OLIGONUCLEOTIDE N3'-]P5' PHOSPHORAMIDATES AS ANTISENSE AGENTS

Uniformly modified oligonucleotide N3'-->P5' phosphoramidates, where e very 3'-oxygen is replaced by a 3'-amino group, were synthesized. Thes e compounds have very high affinity to single-stranded RNAs and thus h ave potential utility as antisense agents, As was shown in this study, the oligonucleotide phosphoramidates are resistant to digestion with snake Venom phosphodiesterase, to nuclease activity in a Hela cell nuc lear extract, or to nuclease activity in 50% human plasma, where no si gnificant hydrolysis was observed after 8 h, These compounds were used in various in vitro cellular systems as antisense compounds addressed to different targeted regions of c-myb, c-myc and bcr-abl mRNAs. C-my b antisense phosphoramidates at 5 mu M caused sequence and dose-depend ent inhibition of HL-60 cell proliferation and a 75% reduction in c-my b protein and RNA levels, as determined by Western blot and RT-PCR ana lysis. Analogous results were observed for anti-c-myc phosphoramidates , where a complete cytostatic effect for HL-60 cells was observed at 1 mu M concentration for fully complementary, but not for mismatched co mpounds, which were indistinguishable from untreated controls, This wa s correlated with a 93% reduction in c-myc protein level, Moreover, co lony formation by the primary CML cells was also inhibited 75-95% and up to 99% by anti-c-myc and anti-bcr-abl phosphoramidate oligonucleoti des, respectively, in a sequence- and dose-dependent manner within a 0 .5 nM-5 mu M dose range. At these concentrations the colony-forming ab ility of normal bone marrow cells was not affected. The presented in v itro data indicate that oligonucleotide N3'-->P5' phosphoramidates cou ld be used as specific and efficient antisense agents.