SEQUENCE-SPECIFIC ANTITUMOR-ACTIVITY OF A PHOSPHOROTHIOATE OLIGODEOXYRIBONUCLEOTIDE TARGETED TO HUMAN C-RAF KINASE SUPPORTS AN ANTISENSE MECHANISM OF ACTION IN-VIVO

To determine the mechanism of action responsible for the in vivo antit umor activity of a phosphorothioate antisense inhibitor targeted again st human C-raf kinase (ISIS 5132, also known as CGP69846A), a series o f mismatched phosphorothioate analogs of ISIS 5132 or CGP69846A were s ynthesized and characterized with respect to hybridization affinity, i nhibitory effects on C-raf gene expression in vitro, and antitumor act ivity in vivo. Incorporation of a single mismatch into the sequence of ISIS 5132 or CGP69846A resulted in reduced hybridization affinity tow ard C-raf RNA sequences and reduced inhibitory activity against C-raf expression in vitro and tumor growth in vivo, Moreover, incorporation of additional mismatches resulted in further loss of in vitro and in v ivo activity in a manner that correlated well with a hybridization-bas ed (i.e., antisense) mechanism of action, These results provide import ant experimental evidence supporting an antisense mechanism of action underlying the in vivo antitumor activity displayed by ISIS 5132 or CG P69846A.