In vitro and in vivo effects of an alpha3 neuronal nicotinic acetylcholine receptor antisense oligonucleotide.

In the mammalian central nervous system (CNS), a family of alpha and beta subunits (alpha2-7, beta2-4) assemble to form both hetero- and homopentameric neuronal nicotinic acetylcholine receptors (nAChRs). In contrast to alpha4beta2 and alpha7, the predominant brain subtypes, far less is known regarding the functional expression and significance of alpha3-containing nAChRs in the CNS. In trying to better understand the role alpha3 in the CNS, an antisense knockdown strategy was utilized in the present studies. Specifically, Isis 106567 was identified out of 80 antisense oligonucleotides (aONs) designed and screened for their ability to reduce alpha3 mRNA expression in PC-12 cells. In addition to reducing alpha3 mRNA by greater than 75%, Isis 106567 attenuated nicotine-induced calcium influx in alpha3-expressing F11 cells. In vivo studies revealed significant reduction of alpha3 mRNA levels in both thalamus and medial habenula, regions known to express alpha3, following continuous (7 days) intracerebroventricular (i.c.v.) infusion of Isis 106567 in rats. Consistent with functional alpha3 knockdown, epibatidine-induced c-Fos expression in the medial habenula was attenuated in aON-treated rats. Known physiological responses elicited by epibatidine, such as hypothermia and micturition, were not affected by alpha3 aON treatment. However, the incidence of epibatidine-induced seizures was reduced in alpha3-antisense aON-treated rats, suggesting that alpha3 may be involved in mediating seizures produced by the nAChR agonist. Results of our studies suggest that Isis 106567 may be a useful in vivo tool for characterizing the functional significance of alpha3 expression in the CNS.

2'-O-[2-[(N,N-dimethylamino)oxy]ethyl]-modified oligonucleotides inhibit expression of mRNA in vitro and in vivo.

Synthesis and antisense activity of oligonucleotides modified with 2'-O-[2-[(N,N-dimethylamino)oxy] ethyl] (2'-O-DMAOE) are described. The 2'-O-DMAOE-modified oligonucleotides showed superior metabolic stability in mice. The phosphorothioate oligonucleotide 'gapmers', with 2'-O-DMAOE- modified nucleoside residues at the ends and 2'-deoxy nucleosides residues in the central region, showed dose-dependent inhibition of mRNA expression in cell culture for two targets. 'Gapmer' oligonucleotides have one or two 2'-O-modified regions and a 2'-deoxyoligonucleotide phosphorothioate region that allows RNase H digestion of target mRNA. To determine the in vivo potency and efficacy, BalbC mice were treated with 2'-O-DMAOE gapmers and a dose-dependent reduction in the targeted C-raf mRNA expression was observed. Oligonucleotides with 2'-O-DMAOE modifications throughout the sequences reduced the intercellular adhesion molecule-1 (ICAM-1) protein expression very efficiently in HUVEC cells with an IC(50) of 1.8 nM. The inhibition of ICAM-1 protein expression by these uniformly modified 2'-O-DMAOE oligonucleotides may be due to selective interference with the formation of the translational initiation complex. These results demonstrate that 2'-O-DMAOE- modified oligonucleotides are useful for antisense-based therapeutics when either RNase H-dependent or RNase H-independent target reduction mechanisms are employed.

Analgesic profile of intrathecal P2X(3) antisense oligonucleotide treatment in chronic inflammatory and neuropathic pain states in rats.

Extracellular adenosine triphosphate (ATP), acting at P2X ionotropic receptors, is implicated in numerous sensory processes. Exogenous ATP has been shown to be algogenic in both animals and humans. Research focus has been directed towards the P2X(3) receptor, as it is preferentially expressed on nociceptive C-fibers and its implication in pain processing is supported by an altered nociceptive phenotype in P2X(3) knock-out mice. In order to further characterize the role of P2X(3) receptor activation in nociception, we evaluated the effects of continuous intrathecal administration of P2X(3) antisense oligonucleotides for 7 days in the rat. P2X(3) receptor antisense oligonucleotide treatment significantly decreased nociceptive behaviors observed after injection of complete Freund's adjuvant (CFA), formalin or alphabeta-methylene ATP into the rat's hind paw. The anti-hyperalgesic effects of the antisense treatment in the CFA model of inflammatory pain were dose related. Similar effects were observed with two distinct P2X(3) antisense oligonucleotides. These behavioral effects were significantly correlated with a decrease in P2X(3) receptor protein expression in the dorsal root ganglia (DRG). In contrast, a decrease in P2X(3) receptor protein expression in the DRG did not affect nociceptive behavior in the carrageenan model of acute thermal hyperalgesia. P2X(3) receptor antisense oligonucleotide treatment also significantly reduced mechanical allodynia observed after spinal nerve ligation. Overall, the present data demonstrate that activation of P2X(3) receptors contribute to the expression of chronic inflammatory and neuropathic pain states and that relief form these forms of chronic pain might be achieved by selective blockade of P2X(3 )receptor expression or activation.