Pre-treatment with elastase improves the efficiency of percutaneous adenovirus-mediated gene transfer to the arterial media.
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The endothelium and internal elastic lamina (IEL) appear to be the main barriers to adenovirus-mediated gene transfer to medial smooth muscle cells (SMC). The present randomized study tested whether controlled incubation with elastase enhanced the efficiency of catheter-based gene transfer to medial SMC by adenoviral vectors. After an initial safety dose ranging study, rabbits underwent balloon abrasion of the iliac endothelium followed by local incubation of either elastase (2 x 10(-7) IU over 5 min) or saline using a double balloon catheter (DBC). Then, adenoviral vectors (5 x 10(9) p.f.u.) carrying Cmv-Luc or RSV-beta gal reporter genes were instilled for 30 min. Three days later, the number of medial SMC expressing lacZ was increased in the elastase-treated arteries compared with saline-treated arteries (7.2 +/- 2.5 versus 2.3 +/- 0.9 cells per section, P = 0.003). Likewise, the amount of luciferase protein product was increased (70 +/- 32 versus 36 +/- 15 pg luciferase/mg tissue, P = 0.03). No vessel enlargement, light or electron microscopic evidence of injury or inflammation was seen in elastase-treated arteries up to 7 weeks. Preincubation with elastase increased transduction efficiency of catheter-based gene delivery of replication-defective adenoviral vectors to rabbit iliac arteries without detectable arterial damage. (+info)
Immune responses limit adenovirally mediated gene expression in the adult mouse eye.
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In order to investigate the immunological consequences of gene transfer to the eye using viral vectors, adenovirus carrying a lacZ reporter gene (AV.LacZ) was injected either subretinally, subconjunctivally or into the anterior chamber of three groups of adult mice: immunocompetent or transiently immunosuppressed BALB/c mice and congenic immunodeficient nude mice. Adenovirally mediated lacZ expression persisted for approximately 3 weeks following injection of the vector into the anterior chamber, retina or extra ocular tissues of the conjuctiva of BALB/c mice. It appears that T cell-mediated immune responses limit the duration of AV-mediated ocular gene expression in adult mice since lacZ gene expression was detected for at least 15 weeks in T cell-deficient BALB/c nude mice, although the level of transgene expression decreased with time. Since intra-ocular AV-mediated gene expression was not significantly longer than extra-ocular expression, it appears that the eye is not normally immune-privileged with respect to viral vectors. Inflammatory cells were detected in the vitreous after anterior chamber injection and in the retina after subretinal injection of adenovirus. The presence of both CD4+ and CD8+ T cells was established by immunophenotyping. Reinjection of BALB/c mice resulted in rapid decline in reporter gene expression, but successful readministration was possible in the case of immunodeficient nude mice. However, after transient depletion of T cells, achieved by intraperitoneal injection of both CD8- and CD4-specific antibodies, the duration of expression in BALB/c mice was longer in the eye (at least 12 weeks, again with decrease in level over time), than in extra-ocular tissues (8 weeks) provided the animal was not reinjected with virus raising the possibility of partial ocular immune-privilege after transient immunosuppression. (+info)
High efficiency gene transfer to the central nervous system of rodents and primates using herpes virus vectors lacking functional ICP27 and ICP34.5.
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The safe and efficient use of herpes simplex virus (HSV)-based vectors to deliver genes of potentially therapeutic benefit to the central nervous system will require their effective disablement by the inactivation of viral genes required for lytic growth. Here we report that viruses lacking functional genes for ICP27 (which is required for growth in all cell types) and ICP34.5 (which is required for growth in nondividing cell types) can deliver a marker gene to both the rodent and primate CNS with high efficiency whilst producing relatively minimal damage and having no effect on sodium currents in dorsal root ganglion neurons. Such viruses paradoxically deliver genes at much higher efficiency than the less disabled single mutant lacking ICP34.5 alone and also, as expected, produce less damage in vivo. Moreover, unlike the single mutant lacking ICP27 the double mutant viruses cannot revert to wild-type by acquistion of complimenting gene sequences during growth of virus stocks in vitro on dividing cells expressing ICP27 since artificial expression of ICP34.5 in these cells is not required. Such ICP27-; ICP34.5- viruses thus offer a platform for the development of vectors which are sufficiently safe for ultimate use in human gene therapy. (+info)
The polysulfonated compound suramin blocks adsorption and lateral difusion of herpes simplex virus type-1 in vero cells.
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Several polysulfonate compounds have been shown to have the potential to inhibit the replication of herpesviruses by blocking binding and penetration of the host cell. We analyzed the actions of the polysulfonate compound suramin on the replication of herpes simplex virus type 1 (HSV-1) and compared them with the actions of heparin. We used the expression of a reporter gene (beta-galactosidase) recombined into the latency-associated transcript region of the 17syn+ strain of HSV-1 to quickly evaluate productive cycle activity and have shown that it can be directly correlated with virus replication under the conditions used. We find that suramin, like heparin, blocks the binding of HSV-1 to the cell membrane. Also, suramin efficiently blocks the cell-to-cell spread of the virus; this effect has not been previously reported. Our control experiments demonstrate that heparin also has some effect on intercellular spread of HSV-1 but to a significantly lesser degree than does suramin. We suggest that suramin and related polysulfonate compounds have potential for developing of antiherpes treatments. (+info)
Enrichment of transiently transfected mesangial cells by cell sorting after cotransfection with GFP.
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Early passage mesangial cells, like many other nonimmortalized cultured cells, can be difficult to transfect. We devised a simple method to improve the efficiency of transient protein expression under the transcriptional control of promoters in conventional plasmid vectors in rat mesangial cells. We used a vector encoding modified green fluorescent protein (GFP) and sterile fluorescence-activated cell sorting (FACS) to select a population consisting of >90% GFP-expressing cells from passaged nonimmortalized cultures transfected at much lower efficiency. Only 10% transfection efficiency was noted with a beta-galactosidase expression vector alone, but cotransfection with GFP followed by FACS and replating of GFP+ cells yielded greater than fivefold enrichment of cells with detectable beta-galactosidase activity. To demonstrate the expression of a properly oriented and processed membrane protein, we cotransfected GFP with a natriuretic peptide clearance receptor (NPR-C) expression vector. Plasmid-dependent cell surface NPR-C density was enhanced by 89% after FACS, though expression remained lower in selected mesangial cells than in the CHO cell line transfected with the same vector. We conclude that cotransfection of rat mesangial cells with GFP, followed by FACS, results in improvement in transient transfection efficiencies to levels that should suffice for many applications. (+info)
A new class of repression modules is critical for heme regulation of the yeast transcriptional activator Hap1.
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Heme plays key regulatory roles in numerous molecular and cellular processes for systems that sense or use oxygen. In the yeast Saccharomyces cerevisiae, oxygen sensing and heme signaling are mediated by heme activator protein 1 (Hap1). Hap1 contains seven heme-responsive motifs (HRMs): six are clustered in the heme domain, and a seventh is near the activation domain. To determine the functional role of HRMs and to define which parts of Hap1 mediate heme regulation, we carried out a systematic analysis of Hap1 mutants with various regions deleted or mutated. Strikingly, the data show that HRM1 to -6, located in the previously designated Hap1 heme domain, have little impact on heme regulation. All seven HRMs are dispensable for Hap1 repression in the absence of heme, but HRM7 is required for Hap1 activation by heme. More importantly, we show that a novel class of repression modules-RPM1, encompassing residues 245 to 278; RPM2, encompassing residues 1061 to 1185; and RPM3, encompassing residues 203 to 244-is critical for Hap1 repression in the absence of heme. Biochemical analysis indicates that RPMs mediate Hap1 repression, at least partly, by the formation of a previously identified higher-order complex termed the high-molecular-weight complex (HMC), while HRMs mediate heme activation by permitting heme binding and the disassembly of the HMC. These findings provide significant new insights into the molecular interactions critical for Hap1 repression in the absence of heme and Hap1 activation by heme. (+info)
Cooperative interaction between GATA-4 and GATA-6 regulates myocardial gene expression.
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Two members of the GATA family of transcription factors, GATA-4 and GATA-6, are expressed in the developing and postnatal myocardium and are equally potent transactivators of several cardiac promoters. However, several in vitro and in vivo lines of evidence suggest distinct roles for the two factors in the heart. Since identification of the endogenous downstream targets of GATA factors would greatly help to elucidate their exact functions, we have developed an adenovirus-mediated antisense strategy to specifically inhibit GATA-4 and GATA-6 protein production in postnatal cardiomyocytes. Expression of several endogenous cardiac genes was significantly down-regulated in cells lacking GATA-4 or GATA-6, indicating that these factors are required for the maintenance of the cardiac genetic program. Interestingly, transcription of some genes like the alpha- and beta-myosin heavy-chain (alpha- and beta-MHC) genes was preferentially regulated by GATA-4 due, in part, to higher affinity of GATA-4 for their promoter GATA element. However, transcription of several other genes, including the atrial natriuretic factor and B-type natriuretic peptide (ANF and BNP) genes, was similarly down-regulated in cardiomyocytes lacking one or both GATA factors, suggesting that GATA-4 and GATA-6 could act through the same transcriptional pathway. Consistent with this, GATA-4 and GATA-6 were found to colocalize in postnatal cardiomyocytes and to interact functionally and physically to provide cooperative activation of the ANF and BNP promoters. The results identify for the first time bona fide in vivo targets for GATA-4 and GATA-6 in the myocardium. The data also show that GATA factors act in concert to regulate distinct subsets of genes, suggesting that combinatorial interactions among GATA factors may differentially control various cellular processes. (+info)
The homeobox gene Msx1 is expressed in a subset of somites, and in muscle progenitor cells migrating into the forelimb.
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In myoblast cell cultures, the Msx1 protein is able to repress myogenesis and maintain cells in an undifferentiated and proliferative state. However, there has been no evidence that Msx1 is expressed in muscle or its precursors in vivo. Using mice with the nlacZ gene integrated into the Msx1 locus, we show that the reporter gene is expressed in the lateral dermomyotome of brachial and thoracic somites. Cells from this region will subsequently contribute to forelimb and intercostal muscles. Using Pax3 gene transcripts as a marker of limb muscle progenitor cells as they migrate from the somites, we have defined precisely the somitic origin and timing of cell migration from somites to limb buds in the mouse. Differences in the timing of migration between chick and mouse are discussed. Somites that label for Msx1(nlacZ )transgene expression in the forelimb region partially overlap with those that contribute Pax3-expressing cells to the forelimb. In order to see whether Msx1 is expressed in this migrating population, we have grafted somites from the forelimb level of Msx1(nlacZ )mouse embryos into a chick host embryo. We show that most cells migrating into the wing field express the Msx1(nlacZ )transgene, together with Pax3. In these experiments, Msx1 expression in the somite depends on the axial position of the graft. Wing mesenchyme is capable of inducing Msx1 transcription in somites that normally would not express the gene; chick hindlimb mesenchyme, while permissive for this expression, does not induce it. In the mouse limb bud, the Msx1(nlacZ )transgene is downregulated prior to the activation of the Myf5 gene, an early marker of myogenic differentiation. These observations are consistent with the proposal that Msx1 is involved in the repression of muscle differentiation in the lateral half of the somite and in limb muscle progenitor cells during their migration. (+info)