Oligonucleotide-europium complex conjugate designed to cleave the 5' cap structure of the ICAM-1 transcript potentiates antisense activity in cells.

The 5' cap structure of mRNA is a N7 methylated guanosine residue that is linked by a 5'-5' triphosphate linkage to the 5'-terminus of cellular and viral RNAs synthesized by RNA polymerase II. This unique structure facilitates several processes of mRNA metabolism, including splicing, nucleocytoplasmic transport,initiation of translation, and degradation. Previous research has demonstrated that the lanthanide macrocycle complex, Eu(THED)3+, effectively cleaves the 5' cap structure of mRNA in solution by nucleophilic attack of the triphosphate linkage via the metal-activated hydroxyethyl group of the THED ligand. This report shows that attachment of a Eu(THED)3+analog to the 3'-terminus of an antisense oligonucleotide, which targets the 5'-terminus of the intercellular adhesion molecule 1 mRNA, potentiates the inhibitory activity of the antisense oligonucleotide in cytokine-treatedendothelial cells.

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.