Gene therapy for the management of pain: part II: molecular targets.

Abstract

Received from the Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins Hospital, Baltimore, Maryland; the Department of Anesthesiology and Pain Management, University of Texas Southwestern Medical Center, Dallas, Texas; and the Departments of Anesthesiology, Pharmacology, and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York.TREATMENT of chronic pain, particularly of neuropathic etiology, is extremely difficult and resistant to many available pharmacologic therapies. Current analgesic agents may be limited with regard to analgesic efficacy or side effects. 1,2Newer and experimental pharmacologic agents may also have significant limitations. 3By targeting a specific receptor or other specific protein targets, a gene therapy approach to the treatment of pain may provide greater analgesic efficacy without the limitations associated with current pharmacotherapy. Advances in the field of gene therapy, along with significant increases in our understanding of the neurobiology of nociception and knowledge of the fundamental genetic structure of many nociceptive targets, have made gene therapy for the management of pain a conceivable reality.In part I of this review, 3Awe introduced the basic concepts of gene therapy with an emphasis on the available tools (e.g. , viral vectors and antisense oligonucleotides) and strategies for upregulating antinociceptive or downregulating pronociceptive targets. In part II, we summarize current knowledge regarding the nociceptive role, molecular biology, and antisense and knockout data of several novel nociceptive targets for gene therapy. We base our selection of the targets included in this review on the three aforementioned criteria. The targets selected are the best characterized and, in our opinion, most likely amenable to the gene therapeutic approach. A simple but feasible strategy and potential gene therapy targets for the management of pain are summarized in figure 1. However, the list is admittedly incomplete, and the readers are referred to other recent reviews cited in part I of this review for a broader perspective on potential targets for the management of pain. 3AAssuming that an ideal gene delivery system is available, viral or otherwise, what would be the therapeutic target? Of the many targets comprising the complex nociceptive cascade, how does one choose the best point for therapeutic intervention? An ideal target should have (1) a well-defined role in the pathogenesis of neuropathic pain, (2) a well-defined pharmacologic profile with specific pharmacologic tools (i.e. , agonists and antagonists) available, and (3) little role in normal physiology, thus limiting the chances of side effects associated with pharmacologic manipulation of the target. No consensus exists on such an ideal target for management of pain.Targets that lend themselves to a genetic approach to pain management should have strong evidence for a role in nociception, a reasonably defined molecular biology and function, and antisense knockdown and knockout data. Space precludes discussion of several traditional and obvious targets, such as opioid and α2-adrenergic receptors, and some potentially promising targets, including N-type calcium channels, voltage-gated Ca2+channels, adenosine triphosphate–sensing purinergic P2X3, acid-sensing ion channel, and neurotrophin receptors. Some of these targets were recently reviewed elsewhere. 4The opioid receptors as a target for a genetic approach to pain management is clearly well supported by existing literature. We chose not to include opioid receptors in our review for the simple reason that many conventional and experimental drugs highly selective for this receptor already exist. In addition, therapeutic limitations imposed by the development of tolerance appear to be a fundamental property of opioid receptors most likely irreconcilable even through a genetic approach. Although our review focuses on receptors as the prime target, nonreceptor targets, such as the enzymes responsible for the synthesis of the neurotransmitter that acts on the receptors or signal transduction molecules that mediate the downstream effect of receptor activation, are also potential targets.In the following sections, we review selected central nervous system (CNS) targets for gene therapy, with particular attention to the evidence of role in nociception, information on the molecular biology, and studies investigating a gene therapeutic approach in altering nociception. Molecular biologic information, including the size of the cDNA encoding the target protein and genomic structure, are critical in designing concrete strategies for gene therapy using viral vectors or oligodeoxyribonucleotides (ODNs). Figure 2shows the protein topology and table 1summarizes the pertinent molecular biologic information for the potential therapeutic targets described below. Table 2lists the conventional agonists and antagonist drugs described in the text for the selected targets. With substance P (SP) as its most selective endogenous ligand, the tachykinin neurokinin 1 (NK-1) receptor is present throughout the CNS and peripheral nervous system and mediates a variety of physiologic activities. 5Of the three neurokinin receptor subtypes, NK-1 is the most prevalent at the spinal cord level and important in enhancing action of excitatory amino acids (EAAs) and mediating secondary hyperalgesia and central sensitization. 5,6The NK-1 receptors have been localized postsynaptically to afferent nerve fibers in dorsal horn laminae I, III, and IV 7–9and are consistent with the role of SP in nociception. 6Neurokinin-1 receptors appear to be expressed mostly in excitatory neurons, as NK-1 receptor immunoreactive neurons are minimally γ-aminobutyric acid (GABA)- or glycine-immunoreactive. 8Chronic inflammatory and sciatic nerve transection models of persistent pain result in upregulation of NK-1 receptor immunoreactivity in the superficial laminae of the dorsal horn. 10Death of SP receptor-containing lamina I spinal neurons after internalization of a SP-coupled neurotoxin results in significant attenuation to noxious stimuli and inhibition of hyperalgesia. 11Data from administration of NK-1 agonists support the role of NK-1 receptors in mediating excitation in noxious stimuli–responsive spinal neurons. Iontophoretic application of SP results in preferential excitation of dorsal horn nociceptive neurons, whereas nonnociceptive neurons are unaffected. 12Intrathecal injection of NK-1 receptor agonists into awake rats and mice elicit biting, scratching, and licking of forelimbs. 13–15After surgical deafferentation, there is an increase in dorsal horn NK-1 binding sites, and intrathecal injection of SP produces significantly increased pain-related behaviors. 9,16Administration of the selective NK-1 agonist, Sar-SP, results in production of c-fos in laminae I, and injection of NK-1 antagonist L-668,169 significantly decreases formalin-induced c-fos expression. 17In addition, increased expression of NK-1 mRNA in the dorsal horn of rat after peripheral noxious stimuli is blocked by the NK-1 receptor antagonist LY-306,740. 18Use of NK-1 antagonists in whole animal behavior models has emphasized the role of NK-1 receptors in mediating nociception after prolonged noxious chemical stimuli as opposed to phasic mechanical stimuli. 19,20NK-1 receptors appear to be important in development of thermal and mechanical hyperalgesia. 21–23The NK-1 antagonists L-733,060 and CP-99,994 inhibit late- but not early-phase response after injection of formalin in mice and gerbils. 19,24Electrophysiologic studies after injection of SP in rats reveal an increase in response to noxious mechanical stimuli that is reduced in a dose-dependent manner by the specific NK-1 receptor antagonist CP-96,345 but not by its inactive enantiomer, CP-96,344. 25Although SP results in neuronal excitation in response to nonnoxious stimuli, NK-1 antagonists do not inhibit responses to nonnoxious mechanical stimuli. 25Previous administration of a selective NK-1 antagonist (RP-67,580), but not its inactive isomer (RP-68,651), produced a dose-related reduction in the number of formalin-evoked c-fos–like immunoreactive spinal neurons. 26Human clinical studies have not supported analgesic action of NK-1-receptor antagonists thus far. Initial studies demonstrated analgesic action of CP-99,994 equivalent to ibuprofen for dental pain. 27Subsequent studies in osteoarthritis, neuropathic pain, and migraine have demonstrated no analgesic effect, although access to CNS by the compounds relatively impermeable to the blood–brain barrier may be a problem. 28In contrast, NK-1 receptor antagonists have demonstrated potent antiemetic effects and offer promising use in treatment of psychiatric disorders. 28,29The reason for the failure of NK-1 receptor antagonists to demonstrate clinical efficacy in humans is not clear but may be because of the pharmacokinetic limitations of the particular antagonists examined or the discordance between painful behavior measured in animals and human clinical pain. 28,30Acting at the NK-1 receptor, tachykinins will facilitate the acute, excitatory effects of EAAs on N -methyl-d-aspartate (NMDA) receptors to produce prolonged enhancement of EAA responses, resulting in sensitization of dorsal horn neurons and secondary hyperalgesia. 5,31,32Co-application of SP and NMDA produces enhancement of responses of primate spinothalamic neurons and increased sensitivity to cutaneous mechanical stimulation. 33SP and glutamate coexist in the same primary afferent terminals in the dorsal horn, and tachykinins may tonically be released to modulate NMDA-mediated glutamatergic transmission. 34,35Administration of selective NK-1 antagonists will block the potentiating effect of SP on EAA actions on spinal neurons. 36,37In addition, co-application of an NMDA receptor and selective NK-1 antagonist produce a supraadditive effect in inhibition of nociception, suggesting an interaction between intracellular signal transduction cascades initiated by the two ligands. 32Thus, the NK-1 receptor appears to play an important role in mediating persistent nociception and may contribute to central sensitization. NK-1 receptors are expressed in spinal cord at locations consistent with processing of afferent nociceptive input and are rapidly downregulated after nociceptive afferent input. Physiologic experimental evidence also supports the role of NK-1 receptors in mediating nociception despite the fact that human clinical data with selective NK-1 receptor antagonists have been disappointing.As with other G protein–coupled receptors, the tachykinin NK-1 receptor has seven hydrophobic transmembrane domains (TM1–TM7) with an extracellular NH2and intracellular COOH terminus. 38–40A functional cDNA encoding rat SP receptor consisted of 407 amino acids with a molecular mass of approximately 46,385 Da. 38,40There is a section of a GU dinucleotide repeat at the 5′ end and CU and CA dinucleotide repeats in the 3′ end that may result in a hairpin loop formation, thus potentially affecting translation or stability of receptor mRNA. 40Like other G protein–coupled receptors, there are potential phosphorylation sites (serine and threonine residues) in the COOH terminus. Mechanisms of interactions between NK-1 receptors and its agonists and antagonists have not been definitively elucidated; however, two popular hypotheses for binding include the volume-exclusion hypothesis (agonist and antagonist binding may overlap) and receptor–ligand interaction, where allosteric alteration in receptor conformation occurs after binding by an agonist or antagonist. 41The NK-1 receptor is encoded by a five-exon and four-intron gene structure. 42,43The NK-1 receptor is approximately 45 kilobases in length, and the five exons consist of 965, 195, 151, 197, and 2,010 base pairs. 43Exon 1 encodes for the NH2terminus to TM3, which is important for SP binding. 44Exons 2 and 3 encode for TM4 and TM5. The genetic code for the COOH terminus and TM6–TM7 is included in exons 6 and 7. 45Putative NK-1 receptor promoter regions include a proximal promoter consisting of six to eight bases located immediately upstream from the conventional TATA sequence and several conserved sequences, including an adenylate cyclase (cAMP)-responsive, phorbol ester, and calcium-activated transcription sites. 42A proposed structure for the NK-1 receptor has been described. 46–48Certain structural areas are important for various NK-1 receptor functions. The third intracellular loop is crucial to downstream second messenger activation, as substitution in this area prevents cAMP production and phosphoinositol turnover after NK-1 agonist application. 49Sections of TM2 (Asn-85, Asn-89, Tyr-92, Asn-96) and TM7 (Tyr-287) are required for high-affinity binding of peptides. 48Other regions, such as Glu-78 in TM2 and Tyr-205 and Tyr-216 in TM5, may be important for G-protein coupling and activation. 48,50Tyrosine-containing sequences in TM7 and COOH terminus interact with the endocytic apparatus and may be important for the process of NK-1 receptor endocytosis. 51The NK-1 receptor is coupled to G proteins that are sensitive (Go, Gi) and insensitive (Gq, G11) to pertussis toxin. 39Several independent second messengers mediate some of the biologic effects after NK-1 receptor activation. 39,52NK-1 agonists may differentially stimulate second messenger pathways, and targeting downstream mediators of NK-1 receptor activation to attain antinociception may be difficult. 53,54Other effects may not require participation of second messengers, such as those mediated through direct modulation of channel activity and SP-induced stimulation of a nonselective inward current. 39Once activated by SP, the NK-1 receptor undergoes receptor internalization in the cell bodies and dendrites of nociceptive spinal neurons as marked by a loss of SP receptor immunoreactivity on cell membranes and increase in SP receptor–positive endosomes. 55,56Within 1 min of SP injection, 60% of SP receptor–immunoreactive neurons show receptor internalization with return to baseline in approximately 60 min. 55Once internalized, the receptor undergoes morphologic reorganization such that the uniform tubular structure of dendrites is transformed into “swollen varicosities” with eventual endosomal SP degradation and possibly receptor and recycling or resynthesis of NK-1 receptor back to the cell membrane. 56–58Shape transformation of the dendrites actually may result in transformation of neural impulses, which may be relevant to development of pain states. 59The number of neurons internalizing NK-1 receptors corresponds to concentration of SP used. 55NK-1 internalization is inhibited by the NK-1 antagonist RP-67,580. 55Thus, internalization of NK-1 receptors may functionally downregulate the receptor response to subsequent stimulation by SP. Enhancement of tachyphylaxis to SP by attenuation of receptor turnover may be a novel approach to antinociception. Alternatively, enhancement of receptor internalization by SP fragments without activation of downstream signal transduction 60may provide analgesia. Viral transduction and expression of an SP fragment and subsequent diffusion of the peptide into the spinal cord proper is an attractive strategy for attaining antinociception. Similar enhancement of receptor internalization could possibly be induced by an overexpression of the intracellular chaperone protein β-arrestin. 61Quartara and Maggi 5,39provided an excellent review of the tachykinin NK-1 receptor. Thus, the genomic structure and receptor function of the NK-1 receptor is well described and will facilitate gene-based expression and regulation for analgesia.Two knockouts against the gene encoding SP and neurokinin A and one knockout against the gene encoding the NK-1 receptor were recently described. 62–64Cao et al. 62disrupted the preprotachykinin A gene, thus producing mice deficient in SP and neurokinin A. No SP or neurokinin A immunoreactivity was noted, and normal levels of all three neurokinin receptor subtypes were present. There was no difference between mutant and wild-type mice in response to low and high intensities of thermal stimuli; however, mutant mice showed a reduced sensitivity at an intermediate temperature. There was no difference between groups in response to the late phase of the formalin test. In tests for mechanical nociception, mutant mice showed reduced response to tail clip, but there was no difference between groups in response to von Frey hairs. When compared with wild-type mice, mutant mice showed a significant reduction in response to chemical and visceral pain. Mutant mice failed to develop neurogenic inflammation after peripheral capsaicin injection. 62Zimmer et al. 63also produced mutant mice deficient in SP but with a different phenotype than that produced by Cao et al. 62Expression of NK-1 receptor was significantly higher in mutant mice, possibly resulting from receptor upregulation in absence of endogenous ligands. There was no difference between mutant and wild-type mice with respect to number and distribution of small-diameter, calcitonin gene-related peptide (CGRP)-immunoreactive dorsal root ganglia (DRG) neurons, suggesting that there was not a significant decrease in primary sensory neurons. There were no differences in response between wild-type and mutant mice after tail-flick assay and acetic acid–induced writhing tests; however, mutant mice were hypoalgesic in hot-plate and formalin tests. 63De Felipe et al. 64developed a mouse deficient in NK-1 receptors. Mutant mice were healthy and fertile and showed normal behavior except for a decrease in aggressive behavior. Absence of NK-1 receptors was confirmed by receptor autoradiography, and normal SP and CGRP distribution were noted. There were no differences in response between wild-type and mutant mice after tail-pinch, paw-withdrawal, tail-flick, or hot-plate assays (acute nociception); however, mutant mice did not develop spinal neuron sensitization.The differences observed between the three knockouts highlight the problem of interpretation of data obtained from conventional knockout mice. Any observed phenotypic alteration may or may not be the result of targeted gene disruption. 65In this case, Zimmer et al. 63and Cao et al. 62both produced a knockout mouse deficient in the gene encoding SP; however, significant differences in the phenotype were demonstrated and possibly reflected differences in either genetic background or experimental parameters. In general, other issues that may contribute to difficulty in data interpretation include pleiotropy (multiple functions of genes), epistasis (genes and their products produce biologic phenomena), and compensation (genetic redundancy). 65Although a detailed review of issues involved in transgenic studies of nociception are beyond the scope of this review, a recent article provides greater depth on this subject. 65Antisense ODNs directed against the NK-1 receptor have been described in two studies. Ogo et al. 66developed an antisense ODN directed against the NK-1 receptor NH2terminus. By the second day of treatment with target antisense ODN, a reduction of 31% in SP/NK-1 receptor binding and 35% in calcium ion influx induced by SP were noted in vitro . Control ODN did not result in any change of either measurement. The investigators then injected antisense ODN–encapsulated liposomes in rat cerebral cortex. When compared with control animals, those with antisense ODNs had a reduction in cortical SP binding sites by approximately 40% 7 days after initial injection. Animals in this study did not show behavioral or neurologic abnormalities. 66Hua et al. 57intrathecally injected several antisense ODNs against NK-1 receptor mRNA of the rat. Administration of antisense ODNs alone did not result in a reduction of pain behavior or spinal NK-1 receptors, as marked by immunostaining, and there was no significant difference between groups with respect to levels of NK-1 receptor mRNA. However, intrathecal injection of SP in rats treated with antisense ODNs resulted in a reduction in pain behavior and spinal NK-1 receptor immunoreactivity. Although the duration of antisense ODN administration (2 days) may not have been enough to diminish functional expression or synthesis of the NK-1 receptor in rats treated with antisense alone, those treated with antisense and SP did demonstrate a significant reduction in surface receptor protein, possibly suggesting that receptors, once depleted, are not replaced by de novo synthesis. Consequently, behavioral data obtained from ODN treatment potentially may not manifest until turnover of existing receptors occurs. There were no motor dysfunction or other behavioral abnormalities noted in rats treated with antisense ODNs. 57Like the previous antisense ODN study, it was difficult to conclude definitively whether inhibitory effects of antisense ODN were a result of mRNA degradation (after internalization) or arrest of mRNA translation. 57,66The long turnover of NK-1 receptors in steady state (> 7 days) may require prolonged administration of antisense ODN to effect NK-1 receptor downregulation. 57,66A thorough knowledge of the target protein turnover is essential in designing a rational antisense ODN–based gene therapy strategy, unfortunately unavailable for the NK-1 receptor.Several approaches to gene-based modification of the NK-1 receptor have been described. Initial antisense ODN studies appear promising; however, further in vivo studies with appropriate controls and a critical evaluation of the duration of ODN treatment are required .Central sensitization is mediated in part by action of EAAs on NMDA receptors. Although NMDA receptor activation alone is insufficient for development of central sensitization, influx of calcium through NMDA receptor channels may induce prolonged changes by way of intracellular second messengers and protein kinases, including protein kinase C (PKC). 67PKC appears to mediate CNS neuronal plasticity after tissue injury. 67–69Rats pretreated with inhibitors of phospholipase C and PKC demonstrate significant reductions in nociceptive behavior. 67Those treated with PKC activators show significantly enhanced persistent nociceptive behavior; however, this behavior is not present in early phase of the formalin response. 70Spinal infusion of the PKC inhibitor NPC15437 prevents sensitization of wide-dynamic-range spinothalamic neurons to mechanical stimuli after intradermal injection of capsaicin. 68Intrathecal injection of GM1 ganglioside, an inhibitor of PKC activation, reduces spontaneous pain behavior and thermal hyperalgesia in rats after chronic constrictive sciatic nerve ligation. 69Thus, the role of PKC in nociception is well supported by classical pharmacologic data.Protein kinase C consists of a number of isoforms that vary in function and distribution. One of these isoenzymes, PKC-γ, is found only in brain and spinal cord. Long-term potentiation or changes in neuronal plasticity, learning, and memory are mediated by the PKC-γ isoform, which appears postnatally and is restricted to CNS. 71–73In the spinal cord, PKC-γ is found in the cytoplasm and restricted to neurons and interneurons in the substantia gelatinosa and axons of the dorsal corticospinal tract. 74,75PKC-γ immunoreactivity in the spinal cord dorsal horn increases in rats after chronic constriction injury but does not increase with intrathecal administration of the NMDA antagonist MK-801. 76Protein kinase C-γ does not appear to mediate acute nociception but is central to regulation and development of neuropathic pain. Although there are no specific agonists or antagonists of PKC-γ that may be used to investigate the role of PKC-γ in nociception, a mutant mouse lacking PKC-γ has been bred. 75PKC-γ–deficient mice show a normal response to the first phase of the formalin test but demonstrate significant reductions to the second phase. Unlike wild-type mice, PKC-γ–deficient mice completely fail to develop neuropathic pain after partial sciatic nerve ligation. 75Furthermore, mutant mice do not develop the typical pattern of neurochemical reorganization in ipsilateral dorsal horn observed after partial nerve injury and complete nerve transection in normal mice. 75Peripheral nerve injury in mutant mice produces an unaltered response of primary afferents, and effects of PKC-γ deletion are manifested postsynaptically. 75Thus, it appears that PKC-γ is essential to the development of neuropathic pain. Further studies are necessary to define the downstream events leading to the development of neuropathic pain subsequent to PKC-γ activation.Protein kinase Cs are a family of phospholipid-dependent, serine-threonine kinase isoenzymes that play a critical role in intracellular signal transduction. Conventional PKCs are activated by second messengers such as diacylglycerol, inositol triphosphate, and calcium. Activated PKC is associated with modulation of ion channels, desensitization of receptors, enhancement of neurotransmitter release, and modulation of synaptic transmission. 77PKC interactions with its substrates are described elsewhere. 78Prolonged activation of PKC may be central in mediating long-term cellular events such as learning, nociception, tumor genesis, or cellular proliferation and differentiation. 75,79,80The precise functions of specific isoenzymes are not clear at this time.Protein kinase C-γ is classified as a calcium-dependent or conventional PKC as it is activated by calcium, diacylglycerol, and phosphatidylserine. 81In general, all PKC isoenzymes, including PKC-γ, consist of four constant regions (C1–C4) separated by five variables regions (V1–V5). 72The V3 region separates the regulatory domains (V1–V2, C1–C2) from catalytic domains (V4–V5, C3–C4). The C1 region contains a sequence motif in the phosphorylation site that appears to be a pseudosubstrate site with autoregulatory characteristics. 72,82In addition, there is a cysteine-rich region in C1 that is necessary for diacylglycerol and phorbol ester binding. 83C2 is important for participation in calcium binding, and C3 contains an adenosine triphosphate binding site. The substrate binding site and phosphate transfer region are located on C4. 72The V3 region is also known as the hinge region because it is sensitive to proteolytic cleavage. Of all the PKC isozymes, the γ isoform is the most proteolytic and may have a shorter half-life, thus potentially allowing for a more rapid turnover of cellular signaling and increased phosphorylation when compared with other PKC isozymes. 83In general, there are several steps in which PKC may be regulated. 81Before activation, PKC must be posttranslationally phosphorylated, which results in a functional form. PKC is initially transphosphorylated, which allows it to undergo autophosphorylation with Thr-641 and become catalytically competent. PKC release into the cytosol occurs after phosphorylation of Ser-660. 81PKC may also be regulated by the cofactors diacylglycerol, phosphatidylserine, and calcium, all of which need to bind appropriately to activate PKC. Once activated, PKC translocates and binds to membranes. Maximal membrane binding and stabilization require the presence of phosphatidylserine, diacylglycerol, and calcium. Finally, inhibitory second messengers, such as sphingolipids, may alter diacylglycerol-mediated activation of PKC. 81Although the genomic structure of PKC-γ has not yet been completely elucidated, the general genomic structure for PKC has been established. 84The first exon and intron of the gene sequence data for PKC-γ is available. 85Promoters and the promoter region for the PKC-γ gene have been identified and may provide information on regulation of PKC-γ expression. 86,87Functional characterization of the PKC-γ receptor promoter region reveals that it is 87 base pairs upstream from the transcriptional initiation site. 86Within the promoter region, several sequence segments of transcriptional factor binding sites that may mediate transcription have been identified. 87Multiple DNA-binding proteins may act in conjunction to modulate PKC-γ expression. There may be positive control of PKC-γ expression by itself or other PKC isozymes. 72,87Further elucidation of the genomic structure and function of PKC-γ will allow new gene-based approaches to provide antinociception through regulation of expression levels.Some of the characteristics of PKC-γ–deficient mice have been described previously. PKC-γ–deficient mice are anatomically normal, which is consistent with postnatal appearance of PKC-γ. 71,75Immunoprecipitable PKC-γ activity and PKC-γ mRNA transcripts are absent in mutant mice. In addition, there is no evidence of upregulation of other PKC isoenzymes. These mice show mild ataxia and deficits in memory and learning but have normal baseline synaptic transmission. 71,75PKC-γ–deficient mice show abnormal long-term potentiation, and responses to nociceptive input are described in Protein Kinase C-γ Isoform: Evidence of Role in Nociception. 88PKC-γ appears to be a key regulatory component of synaptic plasticity. 71Specific inhibitors of PKC isoenzymes are not widely available nor available for the γ isoenyzme. Experiments in our own laboratory indicate successful antisense ODN–mediated knockdown of PKC-γin vitro and in vivo with a parallel decrease in the phase II formalin response. No downregulation of PKC-α and -β isoenzymes was induced by the PKC-γ–specific ODN. 89The restricted distribution of PKC-γ and implication of PKC-γ in development of neuropathic pain lends itself to an ODN-based approach to antinociception. Direct subarachnoid administration of antisense ODN for a selective knockdown of spinal cord PKC-γ is an attractive strategy for preempting central sensitizati