Critical Review. Search for the Ideal Analgesic in Pain Treatment by Engineering the Mu-Opioid Receptor

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1 IUBMB Life, 62(2): , February 2010 Critical Review Search for the Ideal Analgesic in Pain Treatment by Engineering the Mu-Opioid Receptor Pao-Luh Tao 1, Ping-Yee Law 2 and Horace H. Loh 2 1 Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan, Republic of China 2 Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN, USA Summary The myriad of side effects that associate with morphine has been problematic in the clinical use to manage moderate to severe pain. It has been the holy grail of the pharmacologists to develop a compound, or treatment paradigm that could retain the analgesic effect of the drug as eliminating or reducing the side effects, mainly the tolerance and addiction development associates with chronic usage of the drug. In our earlier receptor structure/activities studies, we discovered an unique mutation of a conserved Ser in the fourth transmembrane domain of the opioid receptor that the alkaloid antagonist could activate the receptor. On the basis of this initial finding, we decide to explore the possibility of using virus to deliver the mutant muopioid receptor at the various sites of the nociceptive pathway and induce the antinociceptive responses with the systemic administration of opioid antagonists. In this article, we will summarize the progress of such approach and the probable advantages over the conventional approach of drug development in the treatment of chronic pain. Ó 2009 IUBMB IUBMB Life, 62(2): , 2010 Keywords morphine; pain treatment; mu-opioid receptor; naloxone. Received 7 October 2009; accepted 17 November 2009 Address correspondence to: Horace H. Loh; Frederick Stark Professor and Head, Department of Pharmacology, University of Minnesota, Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA. Tel: Fax: lohxx001@umn.edu INTRODUCTION Opioids have been used very successfully for the treatment of moderate to severe acute and chronic pain. Unfortunately, their uses have been associated with many troublesome side effects, such as, nausea, vomiting, constipation, respiratory depression, sedation, pruritus, tolerance, and dependence. Although with brief, short-term prescription of opioids rarely led to addiction or abuse of the drug (1) and long-term administration of opioids inadvertently had led to addiction or abuse in % of patients (2, 3). With the rise in the number of prescriptions written for opioids in the United States (4), there is a parallel or greater increase in the problems associated with the opioid analgesics (5, 6). For example, emergency departments reported more than 50-fold increase in fentanyl associated incidents from 1994 to 2002, whereas the prescription of this opioid increased 7.2-fold during the same period. With the on-going trend for primary care physicians caring for pain patients rather than physicians who specialize in pain treatment, the risk of diversion and abuse of opioid analgesics will increase. Therefore, there is an urgency in the development of opioids or treatment paradigms that could minimize the side effects, especially the addiction and abuse to drug during longterm treatment. In the past, many drugs have been used to alleviate the side effects as maintaining the analgesic potency of the opioids. Frequently, nonopioid pharmacological strategies are used together with opioid administration. For example, laxative or bulking agents are used to counteract opioid constipative effects. Dopaminergic or serotonergic (5-HT 3 ) antagonists are used for postoperative nausea and vomiting due to opioid analgesics. These agents have their own adverse effects and the resulting polypharmacy also lead to undesirable effects. For example, laxative could cause abdominal cramps, whereas their efficacy in reversing the opioid constipative action has not been established unequivocally (7). The 5-HT 3 antagonists used to treat the postoperative nausea and vomiting often resulted in headache and increase liver enzymes in proportion of patients (8). Such adverse effects of nonopioid drugs have led to the use of peripheral opioid antagonists in the elimination of opioid side effects. Unlike the centrally acting opioid antagonists, such as, naloxone or naltrexone that could reverse the analgesic effects in post-operative patients (9 11), peripheral opioid antagonists, such as, methylnaltrexone (the quaternary derivative of naltrexone) or alvimopan (a zwitterionic form of trans-3,4-dimethyl-4- (3-hydroxyphenyl) piperidine) because of their polarities and ISSN print/issn online DOI: /iub.292

2 104 TAO ET AL. inability to penetrate the blood brain barrier, have been used successfully to prevent the constipation, nausea, or vomiting effects in post-operative pain treatment (12, 13). However, such peripheral antagonists treatment could not prevent the tolerance and addiction to the opioid analgesics during long-term administration. Several approaches were used to address the side effects of tolerance and addiction to the drug during long-term treatment. With the cloning of the three opioid receptor types, l-, d-, and j-opioid receptors (MOR, DOR, and KOR) (14 18), and the subsequent generation of respective receptor null mouse lines (19 22), it is apparent that the in vivo morphine effects were mediated by MOR activation. In the absence of MOR, the rewarding response to morphine was not observed (20). Clinical studies indicated that the mixed agonist antagonists, especially those that could activate KOR and are antagonists in MOR, though have lower patient compliance due to their dysphoric properties, have lower addictive liability (23). Thus, the development of KOR agonists as possible pharmaceutical agents for pain control with low addiction liability has been the focus of many laboratories (24). A latest example is the development of buprenorphine analogs that have partial agonistic properties in KOR and exhibited minimal dependence development in mice (25). Although the development of agonists selective for KOR shows promises, this approach is deficient in the relative lower efficacy in pain relieve exhibited by the KOR agonists when compare with morphine, and the decrease in patient compliance due to the dysphoric effect exhibited. Furthermore, even with mutagenesis and receptor chimeras studies identifying differences in receptor domains involved in MOR, DOR, and KOR ligands binding (26, 27), the high homology among the three opioid receptors, especially within the transmembrane domains that participate in ligand binding (28), has limited the development of ligands that bind to one receptor exclusively. The relative selectivity for the receptors has led to the development of drugs that are mixed agonist antagonist, such as, pentazocine and nalorphine, or the ability of a putative in vitro selective DOR agonist, [D-Pen 2,5 ]-enkephalin (DPDPE) to elicit in vivo antinociceptive responses in a DOR null mice (22). Another approach to develop opioids mediated pain treatment is to develop paradigms taking advantages of the studies on elucidating the molecular mechanisms of drug tolerance and addiction. Numerous studies have pointed to the involvement of glutamatergic transmission in the response to long-term opioid administration. Co-administration of N-methyl-D-aspartate (NMDA) receptor antagonists, such as, MK-801, or the administration of the antisense oligonucleotides to nitric oxide synthase resulted in the attenuation of chronic opioid effect (29 32). The increase expression of specific a-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) receptor subunits, GluR1 within the neurons of the ventral tegmental area in response to opiate administration suggested the involvement of these glutamate receptors in adaptation to the drug (33). The probable increase in the mesolimibic dopamine system activity by the increase in AMPA activity as the mechanism for chronic opiate action was further supported by the sensitization to morphine in animals injected with viral vector expressing the GluR1 subunit (34). With these clear indications of the probable involvement of glutamate receptors in the opioid tolerance and dependence processes, treatment paradigms have been investigated to reduce the long-term morphine effect. The most promising one is the use of dextromethorphan, which is NMDA receptor antagonist, to prevent or reverse morphine tolerance or morphine rewarding effects in animals and to reduce morphine requirement in postoperative pain (31, 35 37). However, the high incidents of adolescent abuse of high doses of dextromethorphan have resulted in psychosis, dependence, and physical withdrawal symptoms due to its sigma and serotonergic receptors activities (38, 39). Hence, the use of the glutamatergic agents for opioid drug addiction has not been translated into desirable clinical results. In the search of opioids that have minimal tolerance and addiction liability, recent approaches have targeted the possible homo- and hetero-dimerization of the opioid receptors. Using either co-immunoprecipitation of the receptors or the bioluminescence studies, the oligomerization of the opioid receptor could be demonstrated (40 43). Intriguingly, the heterodimerization of opioid receptors with each other or with other receptors have resulted in the appearance of unique phenotypes (44 47). The heterodimerization of the d- and j-opioid receptors resulted in the decrease in the affinities of receptor-selective ligands (46, 48). The d- and j-opioid receptors could also heterodimerize with the ß 2 -adrenergic or substance P receptor, resulting in an alteration of the functionalities of these receptors (47, 49). Furthermore, the heterodimerization of MOR and DOR appeared to affect both the in vitro and in vivo activities of MOR-selective agonists (50, 51). The use of DOR selective antagonist could potentiate morphine in vivo antinociceptive responses in wild type but not DOR null mice (51). Such receptor oligomerization resulting in synergism in receptor activities have been implicated in the earlier studies reported by Vaught and coworkers in which Leu5-enkephalin potentiated morphine effects (52, 53). The putative heterodimerization of MOR and DOR resulting in alteration in phenotypes could be the basis for the previous reported observations in which co-administration of DOR selective antagonists, such as, naltrindole, TIPP[w], or DOR antisense oligonucleotides resulted in attenuation of morphine tolerance and dependence development (54 56). The involvement of DOR in the manifestation of chronic morphine effect was demonstrated unequivocally with DOR null animals. Zhu et al. (22) reported in DOR null mice, morphine tolerance development was completely blocked. Such observations have encouraged the development of nonpeptide ligands possessing mixed MOR agonist/dor antagonist activities. Based on their initial observations that bridging of neighboring pharmacophores could result in an increase in ligands potencies with optimal spacer length (57 59), Portoghese and coworkers have developed bivalent ligands with oxymorphamine as MOR-selective agonist pharmacophore and 7 0 -aminonaltrindole as DOR

3 IDEAL ANALGESIC IN PAIN TREATMENT 105 selective antagonist pharmacophore. The efficacies of these compounds in the anitnociceptive tests were shown to be more efficacious than morphine depending on the spacer length (60, 61). The interesting aspect of these bivalent ligands is that the tolerance or dependence responses were not observed in mice chronically treated with these ligands (61). Thus, the pursuit of the approaches such bivalent ligands with different pharmacophores, or the development of opioid ligands cocktails to enhance the recycling and resensitization of MOR during chronic morphine treatment (62), could lead to reduced tolerance and dependence responses in long-term opioids administration. An alternative approach to developing pharmacological strategies in the alleviating the opioid side effects, notably the tolerance and addiction, is to target the opioid receptors. Recently, engineered GPCRs have been designed to delineate the signaling pathways of these receptors. A common property of these GPCR mutants is that the receptors could not be activated by endogenous ligands, but the receptor is activated solely by synthetic ligands (RASSL). The first RASSLs were constructed by Conklin and coworkers, in which the second extracellular loop of KOR was replaced by the same sequence of DOR (Ro1, (63)). A subsequent RASSL was generated by the mutation of Glu 297 of Ro1. These RASSLs exhibited reduced affinities for KOR and DOR endogenous ligands, such as, dynorphin, but could be activated by the synthetic KOR ligand spiradoline. Subsequently, by using tetracycline-inducible system to control tissue specific expression, the RASSL Ro1 was used to demonstrate that Gi activation caused the ventricular conduction delay and a lethal cardiomyopathy (64). RASSLs derived from other GPCRs have been reported (65). One notable example is the mutation of a single amino acid of 5-HT 4 serotoninergic receptor, D100A. This 5-HT 4 mutant was totally insensitive to 5HT, but remained sensitive to synthetic ligands (66). In addition, two of the 5-HT 4 antagonists that have modest activities now could activate the Gs heterotrimers in Cos-7 cells expressing the D100A mutants (66). Such RASSLs distinguish themselves from those in which the synthetic ligands activated both the endogenous receptor and the RASSLs expressed in cell models or tissues (67). One might be able to utilize such property of the RASSLs, antagonist activation, to develop a therapeutic agent for disease treatment. Instead of developing additional RASSLs based on opioid receptors similar to Ro1 and Ro2 that were reported by Conklin and coworkers (63, 67), we have developed a probable therapeutic agent for long-term pain treatment without side effects and reviewed as the following. 1 Effect of the conserved serine residue in TM4 of opioid receptor on antagonists efficacies. Over a decade ago, during the construction of chimeras with l- and d-opioid receptors (MOR and DOR), we reported the discovery of an unexpected receptor mutation in which the classical antagonists, naloxone or naltrexone could activate the receptor chimeras. From sequence analyses, we determined that the mutation conserved serine residue in TM4, Ser 196 in MOR, Ser 177 in DOR, and Ser 187 in j-opioid receptor (KOR) to Leu resulted in the ability of antagonist to activate the G protein-coupled inward rectifying potassium (Kir3) channels expressed in Xenopus oocytes or to inhibit the forskolin-stimulated production of intracellular camp in CHO cells (68). Using the ld2 receptor chimera (i.e., the amino acid residues from the N-terminus to TM2 domain of DOR were replaced with that of MOR), we demonstrated that antagonist, such as, naloxone exhibited full agonistic properties by inhibiting adenylyl cyclase activity, by inducing receptor desensitization and by receptor internalization. Interestingly, the agonistic properties were not changed by the mutation of this conserved Ser residue. Back mutation of the Leu 196 in the ld2 receptor chimera to Ser corresponding to the amino acid residue of wild type receptor resulted in the lost of antagonist ability to activate the receptor. The role of such conserved Ser residue in TM4 of the opioid receptor in controlling ligand s efficacy was established (68). Because the initial observations, we have demonstrated that the ligand s efficacy could be controlled also by the interaction between TM1 and TM7. We noticed in two of our receptor chimeras that have TM1 and TM7 from the same receptor, that is, ld2l7 and ld2l67 (receptor chimera that have TM1, TM2, and TM7 sequences from MOR, and receptor chimera that have TM1, TM2, TM6, and TM7 sequences from MOR, respectively) when co-expressed in Xenopus oocytes with Kir3.1, antagonists could elicit only partial activation of Kir3 channels as compared with the agonists. We could demonstrate similar partial agonistic properties of naloxone or naltrexone in the inhibition of adenylyl cyclase activity in HEK293 cells stably expressing the MORS196L mutant. From the molecular models based on rhodopsin resolved crystal structure, receptor activation requires the disruption of the salt bridge and hydrogen bonding formed between the conserved D/ERY motif within the second intracellular loop and the X 1 BBX 2 X 3 B motif within the third intracellular loop that stabilizes the inactive conformation of the receptor (69 71). Receptor activation also requires the disruption of the van der Waals interaction between TM3 and TM6 that stabilizes the receptor inactive form (72). In addition, the structural domain formed between TM6 and the conserved NPXXY motif in TM7 holding the receptor in its inactive state must be overcome in order for the ligand to activate the receptor (72). Therefore, disruption of intrahelical interaction among the transmembrane domains will facilitate the movement of TM6 and TM7 during agonist activation as predicted by the molecular model proposed by Strahs and Weinstein for the opioid receptor (73). However, such model could not provide an explanation for our observations. In ld2 receptor chimera, TM6 and TM7 are from the same receptor, DOR,

4 106 TAO ET AL. and the antagonists exhibited full agonistic properties in systems expressing this chimera. On the other hand, with ld2l7 orld2l67, the TM6 and TM7 are either from same receptor, MOR, or from two different opioid receptors. Thus, we hypothesized that it was the interaction between TM1 and TM7 that contributed to the determination of ligand s efficacy. In the construction of ld2l7 receptor chimera, the Thr 327 and Cys 330 residues within the TM7 of the MOR sequence corresponded to Ala 309 and Ser 312 of DOR, respectively. By mutating these two amino acid residues in MORS196L to the corresponding DOR residues, we were able to demonstrate full agonistic properties of naloxone and naltrexone in inhibiting the forskolin-stimulated adenylyl cyclase activity (74). Furthermore, by mutating Thr 327 to Ala and Cys 330 to Ser in the MORS196L mutant, naloxone could induce receptor internalization and could suppress the superactivation of adenylyl cyclase activity after chronic agonist treatment. Thus, the interaction of TM1 and TM7 regulates the antagonist efficacy in the Ser 196 mutant (74). 2 The antagonist in vivo activities upon activation of the MORS196A receptor mutant. The ability of opioid antagonists to activate the Ser 196 receptor mutant in vitro could be demonstrated clearly. In order to address whether such mutation would result in any in vivo activity exhibited by the antagonists, we have generated a mouse line in which the Ser 196 residue in MOR was mutated to Ala (75). Because of the presence of the loxp site at the proximity of the spliced junction, the level of this mutant receptor expression in the homozygotic mice was much lower than that observed in wild type, 10%. Nevertheless, morphine produced antinociceptive responses in both the tail-flick from radiant heat source or tail withdrawal from heat source, and acetic acid-induced abdominal constriction. The potency of morphine in producing antinociceptive responses in the MORS196A mutant mice was similar to that of wild type mice. Importantly, naloxone and naltrexone produced partial agonistic responses in the MORS196A mice and not in wild type mice. Partial agonist, such as, nalorphine, behaved like a full-agonist in the MORS196A mice, though the receptor level was much lower in these mice. These in vivo data supported our observations with the in vitro models that mutation of Ser 196 to Ala will result in the ability of classical antagonists to activate the receptor. The efficacies of ligands were increased except those of the opioid full agonists. Chronic morphine treatment resulted in tolerance development, and the appearance of withdrawal signs upon removal of the drug. However, chronic administration of naloxone or naltrexone did not produce tolerance development as indicated by no change in the antagonist potency to produce antinociceptive responses. The withdrawal signs were also reduced in these animals chronically treated with naloxone. Thus, apparently, the activation of the mutant MOR by antagonist and concomitant inhibition of other opioid receptors activities, notably DOR, could block the tolerance development. This was demonstrated clearly by the co-administration of SNC- 80 during chronic naloxone treatment of the MORS196A mutant mice. Under such treatment paradigm, tolerance development to naloxone was observed (76). These data suggested that the in vivo activation of S196A MOR mutant by naloxone or naltrexone could elicit antinociceptive responses with minimal chronic side effects (75, 76). 3 Ability of opioid antagonist to produce antinociceptive responses by the injection of double-stranded adenoassoicated virus (dsaav) which carried the transgene of MORS196A. The generation of the MORS196A knock-in mouse line supported our initial hypothesis that activation of the mutant receptor with antagonist could produce antinociceptive responses with minimal chronic side effects. To utilize such receptor mutants as therapeutic agents, an efficient mean to deliver the mutant receptor within the pain pathway must be resolved. The premise is the expression of the mutant receptor within the nociceptive neurons, those which activities are regulated by MOR, will result in the antinociceptive responses upon mutant receptor activation by antagonists. Because the antagonist will not activate the endogenous opioid receptors, there should be minimal tolerance development or minimal side effects with the systemic antagonist administration. In our initial studies, we have decided to use the dsaav vector system for the delivery of the mutant receptor. The dsaav, generated by a mutation of the inverted terminal repeats (ITRs) leading to nearly exclusive packaging of hairpin-like DNA genome, has the advantage of efficient transduction of viral genome into functional templates post cell entry resulting in high level of transgene expression (77). Furthermore, unlike other dsdna vectors, such as, adenovirus or plasmid DNA, the dsaav-mediated transduction does not rapidly decline with time. Such dsaav properties provide us with the opportunity to investigate the in vivo drug effects after virus injection. To monitor the expression of the transgene, a chimeric protein was constructed by splicing the EGFP onto the C-terminus sequence of MORS196A. The transcription of the fusion protein was under the control of CMV promoter. First, injection of the dsaav within the dorsal horn area was carried out sterotaxically at the S2 S3 region of the spinal cord in MOR2/2 mice and resulted in the expression of green fluorescence at the site of injection. Within 7 days of injection, noticeable green fluorescence was observed. Even 6 months after the injection, cells in the proximity of the injection sites still showed bright fluorescence. Furthermore, it could be shown that the injected fluorescence proteins could retrograde to the dorsal root ganglion. Expression of the green fluorescent protein was observed in small diameter DRG neurons suggesting possible expression of the fusion protein in the nociceptive neurons.

5 IDEAL ANALGESIC IN PAIN TREATMENT 107 Table 1 Relative potencies of morphine and naloxone in MOR 2/2 mice and ICR mice injected with AAV-MORS196AEGFP AD50 (mg/kg, s.c.) MOR 2/2 ICR Morphine Naloxone Morphine Naloxone Sham [40 AAV-EGFP [40 AAV-MOREGFP [40 AAV-MORS196AEGFP DsAAV2 virus with various EGFP constructs were injected into the spinal cord (S2 S3) dorsal horn area of MOR 2/2 or ICR mice stereotaxically. For MOR 2/2 mice, the AD50 values of morphine and naloxone in the tail-flick assays were determined by the up-down method. The tail-flick latencies were measured 14 days after the virus injection. For ICR mice, the various dsaav viruses were injected at the S2 S3 dorsal horn region, and the abilities of morphine and naloxone to produce antinociceptive responses were carried out 28 days after virus injection. The injection of dsaav at the dorsal horn region of MOR2/2 mouse spinal cord demonstrated the feasibility of such approach to deliver the mutant receptor. However, for therapeutic purpose, the delivered mutant receptors must be able to elicit antagonist-mediated antinociceptive responses in animals that express endogenous MOR. Thus, dsaavs with various EGFP fusion proteins, that is, MOREGFP and MORS196AEGFP, were injected at the S2/S3 region of the ICR mice spinal cords. We found neither sham injection nor the injection of dsaav-egfp, dsaav-moregfp, or dsaavmors196aegfp altered the AD50 value of morphine significantly (Table 1). This is not surprising because the dsaav was introduced only at the S2/S3 region of spinal cord, whereas the s.c. injection of morphine could activate the endogenous MOR located throughout the spinal and supraspinal portion of the pain pathway. Interestingly, injection of dsaav-egfp or dsaav-moregfp did not result in naloxone-mediated antinociceptive responses, even at the 40 mg/kg dose. Only in the ICR mice injected with dsaav-mors196aegfp did naloxone elicit an increase in tail-flick latency (Table 1). It was found that MORS196A- EGFP fluorescence colocalized with some calcitonin generelated peptide and neuron-specific protein immunoreactivity in the superficial layers of the dorsal horn after dsaav- MORS196AEGFP injection (Fig. 1), and lasted for at least 6 months. In mice injected with the mutant receptor, morphine induced antinociceptive responses, tolerance development, precipitated withdrawal symptoms and reward effects, similar to those in the control mice (saline injected into the spinal cord). Conversely, in the dsaav2-mors196a-egfp injected mice, naloxone produced antinociceptive effects at the spinal level, but not at the supraspinal level, whereas naloxone had no measurable effect on the control mice. Furthermore, the chronic administration of naloxone to mice injected with dsaav2-mors196a-egfp did not induce tolerance, withdrawal symptoms or reward responses (78). To develop further such mutant MOR into a therapeutic agent, a less invasive method for virus delivery needs to be established. Thus, we have injected the dsaav2-mors196ac- STA-EGFP locally into the subarachnoid space of the spinal cord by intrathecal administration recently. In this receptor mutant, naloxone has been shown to exhibit in vitro full agonistic properties. Similary, after 2 weeks of virus injection, naloxone (10 mg/kg, s.c.) elicited antinociceptive effect (determined by tail-flick test) without tolerance (10 mg/kg, s.c., bid for 6 days), significant withdrawal and rewarding effects. On the other hand subchronic treatment with morphine (10 mg/kg, s.c., bid) for 6 days induced significant tolerance (4.8-fold), withdrawal and rewarding effects (unpublished). CONCLUSIONS The data described thus far suggest that local expression of specific engineered opioid receptors in spinal cord and systemic administration of naloxone could be developed into a new strategy in the management of chronic pain with little side effect of morphine. This approach has the advantage over the conventional approach of drug development. The drugs in question are the opioid antagonists, naloxone and naltrexone, which have shown to devoid of any pharmacological or physiological responses, as long as pre-exposure of opioid agonist has not occurred or the endogenous opioid system has not been activated. The antagonists will only activate the engineered receptor, as they will block the activities of the endogenous ligands and the receptors they activate. In this sense, by introducing the expression of the engineered receptor at specific nociceptive neurons, the many subjective effects due to the activation of the CNS opioid receptor could be avoided, especially the addiction that associates with chronic drug administration. Further, by antagonizing the endogenous receptor, naloxone or naltrexone could block the development of tolerance, thereby the analgesic response elicited by the initial dose could be sustained.

6 108 TAO ET AL. Figure 1. Representative fluorescence micrographs of the spinal cord (panels A and C) and the dorsal root ganglion (panel E) 6 weeks after local injection of dsaav2-mors196a-egfp, with a 320 objective (78). In panel (A), merging the immunofluorescence of MORS196A-EGFP (green) with CGRP (red) at the ipsilateral dorsal horn at S3 spinal cord revealed their co-expression (yellow), as indicated by the arrows. In panel (C), merging the immunofluorescence of EGFP (green) with NeuN (red) at the ipsilateral dorsal horn at S3 spinal cord of a different mouse revealed their co-expression (yellow), as indicated by the arrows. In panel (E), merging the immunofluorescence of EGFP (green) with CGRP (red) at nearby L2 dorsal root ganglion neurons revealed their colocalization (yellow), as indicated by the arrows. Inserts in B, D, and F are shown at higher magnification in A, C, and E, respectively (boxed areas). The scale bars represent 80 lm. By no mean the engineered receptor is the panacea for the treatment of chronic pain. There are many issues that associate with the viral delivery of a transgene into neurons, in particular, the chromosomal integrations could result in the malignant transformation. Furthermore, the immunogenicity and cytoxicity associate with vectors, such as, adenovirus also pose severe concerns in the use of gene therapy approach. Although the use of dsaav could overcome some of these problems, the eventual development of current observations into future therapeutic pain treatment paradigm hinges on the specific expression of the transgene in the sensory and nociceptive neurons that normally express the l-opioid receptor. This could be accomplished if the minimal promoter region of the l-opioid receptor gene is identified and drive the expression of the transgene. Thus far, the minimal length of the promoter region that exhibits brain region specific expression of the transgene is 4.7 kb, a size far exceeds the length of nucleotide sequence that could be cloned into the dsaav vector. Hence, either different gene

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