Vol. 57, Issue 4, 725-731, April 2000

Antisense Inhibition of -Opioid Receptor Gene Function In Vivo by Peptide Nucleic Acids

Graeme L. Fraser, Janna Holmgren, Paul B. S. Clarke, and Claes Wahlestedt

AstraZeneca R & D Montréal, Quebec, Canada (G.L.F., J.H.); Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada (G.L.F., P.B.S.C., C.W.); and Center for Genomics Research, Karolinska Institutet, Stockholm, Sweden (C.W.)

Peptide nucleic acids (PNA) are synthetic analogs of DNA that hybridize to complementary oligonucleotide sequences with exceptional affinity and target specificity. The stability of PNA in biological fluids together with the unique hybridization characteristics of these structures suggests that PNA may have considerable potential as antisense agents for experimental use in vivo. To test this hypothesis, we attempted to modulate supraspinal -opioid receptor function in rats using PNA sequences designed to be complementary to a region of the rat -opioid receptor. Repeated i.c.v. administration of PNA over a period of 5 days significantly inhibited the antinociceptive response and locomotor response to selective -opioid receptor agonists. PNA attenuated -opioid receptor function in a sequence-specific, target-specific, and reversible manner characteristic of the functional inhibition caused by an antisense mechanism. There were no apparent toxicities arising from the PNA treatment based on the behavior of the animals and inspection of the treated tissues. Saturation binding studies on brain homogenates did not reveal any significant difference in receptor B_max between treatment groups. However, [³⁵S]guanosine-5'-O-(3-thio)triphosphate binding assays demonstrated a significant decrease in agonist efficacy in homogenates prepared from antisense-treated rats. Taken together, these results demonstrate that peptide nucleic acids are effective antisense agents in vivo and suggest that PNA may be a useful alternative to phosphodiester or phosphorothioate oligonucleotides, or variants thereof, for determination of gene function in vivo.