Modification of phosphorothioate oligonucleotides yields potent analogs with minimal toxicity for antisense...

There is increasing evidence that phosphorothioate oligonucleotides infused into the brain can cause a host of undesired side effects which compromise the antisense experiment. In studies on the corticotropin releasing factor type-2 receptor, several phosphorothioate oligonucleotides administered intraventricularly produced significant weight loss in rats. Four different phosphodiester and phosphorothioate oligonucleotide analogs were examined to identify molecules which could eliminate these side effects while maintaining good potency for antisense inhibition. Of these, chimeric oligonucleotides consisting of a mixed phosphodiester-phosphorothioate backbone, and having 2'-methoxyribonucleotide modifications in 60% of the oligonucleotide were the most optimal. Rats treated with these chimeric oligonucleotides gained weight at rates identical to that of saline-treated controls. In addition, the antisense oligonucleotide but not the mismatch control sequence reduced corticotropin releasing factor type-2 receptor binding of 125iodo-sauvagine in the lateral septum by 40-60% after 5 daily injections. Increasing the dosing period to 9 days reduced receptor binding by 78%. Reductions in protein binding were accompanied by comparable reductions in the in situ hybridization signal of the corticotropin releasing factor type-2 receptor mRNA. However, when an oligonucleotide analog incapable of supporting ribonuclease H activity was used, neither protein nor RNA binding levels were changed compared to saline-treated controls. These results suggest that ribonuclease H or enzymes with similar activity are critical to the antisense inhibition observed in the lateral septum.