Antisense properties of 2'-O-dimethylaminooxyethyl (2'-O-DMAOE) oligonucleotides.

Antisense oligonucleotides with 2'-O-(2-[N,N-dimethyl)aminooxy]ethyl) or (2'-O-DMAOE) modification were synthesized and evaluated for nuclease resistance and pharmacology both in vitro and in vivo. This modification exhibits very high nuclease resistance and efficacy in various biological (ICAM-1, C-raf and PKC-alpha) targets.

ADAM17 but not ADAM10 mediates tumor necrosis factor-alpha and L-selectin shedding from leukocyte membranes.

The release of tumor necrosis factor-alpha (TNF-alpha) from cellular membranes has been shown by different laboratories to be controlled by a disintegrin and metalloprotease, ADAM10 or ADAM17. In contrast, only ADAM17 has shown to be involved in L-selectin shedding. To determine the specific roles of ADAM10 and ADAM17 in the processing of TNF-alpha and L-selectin shedding, antisense oligonucleotides (ASO) targeting both ADAM10 and ADAM17 were identified. We show that ISIS 16337 reduces ADAM17 mRNA and ISIS 100750 reduces ADAM10 mRNA in a sequence-specific and dose-dependent manner in both Jurkat and THP-1 cells. The ADAM17 ASO (ISIS 16337) inhibited both TNF-alpha secretion in THP-1 cells and L-selectin shedding in Jurkat cells, whereas the ADAM10 ASO (ISIS 100750) did not significantly inhibit release of either protein. These results suggest that ADAM17 is one of the major metalloproteases involved in L-selectin shedding as well as TNF-alpha processing. The biologic substrates for ADAM10 in Jurkat and THP-1 cells remain to be elucidated.

Discovery and analysis of antisense oligonucleotide activity in cell culture.

In the past decade antisense oligonucleotides (ASOs) have proven to be a useful tool for dissection of gene function in molecular cell biology (Koller, E., Gaarde, W. A., and Monia, B. P. (2000) Trends Pharm. Sci., 21, 142-148), and validation of gene targets in animal models (Crooke, S. T. (1998) Biotechnol. Gen. Eng. Rev. 15, 121-157), as well as a means for therapeutic treatment of human diseases (Bennett, C. F. (1999) Exp. Opin. Invest. Drugs 8, 237-253). An important step toward usage of ASOs in the described applications is identification of an active ASO. This article describes the underlying basis and means for achieving this goal in cell culture.

Intercellular adhesion molecule-1 suppression in skin by topical delivery of anti-sense oligonucleotides.

We topically applied 20 nucleotide phosphorothioate intercellular adhesion molecule-1 anti-sense oligodeoxynucleotide in a cream formulation. It effectively inhibited tumor necrosis factor-alpha-induced expression of intercellular adhesion molecule-1 in human skin transplanted on severe compromised immunodeficient mice. The effects were concentration dependent, sequence specific, and resulted from reduction of intercellular adhesion molecule-1 mRNA levels in the skin. Intravenous administration of the drug did not show pharmacologic effects, probably due to insufficient drug concentrations in skin. Topical delivery, however, produced a rapid and a significantly higher accumulation of oligodeoxynucleotide in the epidermis and dermis. The results strongly suggest that topically applied anti-sense oligonucleotides can be delivered to target sites in the skin and may be of considerable value in the treatment of psoriasis and other inflammatory skin disorders.

Regulation of ICAM-1-mediated fibroblast-T cell reciprocal interaction: implications for modulation of gut inflammation.

BACKGROUND & AIMS: Immune-nonimmune cell interactions modulate mucosal immunity. We investigated the expression of adhesion molecules by intestinal fibroblasts, the effect of immune cell-derived factor on fibroblast binding of T cells, and the consequences of interfering with adhesion molecule expression on fibroblast-T cell interaction. METHODS: Expression of fibroblast intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 surface and messenger RNA (mRNA) was measured before and after exposure to immune cell-derived supernatants. Fibroblasts were treated with antibodies to ICAM-1 or VCAM-1, or ICAM-1 antisense oligonucleotide Isis 2302, before a T-cell adhesion assay. RESULTS: Fibroblast activation by immune cell-derived cytokines enhanced ICAM-1 and VCAM-1 surface expression and mRNA as well as adhesiveness for T cells. Blockade with neutralizing antibodies showed that binding was almost exclusively dependent on ICAM-1. Isis 2302 specifically reduced fibroblast ICAM-1 mRNA and dose-dependently inhibited ICAM-1 surface expression and T-cell binding. CONCLUSIONS: ICAM-1 is essential for intestinal fibroblast binding of T cells, a phenomenon that is efficiently and specifically disrupted by ICAM-1 antisense oligonucleotides. These observations emphasize the crucial regulatory role of fibroblasts in mucosal immunity and their potential as targets for therapeutic intervention in intestinal inflammation.

Protection against allograft rejection with intercellular adhesion molecule-1 antisense oligodeoxynucleotides.

BACKGROUND: We designed an antisense phosphorothioate oligodeoxynucleotide (oligo) to specifically inhibit the expression of rat intercellular adhesion molecule-1 (ICAM-1) mRNA (IP-9125). METHODS: IP-9125 oligo was delivered intravenously by osmotic pump alone or in combination with cyclosporine (CsA) to recipients in order to prevent the rejection of kidney or heart allografts. In additional experiments, kidney allografts were perfused with IP-9125 before grafting. RESULTS: IP-9125 inhibited ICAM-1 mRNA and ICAM-1 protein expression in rat aortic endothelial cells; scrambled controls IP-12140 and IP-13944 were ineffective. Untreated ACI (RT1a) recipients rejected Lewis (RT1l) kidney allografts at a mean survival time of 8.5+/-1.1 days. A 14-day intravenous administration of 2.5 mg/kg/day IP-9125 prolonged the survival of kidney allografts to 39.2+/-16.4 days; 5.0 mg/kg/day, to 43.0+/-17.5 days; and 10.0 mg/kg/day, to 50.4+/-21.6 days. In contrast, a scrambled control IP-12140 was not effective. A combination of 10 mg/kg/day IP-9125 and 1.0 mg/kg/day CsA delivered for 14 days synergistically extended kidney allograft survival times 88.5+/-7.5 days. In contrast, the combination of 10.0 mg/kg/day control IP-12140 with CsA was ineffective (20.7+/-3.2 days) when compared with CsA alone (20.2+/-4.0 days). Similar results were obtained for heart transplants in recipients treated with IP-9125 alone or in combination with CsA. Furthermore, in situ immunostaining showed that IP-9125 significantly reduced the expression of ICAM-1 protein in kidney allografts. Finally, perfusion of kidney grafts alone with 20.0 mg per 2 ml of IP-9125 protected kidney allografts from rejection (37.5+/-7.5 days; P < 0.001), whereas perfusion with 20 mg per 2 ml of control IP-12140 was ineffective (12.6+/-5.0 days). CONCLUSIONS: Rat ICAM-1 IP-9125 oligo inhibits ICAM-1 protein expression in vitro and in vivo as well as blocks allograft rejection when used for pretreatment of donors, graft perfusion, or postoperative treatment of recipients.

2'-O-(2-Methoxy)ethyl-modified anti-intercellular adhesion molecule 1 (ICAM-1) oligonucleotides selectively increase the ICAM-1 mRNA level and inhibit formation of the ICAM-1 translation initiation complex in human umbilical vein endothelial cells.

Little is known about the mechanisms that account for inhibition of gene expression by antisense oligonucleotides at the level of molecular cell biology. For this purpose, we have selected potent 2'-O-(2-methoxy)ethyl antisense oligonucleotides (IC50 = 2 and 6 nM) that target the 5' cap region of the human intercellular adhesion molecule 1 (ICAM-1) transcript to determine their effects upon individual processes of mRNA metabolism in HUVECs. Given the functions of the 5' cap structure throughout mRNA metabolism, antisense oligonucleotides that target the 5' cap region of a target transcript have the potential to modulate one or more metabolic stages of the message inside the cell. In this study we found that inhibition of protein expression by these RNase H independent antisense oligonucleotides was not due to effects on splicing or transport of the ICAM-1 transcript, but due instead to selective interference with the formation of the 80 S translation initiation complex. Interestingly, these antisense oligonucleotides also caused an increase in ICAM-1 mRNA abundance in the cytoplasm. These results imply that ICAM-1 mRNA turnover is coupled in part to translation.

Altered mRNA splicing and inhibition of human E-selectin expression by an antisense oligonucleotide in human umbilical vein endothelial cells.

We have characterized the mechanism of action of an antisense oligodeoxynucleotide (ASO) targeting human endothelial leukocyte adhesion molecule, E-selectin. ISIS 4730, a 20-base ASO designed to be complementary to a region in the 3'-untranslated region (3'-UTR) of human E-selectin, is a potent and specific inhibitor of both mRNA and protein expression in human umbilical vein endothelial cells. Following treatment with ISIS 4730, a lower molecular weight mRNA (3300 bases) species was detected by Northern blot analysis with a corresponding decrease in the mature E-selectin transcript (3875 bases). The ASO-induced low molecular weight mRNA is stable and remains in the nucleus. We demonstrate that ISIS 4730 targets E-selectin pre-mRNA in the nucleus and promotes cleavage of the pre-mRNA at the hybridization site, resulting in prevention of splicing of the last intron. The change in molecular weight of the E-selectin transcript is the result of loss of the 3'-UTR due to ASO-mediated RNA cleavage and retention of the last intron. Cleavage of the E-selectin pre-mRNA appears to be due to endogenous RNase H or a related enzyme activity.

Role of the intercellular adhesion molecule-1(ICAM-1) in endotoxin-induced pneumonia evaluated using ICAM-1 antisense oligonucleotides, anti-ICAM-1 monoclonal antibodies, and ICAM-1 mutant mice.

This study examined the effectiveness of antisense oligonucleotides targeted to intercellular adhesion molecule-1 (ICAM-1) to inhibit endotoxin-induced upregulation of ICAM-1 and neutrophil emigration and compared the apparent role of ICAM-1 when examined using antisense oligonucleotides, anti-ICAM-1 antibodies, and ICAM-1 mutant mice. Antisense oligonucleotides inhibited upregulation of ICAM-1 mRNA at 4 and 24 h after instillation of endotoxin in a dose-dependent manner. Neutrophil emigration into the alveolar spaces at 24 h was inhibited by 59%, similar to inhibition using the anti-ICAM-1 antibodies 3E2 (58%) and YN1/1 (75%). No inhibition was observed in the ICAM-1 mutant compared to wild-type mice. These data show that antisense oligonucleotides targeted to ICAM-1 inhibit the endotoxin-induced upregulation of ICAM-1 in the lung and are as effective as anti-ICAM-1 antibodies in preventing neutrophil emigration. The incomplete inhibition by either antisense oligonucleotides or antibodies suggests that alternative adhesion pathways that do not require ICAM-1 are important in neutrophil emigration in the lungs. The disparity in the role of ICAM-1 when evaluated using antisense or antibodies compared to mutant mice suggests that either these inhibitors are exerting additional effects on endothelial cells other than blockade of ICAM-1 or mutant mice have upregulated the ICAM-1-independent pathways to compensate for the long-term loss of ICAM-1.

Blocking of heart allograft rejection by intercellular adhesion molecule-1 antisense oligonucleotides alone or in combination with other immunosuppressive modalities.

Intercellular adhesion molecule-1 (ICAM-1) binds circulating leukocytes through interactions with beta 2 integrins, LFA-1, and macrophage Ag-1. The phosphorothioate antisense oligodeoxynucleotide, IP-3082, specific for ICAM-1 mRNA inhibited ICAM-1, but not vascular cell adhesion molecule-1, mRNA induction and expression of ICAM-1 molecules by mouse endothelioma cells. Scrambled control oligonucleotides were ineffective. Untreated C3H (H-2k) mice rejected C57BL/10 (H-2b) heart allografts with a mean survival time of 7.7 +/- 1.4 days. Administration i.v. of IP-3082 by a 7-day osmotic pump prolonged the survival of heart allografts in a dose-dependent fashion: 1.25 mg/kg, to 11 +/- 0 days; 2.5 mg/kg, to 12 +/- 2.7 days; 5 mg/kg, to 14.1 +/- 2.7 days; and 10 mg/kg, to 15.3 +/- 5.8 days (all p < 0.01). Control IP-1082 (10 mg/kg) was ineffective (7 +/- 0.8 days). Although 7-day anti-LFA-1 mAb (50 micrograms/day; i.p.) prolonged allograft survival to 14.1 +/- 2.7 days, the addition of IP-3082 (5.0 mg/kg x 7 days) induced donor-specific transplantation tolerance (> 150 days). Furthermore, IP-3082 (5 mg/kg x 7 days) acted synergistically with antilymphocyte serum, rapamycin, and brequinar, but not cyclosporin A: a single antilymphocyte serum (0.2 ml) i.p. injection alone prolonged graft survival to 10 +/- 0.5 days (p < 0.01) and in combination with IP-3082 (5 mg/kg), to 32.2 +/- 8.3 days (p < 0.001); rapamycin (0.1 mg/kg x 7 days; i.v.) alone prolonged survival to 13 +/- 7.5 days (p < 0.01), and with IP-3082, to 32.4 +/- 8.9 days (p < 0.03); brequinar (0.5 mg/kg x 7 days; oral gavage) alone to 12 +/- 2.4 days (p < 0.05), and with IP-3082 (5 mg/kg), to 38.8 +/- 30.2 days (p < 0.01); in contrast, cyclosporin A (5 mg/kg x 7 days; i.v.) alone produced graft survival of 9.8 +/- 1.3 days (p < 0.1 and in combination with IP-3082 (5 mg/kg), produced survival of 7.8 +/- 3.5 days (NS). Thus, antisense oligonucleotides may proffer a selective gene-targeted immunosuppressive therapy for organ transplantation.

Inhibition of protein kinase C-alpha expression in human A549 cells by antisense oligonucleotides inhibits induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by phorbol esters.

We have identified 20-mer phosphorothioate oligodeoxynucleotides which potently (IC50 values of 100-200 nM) and specifically inhibit protein kinase C (PKC)-alpha mRNA and protein expression in human lung carcinoma (A549) cells. These oligonucleotides target multiple, diverse sites on PKC-alpha mRNA including the AUG translation codon and 3'-untranslated sequences. 2'-O-Methyl phosphorothioate analogs of these oligonucleotides were without effect on PKC-alpha mRNA levels, suggesting that the reduction in targeted PKC-alpha mRNA is through RNase H-mediated cleavage. One oligonucleotide, however, was effective at inhibiting PKC-alpha protein levels as a 2'-O-methyl phosphorothioate at concentrations 2-3-fold greater than its phosphorothioate/deoxy homolog. These results suggest that the ability to serve as an RNase H substrate, although not required for all oligonucleotides, certainly increases their potency. These oligonucleotides have been used to examine the role played by PKC-alpha in mediating the phorbol ester-induced changes in mRNA levels of the cell adhesion molecule ICAM-1. In A549 cells, ICAM-1 mRNA is increased 10-20-fold by treatment of cells with the phorbol ester phorbol 12-myristate 13-acetate. When PKC-alpha protein levels are depleted by oligonucleotide treatment of A549 cells, the increase in ICAM-1 expression in response to phorbol 12-myristate 13-acetate is greatly reduced, demonstrating that PKC-alpha plays a major role in this process.

Inhibition of endothelial cell adhesion molecule expression with antisense oligonucleotides.

In response to inflammatory stimuli, expression of a group of proteins that bind circulating leukocytes (endothelial-leukocyte adhesion molecules) are induced on the luminal surface of vascular endothelium. A series of phosphorothioate oligonucleotides 18 to 21 bases in length were designed and synthesized to hybridize selectively to the mRNA, which encodes three such endothelial-leukocyte adhesion molecules; human intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Antisense oligonucleotides were identified that selectively inhibited ICAM-1, VCAM-1, and E-selectin expression in HUVEC. Oligonucleotides that hybridized to the 3'-untranslated region of either ICAM-1, VCAM-1, or E-selectin mRNAs promoted a selective reduction in the respective mRNA levels. In contrast, oligonucleotides that hybridized to 5'-untranslated sequences did not significantly reduce target mRNA levels, although they did promote a reduction in protein expression. With the use of flow cytometry to measure cell surface expression, ICAM-1 and E-selectin were selectively inhibited by their respective antisense oligonucleotide. At low concentrations of oligonucleotides, only VCAM-1 antisense oligonucleotides inhibited VCAM-1 expression. However, at an oligonucleotide concentration of 50 nM or greater, phosphorothioate oligonucleotides not predicted to hybridize to VCAM-1 mRNA also reduced VCAM-1 expression. The sequence-independent inhibition of VCAM-1 expression by phosphorothioate oligonucleotides could be the result of a perturbation in the transcriptional regulation of the VCAM-1 gene. ICAM-1, VCAM-1, and E-selectin antisense oligonucleotides reduced adhesion of HL-60 cells to TNF-activated HUVEC. These data demonstrate that phosphorothioate oligonucleotides are capable of selectively inhibiting the expression of ICAM-1, VCAM-1, and E-selectin in HUVEC.

Estrogen modulation of the alpha-1-adrenergic response of hypothalamic neurons.

Intracellular recordings were made from 106 arcuate and cell-poor zone (ARC-CPZ) neurons in sagittal slices prepared from intact, ovariectomized and ovariectomized plus estradiol-benzoate-treated female guinea pigs, and the effects of norepinephrine (NE), the alpha 1-agonist methoxamine (MX) and the beta-agonist isoproterenol were tested. Either bath application or pressure application of 2-100 microM NE reversibly hyperpolarized and inhibited the spontaneous firing of the majority (57%, n = 60) of ARC-CPZ neurons. Isoproterenol also inhibited the majority (75%) of the ARC-CPZ neurons which it was tested on. In addition, 2-100 microM NE depolarized and/or increased the spontaneous activity of 20% (n = 21) of ARC-CPZ neurons, and some of these (n = 8) exhibited bursting activity. Similar doses of MX mimicked the NE excitation (depolarization and/or increased firing) in 48% (n = 14) of the ARC-CPZ neurons tested. Based on the serum levels of 17 beta-estradiol, the three groups of females were divided into high ( greater than 30 pg/ml) and low (less than 30 pg/ml) estrogen groups, and it was found that endogenous or exogenous estrogen significantly increased the number of neurons responding to MX (from 29 to 75%). Using intracellular labeling with procion yellow and immunocytochemistry, we have identified that luteinizing-hormone-releasing hormone neurons respond to NE. Therefore, it is suggested that one mechanism for an increase in the noradrenergic excitatory drive at the time of the preovulatory surge of luteinizing hormone in the mammal is an increase in the neuronal response to alpha 1-adrenergic stimulation.

Episodic luteinizing-hormone release in the ovariectomized female guinea pig: rapid inhibition by estrogen.

Negative feedback of estrogen was investigated in ovariectomized female guinea pigs. Two weeks after ovariectomy, indwelling catheters were inserted into the jugular vein, and 3 days later, blood samples were taken every 10 min to determine the pattern of luteinizing hormone (LH) secretion. LH secretion in these guinea pigs was episodic, with a mean pulse period of 32 min. The mean pulse amplitude was 2.1 ng/ml, with mean plasma LH levels of 1.8 ng/ml. Twenty-five micrograms 17 beta-estradiol (E2), given i.v., caused a pronounced inhibition of pulsatile LH release. Twenty-five microliters of 100% ethanol (vehicle) had no effect on plasma LH values. In a second set of experiments, ovariectomized female guinea pigs were given two injections of luteinizing hormone-releasing hormone (LHRH) (1 microgram/kg BW, i.v.) separated by 30 min. Sharp rises in serum LH values were detected after each injection. A third injection of LHRH was administered after an injection of either 25 micrograms E2 or 25 microliters vehicle. In the presence of E2, the LH response was significantly (p less than 0.005) diminished, whereas the vehicle did not change the LH response to LHRH. These rapid effects of E2 on LH secretion and the pituitary responsiveness to LHRH infusion indicate that in the ovariectomized guinea pig E2 can directly block gonadotropin secretion. These findings are consistent with the hypothesis that negative feedback actions of E2 are directly on the membrane of the gonadotrope.

A unique epitope on human serum albumin recognized by monoclonal antibody HSA-1: a probe for identification of the human origin of blood or tissue.

A panel of monoclonal antibodies was raised against human serum albumin from fusions of BALB/c splenocytes and SP2/0-Ag14 murine myeloma cells. This panel was screened against purified albumins from 21 species including chimpanzee, gorilla, and orangutan. A monoclonal antibody (HSA-1) specific for human albumin was identified. The epitope recognized by HSA-1 was shown to be conserved in all human blood samples tested. A double antibody ELISA assay was developed using biotinylated HSA-1 as the specific probe for human albumin. This assay was capable of detecting as little as 30 nanograms or less albumin/ml. This assay was used to verify the presence of human albumin in blood, tissue extracts, and other body fluids. These results show that the HSA-1 monoclonal antibody can be used in determining the human origin of blood, tissue, and a variety of other body fluids.

Episodic patterns of luteinizing hormone and follicle-stimulating hormone release: differential secretory dynamics and adrenergic control in ovariectomized rats.

Long term (4 weeks) ovariectomized rats were bled sequentially at 5-min intervals for 5 h via indwelling intraatrial cannulas. Plasma LH and FSH secretory patterns were determined from the same plasma samples by RIA. Hormonal profiles were subjected to power spectral analysis to determine periodicities of plasma LH and FSH. Distinct and regular release patterns were observed for LH, with significant periodicities between 20-40 min. In contrast to LH, FSH oscillations were neither as distinct nor as regular. However, significant periodicities in FSH (50-60 min) were often detected. At times, plasma LH and FSH appeared to be synchronized, but there were numerous instances of differential secretion. The effects of intracerebroventricular infusion of norepinephine (NE) and clonidine (an alpha 2-agonist) were tested in another group of animals. After a 2- to 3-h control bleeding period each animal bearing a chronic third ventricle cannula received an intracerebroventricular infusion of 0.3 mumol NE, clonidine, or vehicle. Blood sampling was continued for 2-3 h after infusion. Intracerebroventricular infusion of NE caused rapid and potent inhibition of LH secretion with FSH affected to a lesser extent. NE infusion decreased mean plasma LH levels and LH pulse amplitude while causing a marked increase in pulse period. Although mean FSH levels declined after NE infusion, secretory episodes of FSH were detectable even in the absence of pulsatile LH secretion. Infusion of an equimolar dose of clonidine produced a biphasic response in LH, a transient elevation followed by a decrease in overall plasma levels. In contrast to LH, plasma FSH levels showed only a delayed decrease after clonidine infusion. No significant changes in pulse amplitude or pulse period for either gonadotropin were observed. These data show that plasma FSH, like LH, oscillates in a periodic manner, but when compared with episodic LH secretion there are both quantitative and qualitative differences. Although the neural mechanisms involved in periodic LH release are also involved to a lesser extent in FSH secretion, it appears that independent regulatory mechanisms exist for LH and FSH as well.