ENHANCED ACTIVITY OF AN ANTISENSE OLIGONUCLEOTIDE TARGETING MURINE PROTEIN-KINASE C-ALPHA BY THE INCORPORATION OF 2'-O-PROPYL MODIFICATIONS

We have previously described the characterization of a 20mer phosphoro thioate oligodeoxynucleotide (ISIS 4189) which inhibits murine protein kinase C-alpha (PKC-alpha) gene expression, both in vitro and in vivo . In an effort to increase the antisense activity of this oligonucleot ide, 2'-O-propyl modifications have been incorporated into the 5'- and 3'-ends of the oligonucleotide, with the eight central bases left as phosphorothioate oligodeoxynucleotides. Hybridization analysis demonst rated that these modifications increased affinity by similar to 8 and 6 degrees C per oligonucleotide for the phosphodiester (ISIS 7815) and phosphorothioate (ISIS 7817) respectively when hybridized to an RNA c omplement, In addition, 2'-O-propyl incorporation greatly enhanced the nuclease resistance of the oligonucleotides to snake venom phosphodie sterase or intracellular nucleases in vivo. The increase in affinity a nd nuclease stability of ISIS 7817 resulted in a 5-fold increase in th e ability of the oligonucleotide to inhibit PKC-alpha gene expression in murine C127 cells, as compared with the parent phosphorothioate oli godeoxynucleotide. Thus an RNase H-dependent phosphorothioate oligodeo xynucleotide can be modified as a 2'-O-propyl 'chimeric' oligonucleoti de to provide a significant increase in antisense activity in cell cul ture,