Characterization of a Potent and Specific Class of Antisense Oligonucleotide Inhibitor of Human Protein Kinase C-α Expression

Abstract

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.

Targeting Foxo1 in Mice Using Antisense Oligonucleotide Improves Hepatic and Peripheral Insulin Action

“…All ASOs were synthesized as 20-base phosphorothioate chimeric ASOs, where bases 1-5 and 16 -20 were modified with 2Ј-O-(2-methoxy)-ethyl. This chimeric design has been shown to provide both increased nuclease resistance and mRNA affinity, while maintaining the robust RNase H terminating mechanism utilized by these types of ASOs (25). These benefits result in an attractive in vivo pharmacological and toxicological profile for 2Ј-O-(2-methoxy)-ethyl chimeric ASOs.…”

Antisense oligonucleotide-based therapeutics for cancer

“…However, with progress in siRNA, it is also becoming a popular method for gene functionalization in cells McManus and Sharp, 2002). We routinely use oligonucleotides containing 2 0 -MOE-modified RNase H-dependent oligonucleotides because of their increased potency and longer duration of action (McKay et al, 1999). There have been a large number of published studies using this modification to identify gene functions in multiple systems (for example (Li et al, 1999;Taylor et al, 1999a, b;Bulavin et al, 2001;Gottschalk et al, 2001;Urban et al, 2001;Pedersen et al, 2002).…”

“…The two most advanced modifications are 2 0 -O-methyl and 2 0 -Omethoxyethyl (MOE) (Figure 3), with examples of each currently in clinical trials (Table 1). These modifications result in a 3-10-fold increase in potency in cell-based assays compared to phosphorothioate oligodeoxynucleotides, an increase in nuclease resistance and decreased toxicity (Monia et al, 1993;Zhang et al, 1995;Zhao et al, 1995;Baker et al, 1997;McKay et al, 1999;Henry et al, 2000;Geary et al, 2001). Uniform 2 0 -O-modified-oligonucleotides do not support an RNase H mechanism.…”

Bifunctional apoptosis inhibitor (BAR) protects neurons from diverse cell death pathways

“…Oligonucleotides were synthesized and purified as previously described. 33 A549 cells were plated in 96-well plates and treated with 150 nM oligonucleotide in the presence of 3.75 mg/ml Lipofectin reagent (Life Technologies) for 4 h at 371C. Cells were incubated overnight in the absence of oligonucleotide.…”