1071. Development and Characterization of HSV-1 Vector-Expressed Dominant Negative PKC epsilon for Pain Therapy

Abstract

The vanilloid receptor (TRPV1) is an important pro-nociceptive Ca+2 channel, the inhibition of which attenuates pain. Recent studies have shown that TRPV1 phosphorylation by protein kinase C epsilon (PKCe) causes receptor sensitization and hyperalgesia. Inhibition of PKCe activity in nociceptors can hence be an approach to treat chronic pain. We previously reported replication defective HSV-1 vectors as a means to efficiently deliver analgesic gene products to rat dorsal root ganglion (DRG) neurons in-vivo. In this study, we recombined dominant negative PKCe (DNPKC) deleted for the C2 and C1b domains and the catalytic region into a replication defective HSV-1 vector (called vHDNPKC). DNPKC was fused to a green fluorescent protein (GFP) coding sequence and placed under transcriptional control of the HCMV immediate early promoter. Live cell imaging of vHDNPKC-infected Vero and U2OS cells displayed perinuclear green fluorescence that translocated to the plasma membrane following stimulation with the PKC activator, PMA. Whole-cell electrophysiological recordings of capsaicin-induced TRPV1 currents in vHDNPKC-infected adult rat DRG neurons demonstrated significantly smaller current amplitudes (2.5 fold) that desensitized faster (4 fold) than uninfected or vHG-infected controls. Control capsaicin responses were potentiated by isoform-specific PKCe translocation following stimulation with a specific bradykinin receptor 2 agonist, HYP3-bradykinin. HYP3-bradykinin failed to potentiate capsaicin currents in vHPKCDN-infected neurons, while the non-specific PKC activator PDBu caused a slow drift current that was antagonized by the PKC inhibitor bisindolylmaleimide. This result demonstrates isoform-specific inhibition of PKCe activity. Taken together, the data demonstratean in-vitro inhibition of TRPV1 function in nociceptive neurons by HSV-1 vector-expressed dominant negative PKCe. This vector can therefore be used as a viable approach for the in-vivo induction of analgesia.