HSV Delivery of a Ligand-regulated Endogenous Ion Channel Gene to Sensory Neurons Results in Pain Control Following Channel Activation

original article

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HSV Delivery of a Ligand-regulated Endogenous Ion Channel Gene to Sensory Neurons Results in Pain Control Following Channel Activation James R Goss1, Michael Cascio2, William F Goins1, Shaohua Huang1, David M Krisky1, Richard J Clarke3, Jon W Johnson3, Hitoshi Yokoyama4, Naoki Yoshimura4, Michael S Gold5 and Joseph C Glorioso1 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA; Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, Pennsylvania, USA; 3Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; 4Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA; 5Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA 1 2

Persistent pain remains a tremendous health problem due to both its prevalence and dearth of effective therapeutic interventions. To maximize pain relief while minimizing side effects, current gene therapy–based approaches have mostly exploited the expression of pain inhibitory products or interfered with pronociceptive ion channels. These methods do not enable control over the timing or duration of analgesia, nor titration to analgesic efficacy. Here, we describe a gene therapy strategy that potentially overcomes these limitations by providing exquisite control over therapy with efficacy in clinically relevant models of inflammatory pain. We utilize a herpes simplex viral (HSV) vector (vHGlyRα1) to express a ligand-­regulated chloride ion channel, the glycine receptor (GlyR) in targeted sensory afferents; the subsequent exogenous addition of glycine provides the means for temporal and spatial control of afferent activity, and therefore pain. Use of an endogenous inhibitory receptor not normally present on sensory neurons both minimizes immunogenicity and maximizes therapeutic selectivity. Received 14 July 2010; accepted 18 October 2010; published online 16 November 2010. doi:10.1038/mt.2010.246

Introduction Chronic pain is major medical problem with up to 50% of patients finding little or no relief with currently available treatments. Gene therapy has the potential to revolutionize pain therapy by the targeted, local production of therapeutic transgenes. Herpes simplex viral (HSV)–based vectors have emerged as the frontrunner in an array of available gene delivery platforms, in part due to the virus’s natural ability to infect primary sensory neurons via peripheral inoculation and subsequent efficient transport of the vector genome to the nucleus. This enables strategies designed to disrupt signaling between the primary sensory nociceptor and projection neurons in the spinal cord. HSV-based vectors have been used to overexpress and/or increase the activity of macromolecules that

interact with the same neurological components involved in nociception. These include: neurotransmitters, such as endogenous opioids or γ-aminobutyric acid;1–7 receptors and channels, such as the µ-opioid receptor, γ-aminobutyric acid-B receptor, TRPV1 receptor, or sodium channel;8–11 growth factors and second messenger systems, such as glial-derived neurotrophic factor and protein kinase C-ε;12–14 and mediators of inflammation such as interleukins and tumor necrosis factor-α.15,16 Although vector-based gene therapy is a very attractive system for pain management, there are at least three limitations. First, there is no temporal control over the analgesia produced with the constitutive release or knockdown of pain-modulating molecules. This would become most problematic when larger areas of tissue are affected potentially limiting appropriate sensory feedback necessary for normal daily functioning. Second, constitutive release for pain-modulating molecules may result in tolerance and the ultimate loss in therapeutic efficacy. Third, it is not possible to regulate the “level” of analgesia, with “efficacy” set and determined by the level of transgene expression. In an effort to address each of these limitations while still capitalizing on advantages inherent in an HSV vector, we developed a vector that expressed a ligandregulated (i.e., “druggable”) transgene product, the ionotropic glycine receptor (GlyR), which would enable exquisite control over the duration, area, and magnitude of the resulting analgesia. In the adult central nervous system, GlyR is typically inhibitory as a result of the increase in Cl− conductance associated with receptor activation. The distribution of the receptor is regulated temporally and spatially, and human GlyRs are expressed predominantly in spinal cord and the lower brain. Importantly, the receptor is not expressed normally in primary sensory neurons. In adults, GlyR is believed to typically have a stoichiometry of two α-subunits and three β-subunits.17 Heterologous expression of just the human α1-subunit, however, is sufficient to reconstitute an active glycine-gated channel with pharmacological properties essentially identical to those of native channels.18–20 Given that Cl− channels effectively attenuate activity in the adult nervous system, and the absence of anion selective ionotropic GlyRs

The first two authors have contributed equally for this work. Correspondence: Joseph C Glorioso, Department of Microbiology and Molecular Genetics, University of Pittsburgh, School of Medicine, 428 Bridgeside Point 2, 450 Technology Drive, Pittsburgh, Pennsylvania 15219, USA. E-mail: [email protected]

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in primary afferents, expression of GlyR in afferents innervating painful ­tissue should provide a “druggable” target with which to selectively block pain. We therefore constructed a HSV vector expressing the α1-subunit of GlyR and tested its analgesic efficacy in diverse and clinically relevant models of inflammatory pain.

Results In order to target expression of GlyR to modulate neuronal activity for the treatment of pain, we constructed an HSV vector that expresses the α1-subunit of the GlyR (vHGlyRα1; Figure  1a). a

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Figure 1  HSV vector-mediated GlyRα1 expression in vitro and in vivo. (a) HSV vector constructs illustrating vector vHG (top) and vector vHGlyRα1 (bottom). (b) Glycine-induced whole-cell currents (Im) from vHGlyRα1 infected human embryonic kidney 293T cells. Application of glycine resulted in a dose-dependent inward and rapidly desensitizing current. (c) Expression of GlyR α1-subunit in primary DRG neurons. DRG neurons, which do not normally express GlyRs, were infected with either vHGlyRα1 (left panels) or vHG (right panels). Twenty-four hours after infection, the presence of α1-GlyR was confirmed using immunohistochemistry (top panels). No GlyR expression was detected in DRG neurons infected with vHG even though expression of GFP could readily be observed (bottom right panel). No immunopositive reaction product was observed in cells incubated without primary antibodies (primary delete, bottom left panel). DRG, dorsal root ganglion; GlyR, glycine receptor; vHGlyRα1, herpes simplex viral vector.

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To verify that our vHGlyRα1 construct is capable of expressing active native-like receptors, human embryonic kidney 293T cells were infected with vHGlyRα1 and electrophysiological recordings were conducted. Currents were induced by an 11 second application of 50 µmol/l (gray) or 1 mmol/l (black) glycine while the cell was ­voltage-clamped at −65 mV. At 24 hours postinfection, all vHGlyRα1 infected cells tested (N = 3) displayed glycineinduced whole-cell currents (Figure 1b). Mean peak current values were −3.57 ± 0.59 nA (50 µmol/l glycine) and −9.25 ± 1.47 nA (1 mmol/l glycine). Current activated more quickly [(10–90% rise time of 265.2 ± 79.1 (50 µmol/l) versus 11.4 ± 1.0 ms (1 mmol/l)] and percent desensitization increased [(73.4 ± 7.3% (50 µmol/l) versus 89.8 ± 1.1% (1 mmol/l)] with increasing glycine concentration. In contrast, application of glycine to uninfected human embryonic kidney 293T cells (N = 4) failed to elicit a current response. As expected under the recording conditions used, the glycine-induced currents observed in vHGlyRα1 infected cells were large, inward, and rapidly desensitizing. Consistent with previous reports,18,20 as the glycine concentration applied was increased from 50 µmol/l to 1 mmol/l, current activation became faster and the percentage of desensitization increased. These data demonstrate that vHGlyRα1 is capable of inducing expression of functional GlyRs in nonneuronal cells. In order to confirm expression in neurons, we transduced dissociated rat primary dorsal root ganglion (DRG) neurons with vHGlyRα1 and examined them for GlyR expression using immunofluorescence histochemistry. DRG neurons infected with vHGlyRα1 revealed diffuse localization of α1-subunits throughout the soma and neurites; no α1-subunits were detected in DRG neurons transduced with a control vector without α1-GlyR (vHG) although these neurons demonstrated robust vector-mediated green fluorescent protein expression (Figure 1c). For in vivo studies rats were subcutaneously injected with vHGlyRα1 or vHG into the plantar surface of the right hindpaw. Inoculation with either vHGlyRα1 or vHG did not alter basal thermal or mechanical thresholds. In order to determine the effective dose range of agonist that will activate the GlyR transgene, vector-inoculated rats were injected with 1, 10, or 100 mmol/l glycine (in 50 µl) and their response to a noxious thermal stimulus was examined.21 Injection of 10 and 100 mmol/l glycine into the plantar surface of the hindpaw resulted in a significant increase in the thermal withdrawal latency in vHGlyRα1 but not vHG inoculated rats (Figure 2a). Injection of glycine into the contralateral hindpaw had no effect on withdrawal latency in either paw, thus demonstrating the need for localized application of the glycine agonist (data not shown). Analgesic effects of vHGlyRα1 were assessed using three pain models: the formalin footpad test, complete Freund’s adjuvant (CFA) model of inflammation, and a rodent model of interstitial cystitis/painful bladder syndrome (IC/PBS). The formalin test, in which spontaneous nociceptive behavior is induced with a subcutaneous injection of dilute formalin,2,22 was conducted 1 week following inoculation of the vectors into the foot. The magnitude of the nociceptive behaviors following injection of formalin allowed for qualitative assessment of the effects of exogenous addition of GlyR agonist and antagonist. Infection with vHGlyRα1 alone did not have any effect on nociceptive behavior as injection of 501

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Figure 2  HSV vector-mediated GlyRα1 alters nociception in animal models of inflammatory pain. (a) Exogenously applied glycine increases thermal withdrawal latency in a dose-responsive manner in rats inoculated with vHGlyRα1. Sprague–Dawley rats were infected with either vHGlyRα1 (filled circles, N = 5) or the control vector vHG (open squares, N = 5). One week later glycine (1, 10, 100 mmol/l in 50 µl distilled water) was injected into the inoculated paw and thermal latency response was measured 15 minutes later. (b) Exogenously applied glycine reduces formalin-induced nociceptive behavior in rats inoculated with vHGlyRα1. Sprague–Dawley rats were infected with either vHGlyRα1 or vHG. Formalin testing was performed 1 week later resulting in a typical biphasic response. Rats infected with vHGlyRα1 demonstrated a significant reduction in nociceptive behavior immediately following injection of glycine into the plantar surface of the foot (black circles, N = 7) compared to vHG treated rats (open squares, N = 5). Injection of the GlyR antagonist strychnine reversed the effects of the exogenously applied glycine (gray circles, N = 5). (c) Exogenously applied glycine increases thermal withdrawal latency in CFA-inflamed paws in rats inoculated with vHGlyRα1. Sprague–Dawley rats were infected with either vHGlyRα1 or vHG, 1 week later 50 µl of CFA was injected into the same foot. Rats inoculated with vHGlyRα1 (black bars, N = 7) had significantly increased thermal withdrawal latencies when challenged with glycine compared to vHG inoculated rats (open bars, N = 3), which showed no affect. (d) Representative examples of in vivo continuous cystometrograms of urethane-anesthetized rats, with cystometrogram performed 2 weeks after bladder wall inoculation of either vHGlyRα1 or vHG. (e) RTx causes bladder hyperactivity characterized by a reduced intercontraction intervals. Intravenous injection of glycine (1 mg/kg) increased intercontraction intervals in vHGlyRα1 inoculated rats but not vHG inoculated rats. All data represent average ± SEM. P < 0.05, Statistical differences were determined by nonparametric Kruskal–Wallis test (formalin study) or t-test (dose–response study, CFA study, IC/PBS (interstitial cystitis/painful bladder syndrome) study) using StatView Software (v 5.0.1; SAS Institute, Cary, NC). CFA, complete Freund’s adjuvant; GlyR, glycine receptor; HSV, herpes simplex virus; RTx, resiniferatoxin; vHGlyRα1, herpes simplex viral vector.

formalin resulted in a typical biphasic response (Figure 2b). The second phase of nociceptive behavior was significantly reduced in vHGlyRα1-infected animals following injection of glycine at the 30-minute time point (Figure 2b, black circles), an effect that lasted for the duration of the study. Glycine-induced analgesia was reversed by injection of strychnine, a specific GlyR inhibitor, at 502

60 minutes (Figure 2b, gray circles). Glycine application had no significant influence on nociceptive behavior in vHG transfected animals (Figure 2b, open squares); strychnine also had no effect in these animals (data not shown). The inhibition of glycineinduced analgesia with strychnine in vHGlyRα1-infected animals provides compelling evidence in support of the notion that the www.moleculartherapy.org vol. 19 no. 3 mar. 2011

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analgesia is due to activation of expressed α1 GlyRs, and cannot be attributed to some indirect effect (e.g., glycine acting as a coagonist on N-methyl d-aspartate receptors). Formalin evoked nociceptive behavior, thought to be a model of ongoing pain, has a relatively short duration (1–2 hours). In order to determine the efficacy of vHGlyRα1 in the attenuation of inflammatory hypersensitivity, a model of more persistent inflammation was employed. Injection of CFA into the foot results in inflammation that lasts for several days.23 One week following inoculation of the vectors into the right hind paw we injected 50 µl of CFA into the same foot and examined changes in thermal sensitivity 1, 2, and 3 days later. As expected, CFA-induced inflammation was associated with swelling of the hindpaw and a substantial decrease in thermal withdrawal latency. This thermal hypersensitivity was significantly attenuated in animals inoculated with vHGlyRα1 demonstrated a significant increase in response time following injection of glycine into the footpad (Figure 2c). To further assess the utility of vHGlyRα1 for the “treatment” of inflammatory hypersensitivity, we also explored the effects of vHGlyRα1 in a visceral inflammation model of IC/PBS in rats.24,25

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Figure 3  Glycine-evoked current in vHGlyRα1 infected cutaneous DRG neurons. (a) DRG neurons innervating the site of HSV inoculation (glabrous skin of the hindpaw) were identified by the presence of the retrograde tracer DiI under epifluorescence illumination. (b) Bright-field illumination of the same region of the coverslip highlights the clear distinction between labeled and unlabeled DRG neurons. (c) Glycine (Gly) evoked currents were readily detectable in DiI labeled neurons (6 of 10), but were only observed in a small fraction of unlabeled neurons studied (2 of 26, P < 0.01 Fisher exact test). (d) An example of a negative response to glycine in an unlabeled DRG neuron. (e) High K+ (50 mmol/l) was applied to all neurons as a positive control. (f) Although the magnitude of the Hi K+ evoked current varied from neuron to neuron, it was present on all neurons studied. (g) The algogenic compound capsaicin (CAP) was applied to each neuron in order to identify a sub-population of putative nociceptive neurons expressing the capsaicin receptor TRPV1. The small inward current evoked in g was a movement artifact, as the same current was evoked with puffer application of normal bath solution (data not shown). (h) Typical capsaicin evoked current. Scale bar in a is the same for both a and b (30 μmol/l). Current traces c,e, and g were from the same GlyR expressing neuron and the scale bars shown in c is the same for e and g. Current traces d,f, and h were from the same GlyR negative neuron and the scale bar shown in d are the same for f and h. All test agents were applied for 2 seconds. CAP, capsaicin; DRG, dorsal root ganglion; GlyR, glycine receptor; HSV, herpes simplex virus.

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The vectors were inoculated into the bladder wall and 2 weeks later catheters were placed through the dome of the bladder and cystometrograms were performed following infusion of saline or the capsaicin analogue resiniferatoxin (RTx). RTx causes bladder hyperactivity characterized by decreased intercontraction intervals. Both vHG and vHGlyRα1 inoculated animals showed a similar cystometrogram pattern during saline infusion and significantly decreased intercontraction intervals after infusion of 10 nmol/l RTx, suggesting that vHGlyRα1 alone did not influence the regulation of bladder activity. Because the vectors were inoculated into bladder tissue, which was not easily accessible to peripheral application of glycine, we administered glycine systemically through direct injection into the jugular vein. Injection of 1 mg/kg glycine but not 0.1 mg/kg glycine resulted in a significant reversal of the RTx-induced decrease in intercontraction intervals in the vHGlyRα1 inoculated rats but not vHG inoculated rats (Figure 2d,e). This implies that systemically delivered glycine activated vector-expressed GlyRs in bladder afferents and not endogenous GlyRs located in spinal cord or brain. The antinociceptive efficacy of peripheral glycine administration in vHGlyRα1 inoculated animals was assumed to be due to the expression of GlyR in DRG neurons that innervate the inoculation site because HSV infects sensory neurons and does not persist in dividing cells. To test this assumption we confirmed the presence of functional receptors in these neurons. Cutaneous afferents were labeled with the retrograde tracer DiI and the presence of glycine-evoked currents was assessed in acutely dissociated DRG neurons 14 days after vHGlyRα1/DiI coinoculation. Consistent with the selective HSV-mediated expression of functional GlyR, glycine-evoked currents were detected from cutaneous neurons (Figure 3).

Discussion The goal of this study was to develop and test the feasibility of a ligand-activated gene therapy system to address concerns inherent with commonly used gene transfer vectors and their constitutively expressed transgenes. We constructed a HSV vector expressing the α1-subunit of the GlyR and infected, by peripheral inoculation, primary sensory afferents that innervate the foot or the bladder wall. Using the formalin test and CFA-induced inflammation of the hindpaw, we demonstrated that both spontaneous nociceptive behavior and thermal hypersensitivity were reduced following the local application of glycine. Comparable results were obtained in a model of bladder hypersensitivity following systemic administration of glycine. Importantly, the presence of the vector itself did not appear to induce any biologically discernable effect nor did the application of glycine to a site distal to the vector inoculation site. In addition, glycine application in control vector-inoculated animals had no effect. Other regulatable systems for gene therapy have been developed over the past several years, most of which utilize specific promoters to either restrict expression to particular tissues/cell types or to turn on or off expression in the presence of an activating drug.26 The tetracycline regulatory system, in which the vector transgene is under the transcriptional control of either a Tet-ON or Tet-OFF inducer, is the most popular.27 One problem with this particular approach is that the activating drug if given 503

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systemically, as is the case for tetracycline, can have adverse side effects. Given the relatively slow time course for “on” and “off ” and the limited control over the magnitude of the “response” this approach also fails to address major limitations associated with more traditional gene therapy approaches. Neither of these limitations is circumvented by the use of short acting promoters that would limit transgene expression to a few days or weeks. Although our data confirm that GlyRs are expressed in primary afferents in vector-inoculated animals (see Figures 1 and 3), it is possible that the effects we observed were partially due to some glycine-activated process in nonneuronal cells, that may have been infected following vector inoculation. It is known that some of the analgesic effect of HSV vectors expressing proenkephalin is due to actions at the site of injection rather than in DRG or spinal cord; however, this is because of the vector-derived proenkephalin that is transported from the DRG back to the peripheral site of injection and released by nerve terminals, not vector-infected nonneuronal cells.28 We also have not determined the length of the analgesic effectiveness of the vHGlyRα1 vector, though we suspect that it would last 3–5 weeks. The vector uses the transiently active human cytomegalovirus promoter to drive transgene expression and previous vectors that we have constructed using this promoter have provided analgesia for this length of time .2,3,12,29 Recently it has been recognized that in certain circumstances, such as in the presence of injury and/or following intense activation of nociceptive afferents, activation of Cl− channels in primary afferents can become excitatory rather than inhibitory,30 which is thought to reflect a depolarizing shift in the Cl− equilibrium potential (ECl). Nevertheless, glycine administration was analgesic in both the presence and absence of tissue injury in vHGlyRα1 inoculated rats. There are a number of potential explanations for the persistence of the analgesic efficacy of glycine in the face of a potential depolarizing shift in ECl including: (i) that the shift in ECl is restricted to central terminals proximal to extrasynaptic γ-aminobutyric acid-A receptors as suggested previously;31 (ii) that the widespread distribution of GlyRs, which require a β-subunit for specific membrane targeting,32 enables depolarization-induced inactivation rather than activation of nociceptive afferents; and that (iii) the Cl− selectivity of GlyRs relative to γ-aminobutyric acid-A receptors enables glycine to remain inhibitory despite a depolarizing shift in ECl.30 The desired analgesic effect in all three experimental pain models tested is both vector- and ligand-dependent, thus this system is a viable approach to regulated gene therapy. Stable expression of GlyR in nociceptors offers the ability to repeatedly activate the therapeutic target by glycine administration using noninvasive methods such as transdermal delivery (e.g., patches33), providing a method for externally controlled pain relief. Furthermore, because the approach we have described enables the selective silencing of primary afferent neurons with exquisite temporal and spatial control, it enables a detailed analysis of afferent function in vivo. This is a significant advance over previous approaches used to assess neuronal function that suffer from limitations in selectivity (e.g.,  surgical interventions or local anesthetics), temporal resolution (e.g.,  genetic mutant animals, inducible mutants, siRNA, or antisense knockdown), or are not amenable to in vivo behavioral analyses (e.g., halorhodopsin). In addition, given available 504

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knowledge of the structure and function of pentameric ligand gated ion channels, and α1-GlyR, in particular,34,35 site-directed mutagenesis provides a means to alter ligand sensitivity and the activity of the receptor.36,37 Mutagenesis can also be exploited to alter ion permeation38 or redesign the receptor’s ligand binding pockets to recognize unique, novel agents such as ivermectin,39 an anthelmintic agent approved by US Food and Drug Administration that may be systemically delivered to activate only recombinant GlyRs.40 Such manipulations may enable selective activation or inhibition of targeted neuronal populations. Thus, this relatively simple system is not only an effective way to control pain, but may be more broadly applied to studies of neuronal function and the identification of neuronal pathways relevant to pain and other modalities of somatosensation.

Materials and Methods Recombinant virus. The plasmid pSHB2-GlyR was created by cloning the

GlyR sequences into plasmid pSHB2 at the BamHI site of the polylinker. pSHB2 was generated by ligation of a HCMV-BGHpA expression construct from pRC-CMV into plasmid pSASB3 at the unique BamHI site. pSASB3 was constructed by cloning the Sph I to AflIII (SalI linkered) fragment (1,928 bp) of the HSV-1 KOS strain genome (nucleotides 124485– 126413) into SphI/SalI-digested pSP72 followed by insertion of the 695 bp BglII to BamHI fragment (nucleotides 131931–132626) containing region upstream of the ICP4 promoter including the viral origin present within the short inverted repeat regions into the BglII to BamHI sites of the vector plasmid. The parental virus vHG was created using the same targeting plasmid except that HCMV-eGFP was cloned into the same BamHI site resulting in the plasmid pSAE3. This plasmid was recombined into an ICP4/ICP27 deletion KOS strain HSV-1 mutant to produce parental vHG. For vHGlyRα1, candidate plaques were initially selected for the loss of green fluorescence under fluorescent microscopy. vHGlyRα plaques were purified by three rounds of limiting dilution with the GlyR construct verified by Southern blot analysis. Functional assay of channels in vitro. Human embryonic kidney 293T cells were maintained as previously described.41 For recordings, human embryonic kidney 293T cells were plated onto glass coverslips pretreated with poly-d-lysine (0.1 mg/ml) and rat-tail collagen (0.1 mg/ml; BD Biosciences, San Jose, CA) in 35 mm culture dishes at 1.5 × 105 cells/dish. 24 hours after plating the cells were infected with vHGlyRα (multiplicity of infection = 10) at 37 °C for 1 hour. Following incubation, infected media was removed and 1.5 ml of growth medium (with serum) was added and the cells were incubated for an additional 24 hours at 37 °C. Recordings were then performed 24 hours postinfection at room temperature. Glycine-mediated whole-cell currents were recorded with an Axopatch 200 amplifier (Molecular Devices, Union City, CA) in voltageclamp mode. Solutions were delivered through an in-house fabricated fast perfusion system.42 The external solution contained (in mmol/l): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES; pH was 7.2 ± 0.05, adjusted with NaOH and osmolality 290 ± 10 adjusted with sucrose as needed. The internal solution contained (in mmol/l): 140 CsCl, 1 CaCl2, 1 MgCl2, 2 MgATP, 10 HEPES, and 10 EGTA; pH was 7.3 ± 0.05, adjusted with CsOH and osmolality 300 ± 0.05. Data were recorded with a Digidata 1322A digitizer under the control of pClamp 9.2 software (Molecular Devices) and all analyses were performed with Clampfit 9.2 (Molecular Devices) or Origin 7.0 (OriginLab, Northampton, MA). Percent desensitization was measured as 100 × (1 − ISS/Ipeak), where ISS and IPeak are the amplitudes of current at steady state and peak, respectively. Cell culture and histology. DRGs from 17-day rat embryos were dissociated with 0.25% trypsin, 1 mmol/l EDTA for 30 minutes at 37 °C with www.moleculartherapy.org vol. 19 no. 3 mar. 2011

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constant shaking and then plated on poly-d-lysine-coated coverslips at 105 cells/well in 24-well plates in 500 μl of defined Neurobasal medium containing B27, Glutamax I, Albumax II, and penicillin/streptomycin (Invitrogen, Carlsbad, CA), supplemented with 100 ng/ml of 7.0S nerve growth factor (Sigma, St Louis, MO). 15 days following plating, cells were transduced with either vHGlyRα1 or vHG (multiplicity of infection = 5) for 1 hour, washed with fresh media, and incubated for an additional 24 hours. Cells were fixed in 4% buffered formalin for 10 minutes, washed three times in phosphate buffered saline, and then blocked in 5% normal goat serum, 0.2% Tween-20, in phosphate buffered saline for 1 hour at room temperature. Cells were incubated with a monoclonal antibody that specifically recognizes the GlyR α-subunit (1:500; Synaptic Systems, Goettengen, Germany) overnight at 4 °C. Some cells were incubated without the primary antibody (primary delete). Cells were washed three times in phosphate buffered saline then incubated with AlexaFluor 594 (1:1,000; Invitrogen) for 1 hour at room temperature. Cells were washed three times in phosphate buffered saline, once in deionized water, and mounted onto glass slides with Aqua Poly/Mount (Polysciences, Warrington, PA). Images were acquired on a Zeiss Axiovert 200 microscope using an Axiocam MRC5 high resolution camera and Axiovision software (Zeiss, Thornwood, NY).

beginning of second phase pain varied between animals from 20 to 40 minutes; this variation was independent of the vector used. Glycine was therefore injected immediately after the 30-, 40-, or 50-minute observation period and strychnine was injected immediately after the 60-, 70-, or 80-minute observation period. In order to present the data in a clear manner, this variation was removed from Figure 2 and all animal data are presented with second phase pain beginning at 20 minutes.

Thermal nociceptive testing. Thermal withdrawal latency was determined in ten 3-month-old male Sprague–Dawley rats (225–250 g) by methods similar to those previously described21 using a plantar analgesia meter (IITC Life Sciences, Woodland Hills, CA). Each animal was placed into a 23 × 10 cm plexiglass enclosure and placed on a glass surface maintained at 30 °C. After a 15-minute accommodation period, a light beam was focused onto the midplantar area of each hind paw and the amount of time it took the animal to move its paw from the heat source recorded. The rats were randomized into two groups and, under isoflurane anesthesia, were subcutaneously injected with 200 μl containing 1 × 108 plaque forming units of either vHGlyRα1 (N = 5) or vHG (N = 5) into the middle of the plantar surface of the right hindpaw; the vector was injected as one bolus. One week following vector inoculation, thermal withdrawal threshold was measured again to determine whether vector injection alone had any affect. Over the next 3 days, rats were subcutaneously injected with 50 µl of 1, 10, or 100 mmol/l glycine into the plantar surface of the vector-inoculated foot to determine what dose of exogenously applied glycine would activate vectorexpressed GlyR. Rats were injected with the glycine, allowed to rest for 10 minutes, and then the thermal withdrawal threshold was determined. All data are reported as the average of three trials per test.

Interstitial cystitis/painful bladder study. We used a rat model of IC/PBS

Formalin footpad test. Under isoflurane anesthesia, 3-month-old male Sprague–Dawley rats (225–250 g) were subcutaneously injected with 200 μl containing 1 × 108 plaque forming units of either vHGlyRα1 (N = 7) or vHG (N = 5) into the middle of the plantar surface of the right hindpaw; the vector was injected as one bolus. One week later, spontaneous nociceptive behavior was evaluated using the formalin footpad test.2,22 Briefly, 50 µl of 2.5% formalin was injected in the plantar aspect of the same foot that had received the viral inoculation; the rats were then placed into a 48 × 27 × 20 cm plastic box positioned over a mirror tilted at a 45° angle. Beginning 30 seconds after the injection of formalin, and once every 10 minutes thereafter, nocisponsive behaviors were continuously recorded for 3-minute periods. Different positions of the hind paw were rated continuously over the 3-minute period; the following scale was used: 1 = paw rested normally on floor; 2 = paw rested lightly on floor, toes ventroflexed; 3 = entire paw was elevated; 4 = animal was licking/biting paw. Weighted pain scores were calculated by multiplying the amount of time the rat spent in each position according to the following formula: 0 × t1 ± 1 × t2 ± 2 × t3 ± 3 × t4/180; where t1, t2, t3, and t4 are the duration (in seconds) spent in categories 1, 2, 3, and 4 respectively. To examine the effects of activation of expressed GlyR, 50 μl of 100 mmol/l glycine was injected into the plantar surface of the formalininjected foot. In a subset of animals GlyR channels were inhibited by injection of 50 μl of 10 mmol/l strychnine, a specific GlyR inhibitor. The Molecular Therapy vol. 19 no. 3 mar. 2011

Complete Freund’s adjuvant study. Under isoflurane anesthesia, 3-month-

old male Sprague–Dawley rats (225–250 g) were subcutaneously injected with 200 μl containing 1 × 108 plaque forming units of either vHGlyRα1 (N = 7) or vHG (N = 3) into the plantar surface of the right hindpaw; uninoculated animals served as an additional control group (N = 3). One week later vector-treated animals were injected subcutaneously into the center of the plantar surface of the right hindpaw with 50 μl of CFA (Sigma).23 The injections produced localized inflammation characterized by erythema and edema that lasted 3–4 days. At 1, 2, and 3 days post CFA, thermal threshold testing was performed. Animals were first tested without glycine, 30 minutes later injected with 100 mmol/l glycine (in 50 µl volume), and 10 minutes later retested as described above. previously described.24,25 Briefly, 4-month-old female Sprague–Dawley rats (250–300 g) were placed under isoflurane anesthesia, a low midline incision was performed to expose the bladder and a total of 5 µl containing 1.5 × 108 plaque forming units of either vHGlyRα1 or vHG were injected into four different sites (20 µl total) on the bladder wall. Two weeks later cystometrograms were performed under urethane anesthesia. Saline was infused into the bladder to determine baseline contractions followed by 10 nmol/l RTx to induce bladder hyperactivity. Glycine was administered into the jugular vein to determine whether nonlocalized systemic delivery of glycine could activate vector-expressed GlyRs in visceral tissue.

Measuring evoked current in cutaneous DRG neurons. Cutaneous affer-

ents in adult male Sprague–Dawley rats were DiI (1,1′-dioctadecyl-3,3,3′, 3′-tetramethylindocarbo-cyanine perchlorate, Invitrogen) labeled as previously described.43 Two weeks after DiI and vHGlyRα1 coinjection, L4-6 DRG were harvested, enzymatically treated, mechanically dispersed and plated onto lamin-ornithine coated glass coverslips as previously described.43 DiI labeled neurons were easily detectable under epifluorescence illumination. Whole-cell patch-clamp recordings were performed using an EPC-9 amplifier (HEKA Elektronik, Lambrecht, Germany). Series resistance was compensated (>80%) with amplifier circuitry. Data were acquired at 5 kHz and filtered at 1 kHz. Electrodes (1.8–3.0 MΩ) were filled with pipette solution (mmol/l): 130 KCl, 5 NaCl, 1 CaCl2, 2 MgCl2, 11 EGTA, 10 HEPES, 2 ATP-Mg, 1 GTP-Li (all from Sigma); pH was adjusted to 7.2 with Tris-base and osmolality was adjusted to 310 mosmol/l with sucrose (Invitrogen). Neurons were continuously perfused with a bath solution that contained (mmol/l): 140 NaCl, 2.5 CaCl2, 0.6 MgCl2, 10 HEPES, 10 Glucose; pH was adjusted to 7.4 with Tris-base and osmolality was adjusted to 325 mosmol/l with sucrose. Neurons were held at −60 mV. A standard protocol was then employed to study each neuron. High K+ (a bath solution in which 50 mmol/l KCl was used to replace an equimolar concentration of NaCl), glycine (1 mmol/l) and then capsaicin (500 nmol/l) were applied via a fast perfusion system (ALA Scientific, Farmingdale, NY) at an interstimulus interval of 3 minutes. Each test solution was applied for 2 seconds. Statistical measurements. All data represent average ± SEM. *P ≤ 0.05,

statistical differences were determined by nonparametric Kruskal–Wallis test (formalin study) or t-test (dose–response study, CFA study, IC/PBS study) using StatView Software (v 5.0.1 SAS Institute, Cary, NC).

ACKNOWLEDGMENTS We thank Mingdi Zhang, Rebecca Sullenberger, Justin Montgomery, and Dan Santone for their assistance in the animal studies; Dave Kopp

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Glycine Receptor Gene Transfer to Treat Pain

and Ali Ozuer for the production/purification of all HSV vectors; Rahul Srinivasan for editorial assistance; and Brian Davis and Rahul Srinivasan for comments and discussions during preparation of this manuscript. Work described in this manuscript was supported grants from the NIH (R01s to J.R.G., J.C.G., and M.C., and a P01 to J.C.G.).

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