Antisense Oligodeoxynucleotides for In Vivo Targeting of Corticotropin-Releasing Hormone mRNA: Comparison of Phosphorothioate and 3′-Inverted Probe Performance

 

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Summary

Antisense DNA has been successfully used in vivo to selectively inhibit expression of proteins in the brain. However, stressful side effects after oligodeoxynucleotide (ODN) application have been observed, but not carefully characterized. An attempt was made to establish an animal model of reduced corticotropin-releasing hormone (CRH) activity, using antisense DNA corresponding to the start coding region of rat CRH mRNA with either 3′-3′ inverted internucleotidic linkage or with all-phosphorothioate modification. Probes were injected intracerebroventricularly (i.c.v.) twice, 12-hours apart. After phosphorothioate sense ODN injection serum corticosterone levels were significantly elevated compared to vehicle (aCSF) or 3′-3′ end inverted sense ODN controls. This increase was also apparent but less pronounced in phosphorothioate antisense treated animals compared with the corresponding sense group. After exposure to ether vapour, both phosphorothioate and inverted antisense ODN injected rats showed a markedly diminished stress induced corticosterone secretion compared to the corresponding sense or vehicle injected rats. These results indicate that a) stress induced corticosterone release is suppressed by i.c.v. CRH antisense treatment, b) phosphorothioate ODNs exert an unspecific, chronic stress-like activation of the HPA-axis and c) this effect is partly inhibited by phosphorothioate antisense directed against CRH mRNA.