Current understanding of key transcription factors regulating angiogenesis is limited. Here we show that RNA-cleaving phosphodiester-linked DNA-based enzymes (DNAzymes), targeting a specific motif in ...the 5' untranslated region of early growth response (Egr-1) mRNA, inhibit Egr-1 protein expression, microvascular endothelial cell replication and migration, and microtubule network formation on basement membrane matrices. Egr-1 DNAzymes blocked angiogenesis in subcutaneous Matrigel plugs in mice, an observation that was independently confirmed by plug analysis in Egr-1-deficient animals, and inhibited MCF-7 human breast carcinoma growth in nude mice. Egr-1 DNAzymes suppressed tumor growth without influencing body weight, wound healing, blood coagulation or other hematological parameters. These agents inhibited endothelial expression of fibroblast growth factor (FGF)-2, a proangiogenic factor downstream of Egr-1, but not that of vascular endothelial growth factor (VEGF). Egr-1 DNAzymes also repressed neovascularization of rat cornea. Thus, microvascular endothelial cell growth, neovascularization, tumor angiogenesis and tumor growth are processes that are critically dependent on Egr-1.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Summary Background The nuclear transcription factor c-Jun is preferentially expressed in basal-cell carcinoma. Dz13 is a deoxyribozyme that targets JUN messenger RNA and has inhibited the growth of a ...range of tumours in mice. We did a phase 1 study to assess safety and tolerability in human beings. Methods Adults with nodular basal-cell carcinoma were recruited from Royal Prince Alfred Hospital, Sydney, Australia, between September, 2010, and October, 2011. Patients were assigned to receive one intratumoral injected dose of 10, 30, or 100 μg Dz13, in a 50 μL volume of lipid carrier, and were assessed for adverse effects in the first 24 h then at 7, 14, and 28 days after injection. Treated tumours were surgically excised 14 days after injection and compared with the baseline biopsy samples for expression of c-Jun and tumorigenesis markers. Findings Nine patients were recruited, of whom three received each dose of Dz13. All patients completed the study with no drug-related serious adverse events. No systemic Dz13 exposure was detected. c-Jun expression was reduced in the excised tumours of all nine (100%) patients, compared with baseline, and histological tumour depth had decreased in five (56%) of nine. Proportions of cells positive for caspases 3, 8, and 9 and P53 were increased, but those of cells positive for Bcl-2 and MMP-9 were decreased. Infiltration by inflammatory and immune cells was stimulated. Interpretation Dz13 was safe and well tolerated after single intratumoral injections at all doses. Funding Cancer Institute NSW, Cancer Council Australia, and National Health and Medical Research Council.
Conventional anti-inflammatory strategies induce multiple side effects, highlighting the need for novel targeted therapies. Here we show that knockdown of the basic-region leucine zipper protein, ...c-Jun, by a catalytic DNA molecule, Dz13, suppresses vascular permeability and transendothelial emigration of leukocytes in murine models of vascular permeability, inflammation, acute inflammation and rheumatoid arthritis. Treatment with Dz13 reduced vascular permeability due to cutaneous anaphylactic challenge or VEGF administration in mice. Dz13 also abrogated monocyte-endothelial cell adhesion in vitro and abolished leukocyte rolling, adhesion and extravasation in a rat model of inflammation. Dz13 suppressed neutrophil infiltration in the lungs of mice challenged with endotoxin, a model of acute inflammation. Finally, Dz13 reduced joint swelling, inflammatory cell infiltration and bone erosion in a mouse model of rheumatoid arthritis. Mechanistic studies showed that Dz13 blocks cytokine-inducible endothelial c-Jun, E-selectin, ICAM-1, VCAM-1 and VE-cadherin expression but has no effect on JAM-1, PECAM-1, p-JNK-1 or c-Fos. These findings implicate c-Jun as a useful target for anti-inflammatory therapies.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The past decade has seen the rapid evolution of small-molecule gene-silencing strategies, driven largely by enhanced understanding of gene function in the pathogenesis of disease. Over this time, ...many genes have been targeted by specifically engineered agents from different classes of nucleic acid-based drugs in experimental models of disease to probe, dissect, and characterize further the complex processes that underpin molecular signaling. Arising from this, a number of molecules have been examined in the setting of clinical trials, and several have recently made the successful transition from the bench to the clinic, heralding an exciting era of gene-specific treatments. This is particularly important because clear inadequacies in present therapies account for significant morbidity, mortality, and cost. The broad umbrella of gene-silencing therapeutics encompasses a range of agents that include DNA enzymes, short interfering RNA, antisense oligonucleotides, decoys, ribozymes, and aptamers. This review tracks current movements in these technologies, focusing mainly on DNA enzymes and short interfering RNA, because these are poised to play an integral role in antigene therapies in the future.
Worldwide, one in three cancers is skin-related, with increasing incidence in many populations. Here, we demonstrate the capacity of a DNAzyme-targeting c-jun mRNA, Dz13, to inhibit growth of two ...common skin cancer types-basal cell and squamous cell carcinomas-in a therapeutic setting with established tumors. Dz13 inhibited tumor growth in both immunodeficient and immunocompetent syngeneic mice and reduced lung nodule formation in a model of metastasis. In addition, Dz13 suppressed neovascularization in tumor-bearing mice and zebrafish and increased apoptosis of tumor cells. Dz13 inhibition of tumor growth, which required an intact catalytic domain, was due in part to the induction of tumor immunity. In a series of good laboratory practice-compliant toxicology studies in cynomolgus monkeys, minipigs, and rodents, the DNAzyme was found to be safe and well tolerated. It also did not interfere in more than 70 physiologically relevant in vitro bioassays, suggesting a reduced propensity for off-target effects. If these findings hold true in clinical trials, Dz13 may provide a safe, effective therapy for human skin cancer.
Coronary reperfusion has been the mainstay therapy for reduced infarct size after a heart attack. However, this intervention also results in myocardial injury by initiating a marked inflammatory ...reaction, and new treatments are keenly sought.
The basic-region leucine zipper protein, c-Jun is poorly expressed in the normal myocardium and is induced within 24 hours after myocardial ischemia-reperfusion injury. Synthetic catalytic DNA molecules (DNAzymes) targeting c-Jun (Dz13) reduce infarct size in the area-at-risk (AAR) regardless of whether it is delivered intramyocardially at the initiation of ischemia or at the time of reperfusion. Dz13 attenuates neutrophil infiltration, c-Jun and ICAM-1 expression in vascular endothelium, cardiomyocyte apoptosis, and the generation of reactive oxygen species in the reperfused myocardium. It inhibits infiltration into the AAR of complement 3 (C3), C3a receptor (C3aR), membrane attack complex-1 (Mac-1), or matrix metalloproteinase-2 (MMP-2) positive inflammatory cells. Dz13 also improves cardiac function without influencing myocardial vascularity or fibrosis.
These findings demonstrate the regulatory role of c-Jun in the pathogenesis of myocardial inflammation and infarction following ischemia-reperfusion injury, and inhibition of this process using catalytic DNA.
Plasma von Willebrand factor (vWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury. Only the very large vWF multimers are effective in promoting platelet ...adhesion in flowing blood. A protein disulfide bond reductase in plasma reduces the average multimer size of vWF secreted by endothelial cells. This activity has been isolated from human endothelial cell conditioned medium and shown to be the trimeric glycoprotein, thrombospondin-1 (TSP-1). Incubation of purified TSP-1 with vWF resulted in formation of thiol-dependent complexes of TSP-1 and vWF, generation of new thiols in vWF, and reduction in the average multimer size of vWF. The ratio of the concentrations of TSP-1 and vWF in plasma reflected with average multimer size of vWF. The higher the plasma TSP-1/vWF molar ratio, the smaller the average vWF multimer size. In addition, administration of TSP-1 to mice resulted in reduction in the average multimer size of plasma vWF. Interaction of TSP-1 with vWF is mediated by TSP-1 type 1 properdin domains and the vWF A3 domain. These results indicate that TSP-1 regulates the multimeric size and therefore hemostatic activity of vWF.
Early growth response factor-1 (Egr-1) binds to the promoters of many genes whose products influence cell movement and replication in the artery wall. Here we targeted Egr-1 using a new class of ...DNA-based enzyme that specifically cleaved Egr-1 mRNA, blocked induction of Egr-1 protein, and inhibited cell proliferation and wound repair in culture. The DNA enzyme also inhibited Egr-1 induction and neointima formation after balloon injury to the rat carotid artery wall. These findings demonstrate the utility of DNA enzymes as biological tools to delineate the specific functions of a given gene, and implicate catalytic nucleic acid molecules composed entirely of DNA as potential therapeutic agents.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Severe deficiency of the von Willebrand Factor (VWF)–cleaving proteinase, ADAMTS13, is associated with the development of thrombotic thrombocytopenic purpura (TTP). Several mutations spread across ...the ADAMTS13 gene have been identified in association with a deficiency of VWF-cleaving proteinase activity in patients with congenital TTP. The spread of these dysfunctional mutations and the domain structure of ADAMTS13 are suggestive of a complex interaction between the enzyme and its substrate. We have studied a patient with congenital TTP who is a compound heterozygote for the Thr196Ile mutation in the metalloproteinase domain and a frameshift mutation (4143-4144insA) in the second CUB domain that results in loss of the last 49 amino acids of the protein. The VWF-cleaving proteinase activity of the truncated enzyme was comparable to that of the wild-type enzyme but its secretion from transfected COS-7 cells was about 14% of the wild type.
Cell migration and proliferation that follows injury to the artery wall is preceded by signaling and transcriptional events that converge at the promoters of multiple genes whose products can ...influence formation of the neointima. Transcription factors, such as early growth response factor-1 (Egr-1), with nucleotide recognition elements in the promoters of many pathophysiologically relevant genes, are expressed at the endothelial wound edge within minutes of injury. The mechanisms underlying the inducible expression of Egr-1 in this setting are not clear. Understanding this process would provide important mechanistic insights into the earliest events in the response to injury. In this report, we demonstrate that fibroblast growth factor-2 (FGF-2) is released by injury and that antibodies to FGF-2 almost completely abrogate the activation and nuclear accumulation of Egr-1. FGF-2-inducible
egr-1-promoter-dependent expression is blocked by PD98059, a specific inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)-1/2 (MEK-1/2), as well as by dominant negative mutants of ERK-1/2. Inducible ERK phosphorylation after injury is dependent on release and stimulation by endogenous FGF-2. Antisense oligonucleotides directed at
egr-1 mRNA suggest that Egr-1 plays a necessary role in endothelial repair after denudation of the monolayer. These findings demonstrate that inducible Egr-1 expression after injury is contingent on the release and paracrine action of FGF-2.
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