxygenase isoform-1, which has a different C-terminal sequence from COX-2, was linked to PGI2 synthase and called Trip-cat enzyme-1. Transient expression of recombinant Trip-cat enzyme-1 in HEK293 cells led to 3,5-fold higher expression capacity and better PGI2 -synthesizing activity as compared to that of the previously engineered Trip-cat enzyme-2. Furthermore, an HEK293 cell line that can stably express the active new Trip-cat enzyme-1 and constantly synthesize the bioactive PGI2 was established by a screening approach. In addition, the stable HEK293 cell line, with constant production of PGI2, revealed strong antiplatelet aggregation properties through its unique dual functions (increasing PGI2 production while decreasing TXA2 production) in TXA2 synthase-rich plasma. This study has optimized engineering of the active Trip-cat enzyme, allowing it to become the first to stably upregulate PGI2 biosynthesis in a human cell line, which provides a basis for developing a PGI2 -producing therapeutic cell line for use against vascular diseases. [source] Ionizing radiation-induced long-term expression of senescence markers in mice is independent of p53 and immune statusAGING CELL, Issue 3 2010Oanh N. L. Le Summary Exposure to IR has been shown to induce the formation of senescence markers, a phenotype that coincides with lifelong delayed repair and regeneration of irradiated tissues. We hypothesized that IR-induced senescence markers could persist long-term in vivo, possibly contributing to the permanent reduction in tissue functionality. Here, we show that mouse tissues exposed to a sublethal dose of IR display persistent (up to 45 weeks, the maximum time analyzed) DNA damage foci and increased p16INK4a expression, two hallmarks of cellular senescence and aging. BrdU-labeling experiments revealed that IR-induced damaged cells are preferentially eliminated, at least partially, in a tissue-dependent manner. Unexpectedly, the accumulation of damaged cells was found to occur independent from the DNA damage response modulator p53, and from an intact immune system, as their levels were similar in wild-type and Rag2,/, ,C,/, mice, the latter being deficient in T, B, and NK cells. Together, our results provide compelling evidence that exposure to IR induces long-term expression of senescence markers in vivo, an effect that may contribute to the reduced tissue functionality observed in cancer survivors. [source] The roles of calcium/calmodulin-dependent and Ras/mitogen-activated protein kinases in the development of psychostimulant-induced behavioral sensitizationJOURNAL OF NEUROCHEMISTRY, Issue 1 2003Stephanie C. Licata Abstract Although the development of behavioral sensitization to psychostimulants such as cocaine and amphetamine is confined mainly to one nucleus in the brain, the ventral tegmental area (VTA), this process is nonetheless complex, involving a complicated interplay between neurotransmitters, neuropeptides and trophic factors. In the present review we present the hypothesis that calcium-stimulated second messengers, including the calcium/calmodulin-dependent protein kinases and the Ras/mitogen-activated protein kinases, represent the major biochemical pathways whereby converging extracellular signals are integrated and amplified, resulting in the biochemical and molecular changes in dopaminergic neurons in the VTA that represent the critical neuronal correlates of the development of behavioral sensitization to psychostimulants. Moreover, given the important role of calcium-stimulated second messengers in the expression of behavioral sensitization, these signal transduction systems may represent the biochemical substrate through which the transient neurochemical changes associated with the development of behavioral sensitization are translated into the persistent neurochemical, biochemical and molecular alterations in neuronal function that underlie the long-term expression of psychostimulant-induced behavioral sensitization. [source] Gene transfer for hemophilia: can therapeutic efficacy in large animals be safely translated to patients?JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2005K. HIGH Summary., Gene transfer is a novel area of therapeutics in which the active agent is a nucleic acid rather than a protein or small molecule. As early as 1997, investigators reported long-term expression of therapeutic levels of factor IX using gene transfer techniques in hemophilia B mice, and similar data were thereafter reported in mice with hemophilia A. Efforts to translate these results to hemophilic dog models at first yielded only marginally therapeutic levels (1%,2% normal circulating levels), but within the past few years have achieved levels in the range of 10%,20% through multiple different gene transfer strategies. Early phase clinical testing has revealed that many aspects of gene transfer in humans were accurately predicted by studies in hemophilic dogs, but that other aspects were not, and were only appreciated as a result of clinical testing. Studies in the next few years will determine whether the problems identified in preclinical and early phase clinical testing can be solved to develop a therapeutic gene transfer approach to hemophilia. [source] Exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis in grape berriesPHYSIOLOGIA PLANTARUM, Issue 2 2003Ashraf El-Kereamy The treatment of grape berries (Vitis vinifera L. cv. Cabernet Sauvignon) with the ethylene-releasing compound, 2-chloroethylphosphonic acid (2-CEPA), at veraison is a method known to enhance grape skin colour. We observed that it produced a 6-fold increase, up to 30 pmol g,1 FW, of the cluster internal ethylene compared to untreated controls within the 24 h following treatment. This ethylene upsurge was associated with increased levels of chalcone synthase (CHS) and flavanone 3-hydroxylase (F3H) transcripts, which persisted over the following 20 days. Transcript levels of leucoanthocyanidin dioxygenase (LDOX) and UDP glucose-flavonoid 3- O -glucosyl transferase (UFGT) were similarly enhanced by 2-CEPA, although to a lesser extent. The effect on UFGT was confirmed at the protein level by an immunoblot analysis. The transcript accumulation of dihydroflavonol 4-reductase (DFR) was unaffected by 2-CEPA treatment. Examination of the levels of CHS, F3H and UFGT mRNAs in berries during bunch exposure to ethylene, revealed elevated levels of each transcript within the first 6 h of treatment when compared to nonethylene-treated controls. HPLC analyses of berry skin extracts showed that levels of each of the anthocyanins analysed (delphinidin, cyanidin, petunidin, peonidin and malvidin) increased over the 10 days following the ethylene burst, and decreased thereafter. However, anthocyanin levels at harvest were still higher in ethylene treated grapes than in controls. This data is the first evidence that ethylene triggers gene expression related to anthocyanin synthesis in grapes, and in addition, our results also confirm the existence of other regulatory modes in the anthocyanin biosynthetic pathway. [source] Growth factors improve gene expression after lentiviral transduction in human adult and fetal hepatocytesTHE JOURNAL OF GENE MEDICINE, Issue 2 2007Clare Selden Abstract Background Lentiviral vectors may be vectors of choice for transducing liver cells; they mediate integration in quiescent cells and offer potential for long-term expression. In adult liver, hepatocytes are generally mitotically quiescent. There has been controversy as to the necessity for lentiviral vector target cells to be in the cell cycle; currently, there is consensus that effective transduction can be achieved in quiescent hepatocytes, by using virus at high titre. However, transduction approaches which reduce the multiplicities of infection (MOIs) required provide potential benefit of cost and safety for therapeutic use. Methods We used two late-generation HIV-based lentiviral vector systems (pHR-SIN-cppT SGW and pRRLSIN.cPPT.PGK.WPRE) encoding LacZ/GFP reporter genes to transduce adult and fetal human hepatocytes in vitro + /, growth factors, hepatocyte growth factor (HGF) and epidermal growth factor (EGF). Green fluorescent protein (GFP) expression was observed microscopically, and quantified by fluorescence spectrometry for protein expression, fluorescence-activated cell sorting (FACS) analysis to identify the proportion of cells expressing GFP, and real-time quantitative polymerase chain reaction (PCR) for number of integrations. Results Gene expression following lentiviral transduction of human liver cells in vitro was markedly enhanced by the growth factors HGF and EGF. In adult cells growth factors led to a greater proportion of cells expressing more GFP per cell, from more integration events. In human fetal cells, the proportion of transduced hepatocytes remained identical, but cells expressed more GFP protein. Conclusions This has implications for the design of regimes for liver cell gene therapy, allowing marked reduction of MOIs, and reducing both cost and risk of viral-mediated toxicity. Copyright © 2007 John Wiley & Sons, Ltd. [source] Adenoviral vectors for gene transfer and therapyTHE JOURNAL OF GENE MEDICINE, Issue S1 2004Christoph Volpers Abstract Due to the very efficient nuclear entry mechanism of adenovirus and its low pathogenicity for humans, adenovirus-based vectors have become gene delivery vehicles that are widely used for transduction of different cell types, especially for quiescent, differentiated cells, in basic research, in gene therapy applications, and in vaccine development. As an important basis for their use as gene medicine, adenoviral vectors can be produced in high titers, they can transduce cells in vivo with transgenes of more than 30 kb, and they do not integrate into the host cell genome. Recent advances in the development of adenoviral vectors have brought considerable progress on issues like target cell specificity and tropism modification, long-term expression of the transgene, as well as immunogenicity and toxicity in vivo, and have suggested that the different generations of non-replicative and replicative vectors available today will each suit best for certain applications. Copyright © 2004 John Wiley & Sons, Ltd. [source] Comparison of wild-type and class I integrase mutant-FIV vectors in retina demonstrates sustained expression of integrated transgenes in retinal pigment epitheliumTHE JOURNAL OF GENE MEDICINE, Issue 12 2003Nils Loewen Abstract Background In neonatal and adult rodent retina, substantial lentiviral vector expression has been detected primarily in retinal pigment epithelium (RPE), except in very young animals (2,5 days post-natal). In non-retinal tissues, studies of lentiviral vectors have utilized various controls. Among the most stringent are class I integrase mutants, which selectively block the integration reaction while leaving all other gag/pol -encoded functions intact. For HIV-1 vectors injected into brain, these have been used to simultaneously control for pseudotransduction and verify that long-term expression requires integration. Such experiments compare particles that differ only in a single amino acid within a single enzyme that forms a very small molar fraction of the virion. Class I integrase mutants have not been described for feline immunodeficiency virus (FIV) integrase, or tested in the eye for any lentiviral vector. Methods We compared subretinally and intravitreally injected FIV vectors and followed animals for up to 7 months, a duration that exceeds prior studies. We also compared the wild-type (WT) vector with one incorporating a single class I amino acid mutation in FIV integrase (D66V). A mock vector (packaging construct absent) was an alternative control. All vectors were vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped and were injected on day 7 of life. One group of animals received either subretinal or intravitreal injections of WT vector in the right eyes. Control left eyes were injected with mock vector. These animals were sacrificed at 2 or 7 days post-injection. A second group received subretinal injections of either WT vector or equivalent D66V vector (reverse transcriptase-normalized to WT), and were analyzed after 2, 3 and 7 months. All eyes were scored for marker gene (,-galactosidase) expression by an observer blinded to vector assignments. Results Subretinal FIV vector injections were much more effective than intravitreal injections. The RPE was the principal retinal layer transduced by the WT vector, and at least 50% of the area of the retina expressed the marker gene at 3 and 7 months. Occasional cells in inner retinal layers also expressed ,-galactosidase at these time points. The sustained retinal expression produced by subretinally injected vector was blocked by the D66V mutation. Conclusions These results show that class I integrase mutant FIV vectors are useful control vectors, and that VSV-G-pseudotyped FIV vectors produce extensive retinal expression for at least 215 days, the longest duration yet reported for lentiviral vectors in retina. Transgene expression is mostly restricted to RPE after post-natal day 7 in rats, suggesting that FIV vectors could be used to target RPE for gene therapy. Copyright © 2003 John Wiley & Sons, Ltd. [source] Inhibition of cartilage degradation: A combined tissue engineering and gene therapy approachARTHRITIS & RHEUMATISM, Issue 3 2003Wael Kafienah Objective To determine if tissue-engineered cartilage can be protected from cytokine-induced degradation using a gene therapy approach. Methods Chemical and pantropic retroviral gene transfer methodologies were compared for their ability to introduce a luciferase reporter gene into adult bovine cartilage chondrocytes grown in monolayer. Pantropic retrovirus was then used to transduce these cells with human tissue inhibitor of metalloproteinases 1 (TIMP-1), and the stability of expression in monolayer or pellet culture was monitored for 6 weeks. Untransduced and TIMP-1,transduced cells were also used to tissue engineer 3-dimensional cartilage constructs that were then challenged with interleukin-1 (IL-1) for 4 weeks. Conditioned media and residual cartilage were collected for analysis of matrix components, including type II collagen and proteoglycans, and for TIMP-1 production and matrix metalloproteinase (MMP) activity. Results Chemical transfection of adult bovine chondrocytes gave rise to short-lived reporter expression that was virtually undetectable after 4 weeks of culture. In contrast, pantropic retroviral transduction gave rise to stable expression that persisted at a high level for at least 6 weeks. Pantropic transduction of the cells with TIMP-1 gave rise to similar long-term expression, both in monolayer and pellet cultures. TIMP-1,transduced tissue-engineered cartilage also retained TIMP-1 expression for an additional 4 weeks of culture in the presence of IL-1. Compared with control samples, TIMP-1,transgenic cartilage resisted the catabolic effects of IL-1, with MMP activity reduced to basal levels and a decreased loss of type II collagen. Conclusion Pantropic retroviral transduction permits long-term expression of potentially therapeutic transgenes in adult tissue-engineered cartilage. While TIMP-1 transduction could be used to prevent collagen breakdown, alternative transgenes may be necessary to protect cartilage proteoglycans. [source] Translating Sleeping Beauty transposition into cellular therapies: Victories and challengesBIOESSAYS, Issue 9 2010Zsuzsanna Izsvák Abstract Recent results confirm that long-term expression of therapeutic transgenes can be achieved by using a transposon-based system in primary stem cells and in vivo. Transposable elements are natural DNA transfer vehicles that are capable of efficient genomic insertion. The latest generation, Sleeping Beauty transposon-based hyperactive vector (SB100X), is able to address the basic problem of non-viral approaches , that is, low efficiency of stable gene transfer. The combination of transposon-based non-viral gene transfer with the latest improvements of non-viral delivery techniques could provide a long-term therapeutic effect without compromising biosafety. The new challenges of pre-clinical research will focus on further refinement of the technology in large animal models and improving the safety profile of SB vectors by target-selected transgene integration into genomic "safe harbors." The first clinical application of the SB system will help to validate the safety of this approach. [source] | |||||||||||||