Sp. Ho et al., MODIFICATION OF PHOSPHOROTHIOATE OLIGONUCLEOTIDES YIELDS POTENT ANALOGS WITH MINIMAL TOXICITY FOR ANTISENSE EXPERIMENTS IN THE CNS, Molecular brain research, 62(1), 1998, pp. 1-11
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.