MODIFICATION OF PHOSPHOROTHIOATE OLIGONUCLEOTIDES YIELDS POTENT ANALOGS WITH MINIMAL TOXICITY FOR ANTISENSE EXPERIMENTS IN THE CNS

There is increasing evidence that phosphorothioate oligonucleotides in fused into the brain can cause a host of undesired side effects which compromise the antisense experiment. In studies on the corticotropin r eleasing factor type-2 receptor, several phosphorothioate oligonucleot ides administered intraventricularly produced significant weight loss in rats. Four different phosphodiester and phosphorothioate oligonucle otide analogs were examined to identify molecules which could eliminat e these side effects while maintaining good potency for antisense inhi bition. Of these, chimeric oligonucleotides consisting of a mixed phos phodiester-phosphorothioate backbone, and having 2-methoxyribonucleoti de modifications in 60% of the oligonucleotide were the most optimal. Rats treated with these chimeric oligonucleotides gained weight at rat es identical to that of saline-treated controls. In addition, the anti sense oligonucleotide but not the mismatch control sequence reduced co rticotropin releasing factor type-2 receptor binding of (125)iodo-sauv agine in the lateral septum by 40-60% after 5 daily injections. Increa sing the dosing period to 9 days reduced receptor binding by 78%. Redu ctions in protein binding were accompanied by comparable reductions in the in situ hybridization signal of the corticotropin releasing facto r type-2 receptor mRNA. However, when an oligonucleotide analog incapa ble of supporting ribonuclease H activity was used, neither protein no r RNA binding levels were changed compared to saline-treated controls. These results suggest that ribonuclease H or enzymes with similar act ivity are critical to the antisense inhibition observed in the lateral septum. (C) 1998 Elsevier Science B.V. All rights reserved.