title: Characterization of a potent and specific class of antisense oligonucleotide inhibitor of human protein kinase C-alpha 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-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.
authors.affiliation: Department of Molecular Pharmacology, ISIS Pharmaceuticals, Carlsbad, California 92008, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: McKay
authors.forename: L J
authors.initials: LJ
authors.lastname: Miraglia
authors.forename: L L
authors.initials: LL
authors.lastname: Cummins
authors.forename: S R
authors.initials: SR
authors.lastname: Owens
authors.forename: H
authors.initials: H
authors.lastname: Sasmor
authors.forename: N M
authors.initials: NM
authors.lastname: Dean
mesh_terms: D001483:Base Sequence; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics*; D008969:Molecular Sequence Data; D020319:Oligodeoxyribonucleotides, Antisense / Q000008:administration & dosage / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics*; D051571:Protein Kinase C-alpha; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013816:Thermodynamics; D013873:Thionucleotides / Q000008:administration & dosage / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D020319:Oligodeoxyribonucleotides, Antisense; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D013873:Thionucleotides; C108494:ISIS 3521; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate