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 (IC 50 ‫؍‬ 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.

A completely symmetric DNA segment has been constructed that binds the lactose repressor of Escherichia coli 10-fold more tightly than does the natural lactose operator sequence. This tight-binding operator is an inverted repeat of a 15-base-pair segment from the left half of the natural operator sequence, the inversion being about the point indicated by the arrow shown below: 5' T-G-T-G-T-G-G-A-A-T-T-G-T-G-A-G-C-G-G-A-T-A-A-C-A-A-T-T-T-C-A-C-A-C-where the upper sequence is the natural operator and the lower sequence is the symmetric operator. The increased affinity of repressor for this symmetric sequence supports the idea that the tetrameric repressor is designed for a two-module binding to DNA, presumably via two (or two pairs) of its identical subunits. The natural operator is apparently "flawed" by "incorrect" base pairs in the right operator half and by an "incorrect" spacing between the operator halves with respect to maximal repressor binding.A substantial body of evidence has accumulated that strongly suggests that at least two subunits of the tetrameric lactose repressor contact the lactose operator DNA segment. This evidence includes work with genetically and biochemically altered repressors (1-3) and chemical and physical studies on the specific protein-DNA complex (4, 5). The extensive bilateral symmetry of the lac operator sequence (6) supports the suggestion that the repressor uses two of its subunits to bind in similar ways to the right and left halves of the operator. However, this picture is not supported when one examines the effects of various base substitutions on repressor affinity for operator. For example, the mutations at positions 5 and 17 ( Fig. 1) represent identical base changes at symmetric locations: yet the left mutation reduces repressor affinity 6-fold more than the right mutation. Likewise, the thymine methyl group at position 8 has been shown to provide a 12-fold increment in repressor binding while the methyl group on the symmetric thymine at position 14 plays no significant role in repressor binding. Two other pairs of identical symmetrically disposed base changes are shown in Fig. 1. In each case, the left mutation is more deleterious to repressor binding than the right mutation, suggesting a greater overall contribution to binding by the left operator half. It has been proposed that binding to the right operator half is diminished by "incorrect" base pairs in this sequence, in particular that the A-T pairs at positions 13 and 15 represent "bad" contacts for repressor (7) and that a symmetric sequence in which these positions are C-G pairs would bind repressor more strongly.We report here the construction of a symmetric sequence in which the left 15 base pairs (bp) of the natural operator sequence have been inverted and repeated to give a palindrome. This segment has the proposed A-T -* C-G transversions in the right half but is 1 bp shorter than the natural operator. It binds lactose repressor substantially better than the natural operator both in vivo and in vit...

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-␣ 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-␣ 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°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-␣ 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-␣ 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-␣ 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.

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:The pharmacokinetics of a 2-O-(2-methoxyethyl)-ribose modified phosphorothioate oligonucleotide, ISIS 104838 (human tumor necrosis factor-␣ antisense), have been characterized in mouse, rat, dog, monkey, and human. Plasma pharmacokinetics after i.v. administration exhibited relatively rapid distribution from plasma to tissues with a distribution half-life estimated from approximately 15 to 45 min in all species. Absorption after s.c. injection was high (80-100%), and absorption after intrajejunal administration in proprietary formulations was as high as 10% bioavailability compared with i.v. administration. Urinary excretion of the parent drug was low, with less than 1% of the administered dose excreted in urine after i.v. infusion in monkeys at clinically relevant doses (<5 mg/ kg). ISIS 104838 is highly bound to plasma proteins, likely preventing renal filtration. However, shortened oligonucleotide metabolites of ISIS 104838 lose their affinity to bind plasma proteins. Thus, excretion of radiolabel (mostly as metabolites) in urine (75%) and feces (5-10%) was nearly complete by 90 days. Elimination of ISIS 104838 from tissue was slow (multiple days) for all species, depending on the tissue or organ. The highest concentrations of ISIS 104838 in tissues were seen in kidney, liver, lymph nodes, bone marrow, and spleen. In general, concentrations of ISIS 104838 were higher in monkey tissues than in rodents at body weight-equivalent doses. Plasma pharmacokinetics scale well across species as a function of body weight alone. This favorable pharmacokinetic profile for ISIS 104838 provides guidance for clinical development and appears to support infrequent and convenient dose administration.