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Results for “antisense targeting”

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PubMed2003

Antisense targeting of oxytocinergic hypothalamus neurons induces cytoplasmic triple helix-like immunoreactivity.

Intracerebroventricular injections of oligonucleotide probes complementary to oxytocin mRNA are known to decrease systemic oxytocin levels. In this study we show that immunoreactive oxytocin in the magnocellular hypothalamic perikarya and in their neurohypophysial projections remains unaffected by intracerebroventricular injections with an oxytocin antisense probe in rats. Hybridization signal for oxytocin mRNA was increased in the supraoptic and paraventricular nuclei in these animals. Immunocytochemistry with a monoclonal antibody, raised against triple helical DNA resulted in an accumulation of cytoplasmic reaction product in many of the magnocellular oxytocin immunoreactive neurons and in a fraction of the Herring bodies inthe posterior pituitary lobe in the antisense treated rats. Such immunostaining could be abolished by pretreating sections with RNase H. Animals injected with a mismatch probe instead of the antisense probe were devoid of cytoplasmic or axonal triple helix immunostaining. Our findings indicate that oxytocinergic transcripts in magnocellular hypothalamic neurons form triple helix-like aggregates upon specific antisense targeting rather than being degraded by endogenous RNases. While de novo transcription of oxytocin is probably stimulated, systemic release of the nonapeptide may be impaired.

Animals↗

PubMed2005

Preproenkephalin targeted antisenses cross the blood-brain barrier to reduce brain methionine enkephalin levels and increase voluntary ethanol drinking.

Antisense potentially can manipulate target gene expression in the brain if it can cross the blood-brain barrier (BBB). We designed three (10mer, 17mer, and 19mer) phosphorothioated antisenses (PS-ODNs) directed against the precursor molecule of methionine enkephalin (Met-Enk), an opiate peptide which suppresses voluntary ethanol drinking. We measured the ability of the antisenses to cross the BBB, accumulate in the brain and CSF, decrease levels of Met-Enk in brain and blood, and affect voluntary ethanol drinking. Each antisense readily crossed the BBB, with 0.07-0.16% of the i.v. dose accumulating per gram of brain. Capillary depletion and CSF sampling each confirmed that the antisenses entered the CNS. Gel electrophoresis of radioactivity recovered from brain and serum showed intact antisense and a higher molecular weight form likely representing antisense bound to protein, but no degradation products. Each antisense molecule and a cocktail of all three reduced Met-Enk levels in brain and serum. Met-Enk levels in the brain were reduced more rapidly and for a longer duration than Met-Enk levels in the serum, indicating a degree of selective targeting to the CNS. Additionally, administration of the cocktail was more effective in reducing Met-Enk levels than any of the individual antisenses. Each antisense increased voluntary ethanol drinking by about 20% and the cocktail increased it by about 80%. Taken together, these results used pharmacokinetic, immunochemical, and behavioral methods to show that PS-ODN antisenses that readily cross the BBB can decrease brain levels of Met-Enk and increase voluntary ethanol drinking.

Animals↗

PubMed1999

Suppression of nidogen-1 translation by antisense targeting affects the adhesive properties of cultured astrocytes.

The multidomain glycoprotein nidogen-1 is a common component of basal membranes. Nidogen-1 is produced by the endothelial cells and the mesenchymal cells of the developing central nervous system. Recent results give evidence that nidogen-1 may also be secreted by cultured Schwann cells to basement membranes of peripheral nerves. We were interested in ascertaining whether astrocytes, which have the capacity to produce laminin and fibronectin and are an important source of extracellular matrix (ECM) molecule secretion in the brain, might also produce nidogen-1. Immunocytochemistry, in combination with polymerase chain reaction and in situ hybridization techniques, revealed that astrocytes in culture synthesize nidogen-1. To show the functional significance of the nidogen-1 secretion by astrocytes, antisense targeting techniques were applied. These experiments showed that nidogen-1 may be an essential modulator of astrocytic adhesion to the substrate. The suppression of nidogen-1 synthesis by the application of antisense oligonucleotides induced a morphological transition from a flat, polygonal to a round cell and was accompanied by the detachment of the astrocytes from the substrate. Hence, nidogen-1 might be an important component of the ECM secreted by astrocytes. The suppression of nidogen-1 synthesis may disturb the aggregation of ECM molecules to a functional basement membrane and thus reduce the astrocytic adhesion to the substrate. Nidogen-1 secretion to basement membranes by astrocytes may have important functional implications during blood-brain barrier and scar formation.

Animals↗

PubMed2001

Stochastic kinetic analysis of the Escherichia coli stress circuit using sigma(32)-targeted antisense.

A stochastic Petri net model was developed for simulating the sigma(32) stress circuit in E. coli. Transcription factor sigma(32) is the principal regulator of the response of E. coli to heat shock. Stochastic Petri net (SPN) models are well suited for kinetics characterization of fluxes in biochemical pathways. Notably, there exists a one-to-one mapping of model tokens and places to molecules of particular species. Our model was validated against experiments in which ethanol (inducer of heat shock response) and sigma(32)-targeted antisense (downward regulator) were used to perturb the sigma(32) regulatory pathway. The model was also extended to simulate the effects of recombinant protein production. Results show that the stress response depends heavily on the partitioning of sigma(32) within the cell; that is, sigma(32) becomes immediately available to mediate a stress response because it exists primarily in a sequestered, inactive form, complexed with chaperones DnaK, DnaJ, and GrpE. Recombinant proteins, however, also compete for chaperone proteins, particularly when folded improperly. Our simulations indicate that when the expression of recombinant protein has a low requirement for DnaK, DnaJ, and GrpE, the overall sigma(32) levels may drop, but the level of heat shock proteins will increase. Conversely, when the overexpressed recombinant protein has a strong requirement for the chaperones, a severe response is predicted. Interestingly, both cases were observed experimentally.

Antisense Elements (Genetics)↗

PubMed2000

fhlA repression by OxyS RNA: kissing complex formation at two sites results in a stable antisense-target RNA complex.

OxyS is a small untranslated RNA that is induced in response to oxidative stress in Escherichia coli. This small RNA acts as a global regulator affecting the expression of multiple genes. OxyS represses the translation of fhlA, a transcriptional activator for formate metabolism. Previously, we have shown that fhlA repression by OxyS is mediated through base-pairing with a short sequence overlapping the ribosome binding site. Here we show that the OxyS-fhlA interaction involves a second site residing further downstream, within the coding region of fhlA. Mutations that disrupt pairing at this site affect the ability of OxyS to prevent 30 S ribosomes from binding to fhlA mRNA. Structure probing of fhlA mRNA demonstrates that both sites reside in the loops of two stem-loop structures. OxyS-fhlA pairing analysis shows that OxyS binds wild-type fhlA with an apparent dissociation constant of 25 nM, indicating that kissing complex formation between OxyS and fhlA results in a stable antisense-target complex. Mutations at either site, which disrupt pairing of OxyS to fhlA, decrease the stability of this complex. Our results indicate that kissing complex formation is sufficient to repress fhlA translation by OxyS.

Base Sequence↗

PubMed1995

Antisense targeting of engrailed-1 causes abnormal axis formation in mouse embryos.

Antisense oligonucleotide targeting of engrailed-1 (En-1) in early somite mouse embryos resulted in reduced En protein levels and produced abnormalities of the brain, face, and heart and shortening of the embryonic axis (caudal dysgenesis). Defects of the brain and limbs were consistent with abnormalities observed in null mutant mice described by other investigators. Abnormalities of the face and heart may be related to alterations in neural crest cells. Caudal dysgenesis suggested a role for En-1 in axis formation and this hypothesis was supported by results showing that En-1 protein and mRNA are present in the primitive streak. Thus, in addition to participating in the signaling pathway for brain and limb development, En-1 appears to play a role in patterning the embryonic axis.

Animals↗

PubMed1999

Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations.

We have used three beta-thalassemic mutations, IVS2-654, -705 and -745, that create aberrant 5' splice sites (5' ss) and activate a common cryptic 3' ss further upstream in intron 2 of the human beta-globin gene to optimize a generally applicable exon-skipping strategy using antisense derivatives of U7 small nuclear RNA (snRNA). Introducing a modified U7 snRNA gene carrying an antisense sequence against the cryptic 3' ss into cultured cells expressing the mutant beta-globin genes, restored correct beta-globin mRNA splicing for all three mutations, but the efficiency was much weaker for IVS2-654 than for the other mutations. The length of antisense sequence influenced the efficiency with an optimum of approximately 24 nucleotides. Combining two antisense sequences directed against different target sites in intron 2, either on separate antisense RNAs or, even better, on a single U7 snRNA, significantly enhanced the efficiency of splicing correction. One double-target U7 RNA was expressed on stable transformation resulting in permanent and efficient suppression of the IVS2-654 mutation and production of beta-globin. These results suggest that forcing the aberrant exon into a looped secondary structure may strongly promote its exclusion from the mRNA and that this approach may be used generally to induce exon skipping.

Exons↗

PubMed2003

Ultrasound-targeted antisense oligonucleotide attenuates ischemia/reperfusion-induced myocardial tumor necrosis factor-alpha.

Ultrasound contrast agents are now emerging as effective vehicles for delivering therapeutic agents to target tissues. In the present study, we used ultrasound-targeted, contrast-bound antisense oligonucleotides to inhibit the expression of tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine with negative inotropic effects. We compared the efficacy of left ventricular vs. intravenous administration and determined the optimal time for delivery. WKY rats were treated with perfluorocarbon-exposed sonicated dextrose albumin (PESDA) microspheres incubated with 100 microg of antisense oligonucleotide directed against TNF-alpha. Contrast was infused into either the superior vena cava or the left ventricular cavity along with simultaneous application of ultrasound. Twenty-four hours later, the animals underwent 15 min of ischemia and 2 h reperfusion. Control animals underwent sham operation only, ischemia/reperfusion only, or received PESDA only. A second group received treatment just prior to, or immediately after the onset of ischemia. At the end of the experimental period, hearts were removed and analyzed for TNF-alpha by northern and western blotting. While no TNF-alpha expression was detected in sham-operated animals, robust expression of TNF-alpha mRNA and protein was seen in controls treated with ultrasound and PESDA alone. In contrast, intravenous or left ventricular administration of antisense oligonucleotides significantly inhibited ischemia/reperfusion-induced TNF-alpha expression. Direct delivery into the left ventricular cavity was more effective than intravenous administration, and delivery just prior to ischemia was most effective in attenuating TNF-alpha expression. Furthermore, attenuation of TNF-alpha expression also significantly inhibited other post-ischemic inflammatory mediators including IL-1beta and intercellular adhesion molecule-1 (ICAM-1). Thus, ultrasound-targeted antisense oligonucleotides can effectively attenuate post-ischemic cytokine expression when delivered in a clinically relevant time frame, obviating the need for pretreatment.

Myocardium↗

PubMed2002

Knockdown of spinal opioid receptors by antisense targeting beta-arrestin reduces morphine tolerance and allodynia in rat.

The development of morphine tolerance and sciatic nerve injury-induced allodynia after functional knockdown of spinal opioid receptors using antisense oligonucleotides targeting beta-arrestin was investigated. Ineffectiveness of morphine in neuropathic pain suggests an implication of the same mechanism in these two processes. The development of morphine tolerance (10 microg intrathecally (i.th.), every 12 h) was significantly inhibited in rats, which received i.th. beta-arrestin antisenses (2 nM). Acute and chronic (6 days) i.th. administration of antisenses antagonized the allodynia in the rat model of neuropathic pain. Our results demonstrated that i.th. administration of beta-arrestin antisenses delayed development of tolerance to morphine and nerve injury-induced cold allodynia, which suggest that both of the investigated phenomena may be mediated by a similar mechanism, e.g. receptor desensitization. Moreover, the antisense oligonucleotides targeting beta-arrestin may constitute a new approach to the therapy of neuropathic pain.

Analgesics, Opioid↗

PubMed2001

Bulged residues promote the progression of a loop-loop interaction to a stable and inhibitory antisense-target RNA complex.

In several groups of bacterial plasmids, antisense RNAs regulate copy number through inhibition of replication initiator protein synthesis. These RNAs are characterized by a long hairpin structure interrupted by several unpaired residues or bulged loops. In plasmid R1, the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by a four-way junction structure and a side-by-side helical alignment. This topology facilitates the formation of a stabilizer intermolecular helix between distal regions of both RNAs, essential for in vivo control. The bulged residues in CopA/CopT were shown to be required for high in vitro binding rate and in vivo activity. This study addresses the question of why removal of bulged nucleotides blocks stable complex formation. Structure mapping, modification interference, and molecular modeling of bulged-less mutant CopA-CopT complexes suggests that, subsequent to loop-loop contact, helix propagation is prevented. Instead, a fully base paired loop-loop interaction is formed, inducing a continuous stacking of three helices. Consequently, the stabilizer helix cannot be formed, and stable complex formation is blocked. In contrast to the four-way junction topology, the loop-loop interaction alone failed to prevent ribosome binding at its loading site and, thus, inhibition of RepA translation was alleviated.

Base Pairing↗

PubMed1994

An approach for new anticancer drugs: oncogene-targeted antisense DNA.

BACKGROUND: Ras oncogenes owe their transforming properties to single point mutations in the sequence coding for the catalytic part of the p21 protein. These mutations lead to changes in cellular proliferation and tumorigenic properties. Point mutations represent a defined target for antisense oligonucleotides which specifically suppress translation of the targeted mRNA. However, the use of oligonucleotides in vivo has, until now, been limited by their instability in serum. NEW TECHNIQUES: Different strategies have been developed to protect the oligonucleotides and increase their transport into the target cell. Linking intercalating agents, hydrophobic groups or polycations to oligonucleotides or encapsulating them in liposomes resulted in a higher resistance to exonucleases and increased oligonucleotide penetration into cells. The stability and cellular uptake of antisense oligonucleotides can also be improved by associating them with polyalkylcyanoacrylate nanoparticles. The polymeric nature renders these small particles more stable than liposomes in biological fluids and during storage. METHOD: Antisense oligonucleotides directed to the point mutation (G to T) in codon 12 of the Ha-ras mRNA were adsorbed to nanoparticles in the presence of hydrophobic cations. RESULTS: These stabilized oligonucleotides selectively inhibited the proliferation of cells expressing this point mutation and partially reversed their tumorigenicity in nude mice.

Antineoplastic Agents↗

PubMed2002

In silico determination of potential antisense targets for human beta-globin variants.

The functional characterization of available genomic sequences is the major task of the research in the post-genome era. This complex task requires an integrative approach of high-throughput systems with in vitro and in vivo models in order to have a reliable evaluation of the biological function. The oligonucleotide antisense technology is one of the most promising approaches for the investigation of gene function; the crucial point of antisense experiments is the identification of optimal target sites for hybridisation. In this paper we have applied a bioinformatic tool for the recognition of optimal antisense targets. In order to evaluate the effect of mutational events on target selection we have tested the program on a sample of human beta-hemoglobin variants. The proposed algorithm software will be integrated in a web based tool at the site: http://www.nettab.org/agewa.

Algorithms↗

PubMed1998

Corticotropin-releasing hormone receptor (type I) antisense targeting reduces anxiety.

Two brain-derived corticotropin-releasing hormone receptors have been cloned, termed corticotropin-releasing hormone receptors type I and type 2. Antisense oligodeoxynucleotides targeted to the cloned rat and mouse corticotropin-releasing hormone receptors type I messenger RNA reduced the binding of the natural ligand of the corticotropin-releasing hormone receptors type I and also the release of adenocorticotrophic hormone in primary rat anterior pituitary cells and in clonal mouse pituitary cells (AtT-20) by up to 60% in an application time-dependent manner. Studies on intracellular uptake of fluorescence-labelled oligodeoxynucleotides indicated a cytoplasmic accumulation starting within two to four hours after application of oligodeoxynucleotides in vitro. In vivo, antisense oligodeoxynucleotides infused intra-cerebroventricularly reduced binding of radiolabelled corticotropin-releasing hormone receptors in central sites of the rat brain. Anxiety induced by i.c.v. administration of corticotropin-releasing hormone was attenuated by corticotropin-releasing hormone receptors type I antisense treatment as determined in the elevated plus maze and in the novel open field test. The corticotropin-releasing hormone-induced behavioural changes were absent in corticotropin-releasing hormone receptors type I antisense-pretreated animals. These results show that the selected antisense probes used were able to suppress corticotropin-releasing hormone receptors type I function in vitro as well as in vivo and suggest that the development of drugs blocking this specific receptor might lead to a novel class of anxiolytics.

Adrenocorticotropic Hormone↗

PubMed1995

Sense- and antisense-targeting of oxytocinergic systems in the rat hypothalamus.

3' phosphorothioate modified sense or antisense oligonucleotides to oxytocin transcripts were used for in vivo targeting of oxytocin (OT) neurons in the rat hypothalamus. Intracerebroventricular injections of antisense probe resulted in a loss of systemic OT. However, abundant immunoreactive OT as well as oxytocin mRNA hybridization signal was visualized in the hypothalamo neurohypophysial system (HNS) of these animals. RT-PCR of hypothalamic homogenates revealed clearly detectable amounts of cytoplasmic OT mRNA in spite of sense or antisense treatment. Immunostaining with an antibody to DNA-RNA triple helix resulted in cytoplasmic reaction product in the HNS in the antisense group, which was not found when tissue sections had been pretreated with RNase. Animals injected with the sense probe showed a less pronounced but significant loss of systemic OT while immunoreactivity for this peptide in the posterior lobe seemed to be unaffected. RT-PCR of OT encoding mRNA extracted from sense injected rats indicated that these transcripts were of smaller size than samples from antisense treated animals or controls. Immunostaining with the triple helix antibody revealed distinct immunoreactive dots in cellular nuclei throughout the brain in the sense group. Our findings suggest that sense and antisense probes may not readily be employed as "functional antagonists" since peptidergic neurons are probably capable of responding in various ways to the treatment. RNase H may be less important in hypothalamic neurons as commonly suggested. Targeted transcripts are likely to form complexes which may somehow interact with secretion. Triple helix formation in the nucleus may not be able to induce an efficient transcriptional arrest. Although endocrine and behavioral changes observed in antisense treated animals seem to confirm the hypothesis that a selective translational "knock out" can be achieved with in vivo hybridization strategies, the actual underlying molecular events are far from being understood. On the other hand, sense or antisense strategies may provide valuable insights into molecular and cellular events associated with neurosecretion.

Animals↗

PubMedDate not supplied

Antisense targeting of the urokinase receptor blocks urokinase-dependent proliferation, chemoinvasion, and chemotaxis of human synovial cells and chondrocytes in vitro.

Proliferation and invasion of synovial pannus in rheumatoid arthritis and cartilage remodeling in osteoarthritis are key events in development of disability of arthritic joints. The mechanisms that trigger these events are still poorly understood. The production of urokinase-type plasminogen activator (u-PA) by synovial cells and chondrocytes and the subsequent interaction of u-PA with its membrane receptor (u-PAR) is under the control of a variety of growth factors and cytokines released within the inflamed joints. Here we show that u-PA, on interaction with the specific receptor, regulates movement and invasion as well as proliferation of human synovial cells and chondrocytes. Targeting the urokinase receptor with an antisense oligonucleotide blocks the u-PA-dependent synoviocyte and chondrocyte proliferation and chemoinvasion, suggesting a possible use for this new class of drugs in the progression of the disease in rheumatoid arthritis and osteoarthritis.

Cartilage, Articular↗

PubMed2001

Statistical prediction of single-stranded regions in RNA secondary structure and application to predicting effective antisense target sites and beyond.

Single-stranded regions in RNA secondary structure are important for RNA-RNA and RNA-protein interactions. We present a probability profile approach for the prediction of these regions based on a statistical algorithm for sampling RNA secondary structures. For the prediction of phylogenetically-determined single-stranded regions in secondary structures of representative RNA sequences, the probability profile offers substantial improvement over the minimum free energy structure. In designing antisense oligonucleotides, a practical problem is how to select a secondary structure for the target mRNA from the optimal structure(s) and many suboptimal structures with similar free energies. By summarizing the information from a statistical sample of probable secondary structures in a single plot, the probability profile not only presents a solution to this dilemma, but also reveals 'well-determined' single-stranded regions through the assignment of probabilities as measures of confidence in predictions. In antisense application to the rabbit beta-globin mRNA, a significant correlation between hybridization potential predicted by the probability profile and the degree of inhibition of in vitro translation suggests that the probability profile approach is valuable for the identification of effective antisense target sites. Coupling computational design with DNA-RNA array technique provides a rational, efficient framework for antisense oligonucleotide screening. This framework has the potential for high-throughput applications to functional genomics and drug target validation.

Algorithms↗

PubMed1997

Antisense targeting of delta opioid receptors in NG 108-15 cells: direct correlation between oligodeoxynucleotide uptake and receptor density.

Antisense oligodeoxynucleotides (ODN) have been used to inhibit the function of a number of structurally defined neurotransmitter receptors in vivo by transiently disrupting their expression in the CNS. However, issues concerning the cellular and molecular mechanisms of these ODN often raise questions about the specificity of such ODN-mediated "knock-down" of target proteins. This study sought to extend our in vivo "knock-down" of the delta opioid receptor (DOR) by targeting this receptor in the NG 108-15 cells with an antisense ODN for the DOR and by using a polyclonal antibody raised against this receptor to determine the efficiency and selectivity of the antisense ODN in inhibiting expression of the DOR. By fluorescence tagging the ODN and immunofluorescence labeling the DOR, we monitored the uptake efficiency of the ODN and the DOR density in individual cells that had been treated with the antisense ODN or with a mismatch control. Quantitative fluorescence image analysis showed that the uptake of ODN by NG 108-15 cells was time- and concentration-dependent and that it was not uniform within a population. Treatment with the antisense ODN elicited an inverse correlation between DOR immunoreactivity and the ODN fluorescence in individual cells. No correlation was found in cells treated with the mismatch control. These findings suggest that the antisense ODN-mediated "knock-down" of the DOR is governed by the sequence specificity of the ODN and the efficiency of its uptake by the target cells in a time- and concentration-dependent manner. These data provide further evidence in support of the selectivity of antisense ODN targeting and the utility of these molecules as an effective tool in neuropharmacological studies.

Animals↗

PubMed2006

miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting.

Current understanding of microRNA (miRNA) biology is limited, and antisense oligonucleotide (ASO) inhibition of miRNAs is a powerful technique for their functionalization. To uncover the role of the liver-specific miR-122 in the adult liver, we inhibited it in mice with a 2'-O-methoxyethyl phosphorothioate ASO. miR-122 inhibition in normal mice resulted in reduced plasma cholesterol levels, increased hepatic fatty-acid oxidation, and a decrease in hepatic fatty-acid and cholesterol synthesis rates. Activation of the central metabolic sensor AMPK was also increased. miR-122 inhibition in a diet-induced obesity mouse model resulted in decreased plasma cholesterol levels and a significant improvement in liver steatosis, accompanied by reductions in several lipogenic genes. These results implicate miR-122 as a key regulator of cholesterol and fatty-acid metabolism in the adult liver and suggest that miR-122 may be an attractive therapeutic target for metabolic disease.

AMP-Activated Protein Kinases↗