Characterization of a potent and specific class of antisense oligonucleotide inhibitor of human protein kinase C-alpha expression

The use of antisense oligonucleotides to inhibit the expression of targeted mRNA sequences is becoming increasingly commonplace. Although effective, the most widely used oligonucleotide modification (phosphorothioate) has some limitations. In previous studies we have described a 20-mer phosphorothioate oligodeoxynucleotide inhibitor of human protein kinase C-alpha expression. In an effort to identify improved antisense inhibitors of protein kinase C expression, a series of 2' modifications have been incorporated into the protein kinase C-alpha targeting oligonucleotide, and the effects on oligonucleotide biophysical characteristics and pharmacology evaluated. The incorporation of 2'-O-(2-methoxy)ethyl chemistry resulted in a number of significant improvements in oligonucleotide characteristics. These include an increase in hybridization affinity toward a complementary RNA (1.5 degrees C per modification) and an increase in resistance toward both 3'-exonuclease and intracellular nucleases. These improvements result in a substantial increase in oligonucleotide potency (>20-fold after 72 h). The most active compound identified was used to examine the role played by protein kinase C-alpha in mediating the phorbol ester-induced changes in c-fos, c-jun, and junB expression in A549 lung epithelial cells. Depletion of protein kinase C-alpha protein expression by this oligonucleotide lead to a reduction in c-jun expression but not c-fos or junB. These results demonstrate that 2'-O-(2-methoxy)ethyl-modified antisense oligonucleotides are 1) effective inhibitors of protein kinase C-alpha expression, and 2) represent a class of antisense oligonucleotide which are much more effective inhibitors of gene expression than the widely used phosphorothioate antisense oligodeoxynucleotides.     

Elevated Fos expression in the nucleus tractus solitarii is associated with reduced baroreflex response in spontaneously hypertensive rats

We delineated the functional role of Fos protein at the nucleus tractus solitarii in the manifestation of reduced baroreceptor reflex control of heart rate during hypertension, using spontaneously hypertensive rats (SHR), stroke-prone SHR, Wistar-Kyoto rats, or Sprague-Dawley rats. Microinjection into the bilateral nucleus tractus solitarii of an antisense oligonucleotide that targets against the initiation codon of c-fos mRNA significantly potentiated the baroreceptor reflex in response to 30 minutes of sustained increase in blood pressure. Of particular note was the restoration of both the impaired sensitivity and capacity of baroreceptor reflex in SHR and stroke-prone SHR to levels comparable to those in normotensive rats. Likewise, the number of Fos-immunoreactive nuclei evoked by the sustained increase in blood pressure in the caudal nucleus tractus solitarii of SHR and stroke-prone SHR was reduced, after this antisense c-fos treatment, to the basal level exhibited by the normotensive animals. Control treatment with the corresponding sense oligonucleotide, an antisense oligonucleotide that targets against a different portion of the coding sequence of the c-fos mRNA or artificial cerebrospinal fluid, on the other hand, elicited no discernible effect on either the baroreceptor reflex response or the induced expression of Fos protein in the nucleus tractus solitarii by baroreceptor activation. We also found that the basal level of Fos expression in the caudal nucleus tractus solitarii was significantly elevated in the SHR and stroke-prone SHR. Together, these novel findings suggest that an elevated expression of basal Fos protein in the NTS during hypertension may be associated with the dysfunction in baroreceptor reflex control of heart rate.     

Suppression of postischemic hippocampal nerve growth factor expression by a c-fos antisense oligodeoxynucleotide

We examined the uptake and distribution of an antisense phosphorothioated oligodeoxynucleotide (s-ODN) to c-fos, rncfosr115, infused into the left cerebral ventricle of male Long-Evans rats and the effect of this s-ODN on subsequent Fos, NGF, neurotrophin-3 (NT-3), and actin expression. To establish the uptake and turnover of s-ODN in the brain, we studied the copurification of the immunoreactivity of biotin with biotinylated s-ODN that was recovered from different regions of the brain. A time-dependent diffusion and the localization of s-ODN were further demonstrated by labeling the 3'-OH terminus of s-ODN in situ with digoxigenin-dUTP using terminal transferase and detection using anti-digoxigenin IgG-FITC. Cellular uptake of the s-ODN was evident in both the hippocampal and cortical regions, consistent with a gradient originating at the ventricular surface. Degradation of the s-ODN was observed beginning 48 hr after delivery. The effectiveness of c-fos antisense s-ODN was demonstrated by its suppression of postischemic Fos expression, which was accompanied by an inhibition of ischemia-induced NGF mRNA expression in the dentate gyrus. Infusion of saline, the sense s-ODN, or a mismatch antisense s-ODN did not suppress Fos expression. That this effect of c-fos antisense s-ODN was specific to NGF was demonstrated by its lack of effect on the postischemic expression of the NT-3 and beta-actin genes. Our results demonstrate that c-fos antisense s-ODN blocks selected downstream events and support the contention that postischemic Fos regulates the subsequent expression of the NGF gene and that Fos expression may have a functional component in neuroregeneration after focal cerebral ischemia-reperfusion.     

Lipopeptide phytotoxins produced by Pseudomonas syringae pv. syringae: comparison of the biosurfactant and ion channel-forming activities of syringopeptin and syringomycin

The maturational status of Adelta and C-fibers in the fetal rat spinal cord was examined using formalin-induced c-fos expression as a marker for neuronal activities. Awake 19-, 20-, and 21-day fetuses (FD) were injected ex utero with 5 microl of 10% formalin either into the ventral aspect of the forepaw or the hindpaw. FD 19 fetuses showed little response to the injection, but with increasing age, the fetuses exhibited more specific behaviors following injury of the paw. By FD 21, fetuses treated with formalin injection showed body curls and twitches, mouth opening, face wiping, and withdrawal of the injected paw. The anatomical data paralleled that of behavior; FD 19 animals expressed a small number of Fos labeled nuclei following the formalin injection that was not statistically different from control animals. The formalin-induced increase in Fos staining was first observed at FD 20 with a large increase in the number of Fos labeled cell occurring between FD 20 and 21. By FD 21, the pattern of Fos stained nuclei resembled that found in neonatal rats. There was constitutive bilateral staining in all untreated, saline and formalin injected fetuses that is unique to prenatal animals. Formalin treated fetuses showed constitutive level of staining in addition to the increase in the c-fos expression caused by formalin. We have thus demonstrated that, as indexed both by behavioral response and by Fos immunoreactivity, rat fetuses are capable of transmitting and responding to noxious input before birth.     

Induction of c-fos expression in hypothalamic magnocellular neurons requires synaptic activation and not simply increased spike activity

Magnocellular neurons of the hypothalamic supraoptic nucleus have been shown to express the immediate-early gene c-fos in a number of experimental and physiological circumstances. In each case the induction of the immediate-early gene followed the increase in the spike activity of the cells. Since an increase in the intracellular concentration of calcium following influx through voltage-sensitive calcium channels is a known stimulus for c-fos expression and since the action potentials of these neurons have a large calcium component, we hypothesized that c-fos induction in these neurons could be attributed to calcium influx during spike activity. In the present experiments we use extracellular recording and immunocytochemistry for Fos, the protein product of c-fos, to demonstrate the activation of the cells following intracerebroventricular administration of the muscarinic agonist, carbachol. Fos expression following carbachol injection was then compared with that induced by a similar number of antidromically evoked action potentials. Antidromic activation, unlike the activation induced by carbachol, did not lead to the induction of Fos. We conclude that Fos induction in these neurons requires receptor activation rather than spike activity.     

Analysis of an oligonucleotide N3'-->P5' phosphoramidate/phosphorothioate chimera with capillary gel electrophoresis

N3'-->P5' phosphoramidate/phosphorothioate chimeric oligonucleotides (ODNs) are presently under investigation as potential antisense drugs. Within the field of antisense research, "second generation" chimeric ODNs have exhibited improved characteristics relative to oligonucleotides with uniformly modified backbones. The ODN of interest for this study consisted of a chemically synthesized 18-mer of mixed nucleotide base sequence with a backbone consisting of eight central phosphorothioate linkages flanked by four N3'-->P5' phosphoramidate (amidate) linkages on the 5'-end and five amidate linkages ont he 3'-end. This chimera presents analytical challenges due to the central phosphorothioate region. Here, we present a capillary gel electrophoresis (CGE) method for the analysis of the above N3'-->P5' phosphoramidate/phosphorothioate chimera. CGE was used to analyze the product prior to its purification by reversed phase - high performance liquid chromatography (RP-HPLC), and each fraction collected from the purification was similarly analyzed. An internal standard was utilized to determine the relative mobility of our product, and polyacrylamide gel electrophoresis (PAGE) analysis was used to verify CGE results.     

A serum-resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA

Development of antisense technology has focused in part on creating improved methods for delivering oligodeoxynucleotides (ODNs) to cells. In this report, we describe a cationic lipid that, when formulated with the fusogenic lipid dioleoylphosphatidyliethanolamine, greatly improves the cellular uptake properties of antisense ODNs, as well as plasmid DNA. This lipid formulation, termed GS 2888 cytofectin, (i) efficiently transfects ODNs and plasmids into many cell types in the presence or absence of 10% serum in the medium, (ii) uses a 4- to 10-fold lower concentration of the agent as compared to the commercially available Lipofectin liposome, and (iii) is > or = 20-fold more effective at eliciting antisense effects in the presence of serum when compared to Lipofectin. Here we show antisense effects using GS 2888 cytofectin together with C-5 propynyl pyrimidine phosphorothioate ODNs in which we achieve inhibition of gene expression using low nanomolar concentrations of ODN. This agent expands the utility of antisense ODNs for their use in understanding gene function and offers the potential for its use in DNA delivery applications in vivo.     

Effect of cisplatin and c-myb antisense phosphorothioate oligodeoxynucleotides combination on a human colon carcinoma cell line in vitro and in vivo

We investigated the effect of c-myb antisense phosphorothioate oligodeoxynucleotides [(S)ODNs] and cisplatin (CDDP) combination on the human colon carcinoma cell line LoVo Dx both in vitro and in nude mice bearing LoVo Dx solid tumour. We show that antisense (S)ODN treatment decreases c-myb mRNA and protein expression, induces growth arrest in the G1 phase of the cell cycle, and inhibits cell proliferation. In vivo treatment with c-myb antisense (S)ODNs results in a reduction in tumour growth. A greater inhibition of cell proliferation in vitro and a higher increase of tumour growth inhibition and growth delay in vivo were obtained with the combination of (S)ODNs and CDDP than when the two agents were administered separately. This comparative study, using the same tumour cell line in vitro and in vivo, suggests that c-myb antisense (S)ODNs might be useful in the therapy of colon cancer in combination with antineoplastic drugs.     

Synthetic antisense oligodeoxynucleotides as potential drugs against hepatitis C

Antisense oligodeoxynucleotides (ODNS) can be used to specifically inhibit hepatitis C viral gene expression. Due to its high degree of conservation and its important function as internal ribosomal entry site, the 5'-non-coding region of the hepatitis C virus has been the most effective target to inhibit translation so far. Inhibition of luciferase reporter gene expression of up to 96 +/- 2% has been achieved. Modifications of ODNs like phosphorothioate, methylphosphonate or benzylphosphonate modification of terminal or intramolecular internucleotide phosphates lead to altered lipophilicity and binding stability to its RNA target and resistance against serum nucleases. The mode of action of ODNs is mainly dependent on an efficient induction of RNase H activity. The uptake of ODNs occurs via receptor-mediated or absorptive and fluid-phase endocytosis. After release from the endosomes, ODNs may exert their effects by interaction with cytosolic or nuclear structures. Side effects can occur when this interaction affects intra- or extracellular targets essential for biological cell function. If these problems can be solved, antisense technology has the potential for future therapy of human disease.     

Differential migration of Th1 and Th2 cells--implications for vaccine and infection studies

We previously reported a synergistic interaction between leptin and cholecystokinin (CCK) to reduce food intake through CCK-A receptors in lean mice fasted for 24 h. To identify the activated neuronal pathways, we investigated changes in Fos expression in brain nuclei 2 h after single or combined intraperitoneal (i.p.) injections of leptin (120 microg/kg) and sulfated CCK-8 (3.5 microg/kg) in male lean mice (C57BL/6) fasted for 24 h using immunohistochemistry for Fos, the protein product of the early gene, c-fos. Leptin did not increase Fos expression in the brain compared with vehicle-treated mice. CCK increased the numbers of Fos-positive neurons in the nucleus of the solitary tract (NTS)/area postrema (AP), central nucleus of the amygdala (CeA) and, to a smaller extent, in the paraventricular nucleus of the hypothalamus (PVN) (5.2-, 2.3- and 0. 3-fold respectively). Injections of leptin-CCK further enhanced Fos expression by 40% in the PVN compared with that induced by CCK alone, but not in the other nuclei. Devazepide (a CCK-A receptor antagonist, 1 mg/kg, i.p.) prevented the increase in Fos expression induced by leptin-CCK in the PVN and by CCK alone in the PVN, CeA and NTS/AP. These results indicate that in fasted mice, i.p. injection of CCK increases Fos expression in specific brain nuclei through CCK-A receptors while leptin alone had no effect. Leptin in conjunction with CCK selectively enhanced Fos expression in the PVN. The PVN may be an important site mediating the synergistic effect of leptin-CCK to regulate food intake.     

Continuous neurogenesis in the olfactory brain of adult shore crabs, Carcinus maenas

To scrutinize the common belief that the number of neurons in the CNS of adult decapod crustaceans stays constant, in spite of their dramatic postlarval increase in size, I counted olfactory projection neurons (OPNs) in the brains of differently-sized postlarval shore crabs, Carcinus maenas, and performed in vivo labeling of proliferating cells with 5-bromo-2'-deoxyuridine (BrdU) on brains of adults. The number of OPNs increases continuously throughout the postlarval life of shore crabs and approximately doubles from the very young to the oldest animals. Brain sections from adult crabs labeled with BrdU revealed ongoing proliferation of cells in the lateral soma cluster, which consists of OPN cell bodies, and in the cluster of somata of hemiellipsoid body local interneurons, which are the targets of the OPNs. Post-injection survival times from 5.5 to 120 h revealed a small but relatively constant number of labeled nuclei with neuronal morphology in both soma clusters of all specimens (31.3 +/- 9.5 S.D. nuclei per lateral cluster, n = 29; 20.1 +/- 4.5 S.D. nuclei per hemiellipsoid body cluster, n = 10). The labeled nuclei were located in a distinct proliferative zone in each cluster. There were significantly more labeled nuclei in both soma clusters after a prolonged post-injection survival time of 1 month (71.3 +/- 7.8 S.D. nuclei per lateral cluster, n = 4; 38.2 +/- 7.1 nuclei per hemiellipsoid body cluster, n = 6). In both soma clusters the labeled nuclei formed a compact group that was dislocated from the proliferation zone towards the outer edge of the cluster. In the proliferation zone of the lateral cluster histological stainings revealed cell bodies of typical neuronal shape that are slightly smaller and more intensely stained than the surrounding OPN somata. Some of these cell bodies were captured in various stages of mitosis. Collectively, these data indicate that continuous neurogenesis occurs in the central olfactory pathway of the brain of shore crabs throughout their entire adult life. This unexpected structural plasticity may enable long-lived decapod crustaceans to adapt to ever-changing olfactory environments.     

Antisense nucleic acid therapy of influenza virus

We have demonstrated that Antisense phosphodiester (ODNs) and phosphorothioate oligonucleotides (S-ODNs) inhibit CAT (chloramphenicol acetyltransferase) protein expression in the clone 76 cell line, which is a derivative of the murine C127 cell line. This cell line expresses the influenza virus RNA polymerase and nucleoprotein (NP) genes in response to treatment with dexamethasone. Phosphodiester, phosphorothioate, and liposomally encapsulated oligonucleotides with four target sites (PB1, PB2, PA, and NP) were synthesized and tested for inhibitory effects by a CAT-ELISA assay using the clone 76 cell line. The liposomally encapsulated ODNs and S-ODNs complementary to the sites of the PB2-AUG and PA-AUG initiation codons showed highly inhibitory effects. On the other hand, the inhibitory effect of the S-ODNs targeted to PB1 was considerably decreased in comparison with the other three target sites. Liposome encapsulation afforded oligomer protection in serum-containing medium and substantially improved cellular accumulation. The liposomally encapsulated oligonucleotides exhibited higher inhibitory activity than the free oligonucleotides. Liposomal preparations of oligonucleotides facilitate release from endocytic vesicles, and thus, cytoplasmic and nuclear localization are observed following cell treatment. The activities of the unmodified oligonucleotides are effectively enhanced by using the liposomal carrier. In the observation of the endocapsulated antisense phosphodiester oligonucleotide, FITC-ODN-PB2-as treated clone 76 cells by a confocal laser scanning microscope, diffuse fluorescence was apparently observed in the cytoplasm. Interestingly, the endocapsulated antisense phosphorothioate oligonucleotide, FITC-S-ODN-PB2-as accumulated in the nuclear region of clone 76 cells. However, weak fluorescence was observed on the endosomes and in the cytoplasmes of the free antisense phosphorothioate oligonucleotides treated clone 76 cells.     

Injection of corticotropin-releasing hormone into the locus coeruleus or foot shock increases neuronal Fos expression

Previous research suggests that corticotropin-releasing hormone can act in the locus coeruleus to increase the firing of locus coeruleus neurons and elicit physiological responses resembling those associated with stress. The present study used immunocytochemical detection of Fos as a measure of neuronal activation to identify brain areas that were activated by bilateral injections of corticotropin-releasing hormone into the locus coeruleus of rats. Injection of corticotropin-releasing hormone into the locus coeruleus increased the expression of Fos in the locus coeruleus as compared with injection of vehicle into the locus coeruleus or injection of corticotropin-releasing hormone into neighbouring pontine sites. The pattern of Fos expression throughout the brain after injections of corticotropin-releasing hormone into the locus coeruleus was generally consistent with the anatomical organization of efferent projections arising from the locus coeruleus; increased Fos expression was observed in many brain areas including the ventral lateral septum, septohypothalamic nucleus, bed nucleus of the stria terminalis, the central amygdaloid nucleus, the dorsomedial nuclei of the hypothalamus, and the thalamic paraventricular and rhomboid nuclei. Foot shock also increased Fos expression in the locus coeruleus and the other brain regions that expressed Fos after corticotropin-releasing hormone injections into the locus coeruleus. A few brain regions, most notably the hypothalamic paraventricular nucleus, expressed Fos in response to foot shock but not corticotropin-releasing hormone. These results indicate that local injection of corticotropin-releasing hormone into the locus coeruleus stimulates the activity of the locus coeruleus neurons. However, the pattern of Fos expression throughout the brain evoked by injection of corticotropin-releasing hormone into the locus coeruleus does not fully replicate the effects of foot shock.     

Biodistribution, stability, and antiviral efficacy of liposome-entrapped phosphorothioate antisense oligodeoxynucleotides in ducks for the treatment of chronic duck hepatitis B virus infection

This study investigated the feasibility of using liposomes to increase the hepatic delivery and antiviral efficacy of phosphorothioate antisense oligodeoxynucleotides (PS-ODN) for the in vivo treatment of hepatitis B virus (HBV) infection. Ducks infected with duck hepatitis B virus (DHBV) were used as the model. We studied the stability of an antisense PS-ODN in duck plasma, its integrity during the process of liposome entrapment, its in vivo biodistribution, plasma clearance, and excretion. In addition, the intrahepatic distribution of a labeled free and liposome-entrapped ODN was also investigated. The results of our studies show that: 1) phosphorothioate ODN remain stable during the process of liposome entrapment; 2) are stable in duck plasma for many hours; 3) are rapidly cleared from the plasma when injected intravenously; 4) intravenous injection of antisense ODNs entrapped within liposomes enhances delivery of the ODN to the liver; and 5) inhibit DHBV replication. Serum DHBV DNA levels fell rapidly, with a corresponding decrease in intrahepatic viral replicative intermediates at the end of the 5-day study period. Although inhibition of viral replication and a fall in the target protein was observed, a marked inhibition of viral replication was also observed with high doses of a random-sequence ODN. Thus, it is not certain that inhibition of viral replication was entirely through an antisense mechanism. Therefore, liposomes may be effective vehicles to improve the delivery of antisense oligonucleotides to the liver for the therapy of hepatotropic viruses.