Specific Inhibition of the Rat Ligand-Gated Ion Channel P₂X₃Function Via Methoxyethoxy-Modified Phosphorothioated Antisense Oligonucleotides

Abstract

P2X3 is one receptor of a family of seven ligand-gated ion channels responding to purines. Increasing evidence indicates its involvement in neuronal signaling and in pain. However, there is currently no selective inhibitor known for this subtype. In order to obtain such a specific inhibitor, a variety of antisense oligonucleotides (ASO) against rat P2X3 was tested, and dose-dependent, sequence-specific downregulation of the rat P2X3 receptor (expressed in a Chinese hamster ovary cell line [CHO-K1]) on the mRNA, protein, and functional levels was observed. Using real-time quantitative PCR, a dose-dependent downregulation of P2X3 mRNA by ASO, as compared with untreated and mismatch controls, was demonstrated. Subsequently, downregulation by the two most potent ASO was confirmed at the protein level by Western blot. Sequence specificity was shown by titration of mismatches to the original selected oligonucleotide, and this correlated with progressive loss of P2X3 inhibition. The functional response of the P2X3 receptor was examined using whole-cell voltage clamping. Upon application of 10 microM of a nonspecific agonist, alpha,beta-methylene-ATP (alphabeta meATP), pretreatment with increasing amounts of the most active ASO 5037 correlated with a decrease in depolarization. The ability to specifically downregulate the P2X3 receptor by ASO treatment will allow investigation of the biologic role of this receptor in neuronal tissues and eventually in in vivo models of chronic pain.

Current and Future Issues in the Development of Spinal Agents for the Management of Pain

“…Intrathecal antisense has been employed to reduce expression of a variety of proalgesic mediators, including growth factor cellular matrix proteins (thrombospondin) [404] and receptors relevant to nociceptive processing, including the NK1 receptor for substance P [405], purines [372]; GFRα1, receptor for GDNF [406], NMDA receptors [407]; glutamate metabotrophic receptors [408-410], P2X3 [411, 412], TrpV1 receptors [413], TrpV1 receptors [413], CCR2 (canonical receptor for MCP-1) [414], synaptic scaffolding proteins [415], a variety of kinases such as P38 MAPK [416, 417], phospholipases [418], and a variety of channels including sodium (Na v 1.8 [419, 420]: and calcium channels (T-type [421, 422]:). Inactivating transcription factors by employing an oligonucleotide decoy protein represents an alternate approach to prevent activation otherwise initiated by a pain state [423].…”

Flexible subunit stoichiometry of functional human P2X2/3 heteromeric receptors

“…In consequence, the blockade of these receptors by pharmacological antagonists (Jarvis, 2003), their down regulation by antisense oligonucleotides (Dorn et al, 2001) or short interfering (si)RNAs (Hemmings-Mieszczak et al, 2003) or their genetic deletion (Cockayne et al, 2005) has been shown to inhibit acute pain or chronic inflammatory and neuropathic pain. Sensory neurons, such as those of the dorsal root ganglia (DRG), contain the cell bodies of the nociceptive C fibre terminals, which display a variety of current responses to activation by a,b-meATP (Petruska et al, 2000;Pankratov et al, 2001).…”

Current and Future Issues in the development of spinal agents for the management of pain

“…i) Intrathecal antisense has been employed to reduce expression of a variety of proalgesic mediators, including growth factor cellular matrix proteins (thrombospondin) [404] and receptors relevant to nociceptive processing, including the NK1 receptor for substance P [405], purines [372]; GFRα1, receptor for GDNF [406], NMDA receptors [407]; glutamate metabotrophic receptors [408][409][410], P2X3 [411,412], TrpV1 receptors [413], TrpV1 receptors [413], CCR2 (canonical receptor for MCP-1) [414], synaptic scaffolding proteins [415], a variety of kinases such as P38 MAPK [416,417], phospholipases [418], and a variety of channels including sodium (Na v 1.8: [419,420] and calcium channels (T-type: [421,422]). Inactivating transcription factors by employing an oligonucleotide decoy protein represents an alternate approach to prevent activation otherwise initiated by a pain state [423].…”