WO2005042711A2 - Pain-associated gene pnpg1 - Google Patents

Abstract

The present invention provides a novel gene, designated herein as “PNPG1”, that is associated with pain. The invention further provides the use of PNPG1 gene and its corresponding protein to diagnose a pain state in a cell or tissue and to screen for novel therapeutic compounds useful for treating pain as well as for other indications.

Description

PAIN-ASSOCIATED GENE PNPGl This application claims priority from U.S. Provisional Application

Serial No. 60/515,224, filed October 28, 2003, the disclosure of which is incorporated herein by reference in its entirety.

1. FIELD OF THE INVENTION [001] The present invention provides a novel gene that is associated with pain and related disorders, in particular, neuropathic pain. This gene, designated herein as the "PNPGl" gene, and its corresponding protein, can be used in screening methods to identify modulators for the gene or protein as potential therapeutic analgesic candidates and for other indications. The PNPGl protein can be used to generate antibodies for detection of PNPGl expression in a biological sample, or as therapeutics. The invention also relates to using the PNPGl gene to generate animal models of pain using transgenic and recombinant technology for screening purposes. 2. BACKGROUND OF THE INVENTION [002] Pain is the most common symptom for which patients seek medical help, and can be classified as either acute or chronic. Acute pain is precipitated by immediate tissue injury (e.g. , a burn or a cut), and is usually self-limited. This form of pain is a natural defense mechanism in response to immediate tissue injury, preventing further use of the injured body part, and withdrawal from the painful stimulus. It is amenable to traditional pain therapeutics, including non-steroidal anti-inflammatory drugs (NSAIDs) and opioids. In contrast, chronic pain is present for an extended period, e.g. , for 3 or more months, persisting after an injury has resolved, and can lead to significant changes in a patient's life (e.g. , functional ability and quality of life) (Foley, Pain, In: Cecil Textbook of Medicine, pp.100- 107, Bennett and Plum eds. , 20^th ed. , 1996).

[003] Chronic, debilitating pain represents a significant medical dilemma.

In the United States, approximately 40 million people suffer from chronic recurrent headaches, 35 million people suffer from persistent back pain, 20 million people suffer from osteoarthritis, 2.1 million people suffer from rheumatoid arthritis, and 5 » million suffer from cancer-related pain (Brower, Nature Biotechnology 2000; 18: 387-391). Cancer-related pain results from both inflammation and nerve damage. In addition, analgesics are often associated with debilitating side effects such as nausea, dizziness, constipation, respiratory depression and cognitive dysfunction (Brower, Nature Biotechnology 2000; 18: 387-391).

[004] Pain can be classified as either "nociceptive" or "neuropathic", as defined below. 2.1. Nociceptive Pain

[005] "Nociceptive pain" is due to activation of pain-sensitive nerve fibers, either somatic or visceral. Nociceptive pain is generally a response to direct tissue damage. The initial trauma typically causes the release of several chemicals including bradykinin, serotonin, substance P, histamine, and prostaglandin. When somatic nerves are involved, the pain is typically experienced as an aching or pressure-like sensation.

[006] Nociceptive pain has traditionally been managed by administering non-opioid analgesics. These analgesics include acetylsalicylic acid, choline magnesium trisalicylate, acetaminophen, ibuprofen, fenoprofen, diflusinal, and naproxen. Opioid analgesics , such as morphine, hydromorphone, methadone, levorphanol, fentanyl, oxycodone and oxymorphone, may also be used (Foley, Pain, In: Cecil Textbook of Medicine, pp.100-107, Bennett and Plum eds., 20^th ed. , 1996).

2.2. Neuropathic Pain [007] The term "neuropathic pain" refers to pain that is due to injury or disease of the central or peripheral nervous system (McQuay, Acta Anaesthesiol. Scand. 1997; 41(1 Pt 2): 175-83; Portenoy, J. Clin. Oncol. 1992; 10: 1830-2). In contrast to the immediate pain caused by tissue injury, neuropathic pain can develop days or months after a traumatic injury. Furthermore, while pain caused by tissue injury is usually limited in duration to the period of tissue repair, neuropathic pain frequently is long-lasting or chronic. Moreover, neuropathic pain can occur spontaneously or as a result of stimulation that normally 'is not painful.

[008] Neuropathic pain is associated with chronic sensory disturbances, including spontaneous pain, hyperalgesia (i.e. , sensation of more pain than the stimulus would warrant), and allodynia (i.e. , a condition in which ordinarily painless stimuli induce the experience of pain). In humans, prevalent symptoms include cold hyperalgesia and mechanical allodynia. Sensitivity to heat is rarely reported. Descriptors that are often used to describe such pain include "lancinating," "burning," or "electric". It is estimated that approximately 4 million people in North America suffer from chronic neuropathic pain, and of these no more than half achieve adequate pain control (Hansson, Pain Clinical Updates 1994; 2(3)).

[009] Examples of neuropathic pain syndromes include those due to disease progression, such as diabetic neuropathy, multiple sclerosis, or post-herpetic neuralgia (shingles); those initiated by injury, such as amputation (phantom-limb pain), or injuries sustained in an accident (e.g., avulsions); and those caused by nerve damage, such as from chronic alcoholism, viral infection, hypothyroidism, uremia, or vitamin deficiencies. Traumatic nerve injuries can also cause the formation of neuromas, in which pain occurs as a result of aberrant nerve regeneration. Stroke (spinal or brain) and spinal cord injury can also induce neuropathic pain. Cancer-related neuropathic pain results from tumor growth compression of adjacent nerves, brain, or spinal cord. In addition, cancer treatments, including chemotherapy and radiation therapy, can also cause nerve injury. [0010] Unfortunately, neuropathic pain is often resistant to available drug therapies. Treatments for neuropathic pain include opioids, anti-epileptics (e.g., gabapentin, carbamazepine, valproic acid, topiramate, phenytoin), NMDA antagonists (e.g. , ketamine, dextromethorphan), topical Lidocaine (for post-herpetic neuralgia), and tricyclic anti-depressants (e.g. , fluoxetine (Prozac^®), sertraline (Zoloft^®), amitriptyline). However, none of these treatments is particularly effective, achieving clinical significance of less than 50% (Bridges et al. , Br. J. Anaes. 2001; 87: 12-26). Neuropathic pain is frequently only partially relieved by high doses of opioids, which are the most commonly used analgesics (Cherny et al. , Neurology 1994; 44: 857-61.; MacDonald, Recent Results Cancer Res. 1991; 121: 24-35.; McQuay, 1997, supra). Current therapies may also have serious side effects such as cognitive changes, sedation, and nausea. Many patients suffering from neuropathic pain are elderly or have other medical conditions that limit their tolerance of such side-effects. The inadequacy of current therapies in relieving neuropathic pain (and without producing intolerable side effects) frequently manifests in depression and increased suicide rates of chronic pain sufferers. 2.3. Inflammatory Pain [0011] Chronic somatic pain generally results from inflammatory responses to tissue injury such as nerve entrapment, surgical procedures, cancer or arthritis

(Brower, Nature Biotechnology 2000; 18: 387-391). Although many types of inflammatory pain are currently treated with NSAIDs, there is much room for improved therapies.

[0012] The inflammatory process is a complex series of biochemical and cellular events activated in response to tissue injury or the presence of foreign substances (Levine, Inflammatory Pain, In: Textbook of Pain, Wall and Melzack eds., 3^rd ed., 1994). Inflammation often occurs at the site of injured tissue, or foreign material, and contributes to the process of tissue repair and healing. The cardinal signs of inflammation include erythema (redness), heat, edema (swelling), pain and loss of function (ibid.). The majority of patients with inflammatory pain do not experience pain continually, but rather experience enhanced pain when the inflamed site is moved or touched.

[0013] Tissue injury induces the release of inflammatory mediators from damaged cells. These inflammatory mediators include ions (H⁺, K⁺), bradykinin, histamine, serotonin (5-HT), ATP and nitric oxide (NO) (Kidd and Urban, Br. J. Anaesthesia 2001, 87: 3-11). The production of prostaglandins and leukotrienes is initiated by activation of the arachidonic acid (AA) pathway. Via activation of phospholipase A2, AA is converted to prostaglandins by cyclooxygenases (Cox-1 and Cox-2), and to leukotrienes by 5-lipoxygenase. The NSAIDs exert their therapeutic action by inhibiting cyclooxygenases. Recruited immune cells release further inflammatory mediators, including cytokines and growth factors, as well as activating the complement cascade. Some of these inflammatory mediators (e.g. , bradykinin) activate nociceptors directly, leading to spontaneous pain. Others act indirectly via inflammatory cells, stimulating the release of additional pain-inducing (algogenic) agents. Application of inflammatory mediators (e.g., bradykinin, growth factors, prostaglandins) has been shown to produce pain, inflammation and hyperalgesia (increased responsiveness to normally noxious stimuli). 2.4. Genetics [0014] Recent efforts to treat neuropathic pain have focused on identification of genes that are differentially regulated in response to pain stimuli. Using rat models of neuropathic pain, changes in gene and protein expression in the injured part of DRG neurons (ipsilateral) compared with the uninjured side (contralateral) or uninjured neurons have been reported (Wang et al , Neuroscience 2002; 114: 520- 46; Kim et al , NeuroReport 2001; 12: 3401-05; Xiao et al , Proc. Natl. Acad. Sci. USA 2002; 99: 8361-65; Costigan et al , BMC Neuroscience 2002; 3: 16; and Sun et al , BMC Neuroscience; 2002; 3: 11). Genes that were found to be up-regulated in injured neurons include those that encode cell-cycle and apoptosis-related proteins; genes encoding pro-inflammatory cytokines or lymphokines, including complement proteins; genes encoding ion channels and their receptors; genes encoding transcription factors; and genes encoding structural or glycoproteins involved in tissue remodeling (Wang et al , supra). Genes that were down- regulated compared with uninjured neurons include: neuropeptides such as somatostatin and Substance P; the serotonin 5HT-3 receptor; the glutamate receptor 5 (GluR5); sodium and potassium channels; calcium signaling molecules; and synaptic proteins (Wang et al , supra).

[0015] Neuronal transcription factors are also differentially regulated in injured neurons. Transcription factors determined to be differentially expressed include JunD, NGF1-A and MRG1 (Xiao et al. , supra; Sun et al , supra). [0016] Despite the identification of certain genes that are differentially regulated in models of pain, there remains a need to identify other pain-related genes, and to develop more effective therapies to treat pain, particularly neuropathic pain. 3. SUMMARY OF THE INVENTION [0017] The present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a mammalian PNPGl protein or a fragment thereof. More particularly, the mammalian PNPGl protein is a rat, mouse or human PNPGl protein. More particularly, the rat PNPGl protein comprises the amino acid sequence of SEQ ID NO:2; the mouse PNPGl protein comprises the amino acid sequence of SEQ ID NO:4; and the human PNPGl protein comprises the amino acid sequence of SEQ ID NO: 6.

[0018] In one embodiment, the nucleotide sequence encoding the rat PNPGl protein comprises the nucleotide sequence of SEQ ID NO: l (cDNA sequence), or SEQ ID NO: 11 (rat genomic sequence), or a degenerate variant thereof.

[0019] In another embodiment, the nucleotide sequence encoding the mouse PNPGl protein comprises the nucleotide sequence of SEQ ID NO: 3 (cDNA sequence), or SEQ ID NO: 12 (mouse genomic sequence), or a degenerate variant _ thereof. [0020] In another embodiment, the nucleotide sequence encoding the human PNPGl protein comprises the nucleotide sequence of SEQ ID NO: 5 (cDNA sequence), or SEQ ID NO: 13 (human genomic sequence), or a degenerate variant thereof.

[0021] In an alternative embodiment, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a mammalian PNPGl protein, with the proviso that the nucleotide sequence does not comprise the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 5. In another embodiment, the mammalian PNPGl protein encoded by the isolated nucleic acid molecule does not comprise the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO:6.

[0022] The present invention further provides an isolated nucleic acid molecule comprising a nucleotide sequence that is homologous to the nucleotide sequence of one of the aforementioned nucleic acid molecules.

[0023] The present invention further provides an oligonucleotide molecule that hybridizes to a nucleic acid molecule of the present invention or to its complement.

[0024] The present invention further provides a recombinant vector (e.g. , a cloning vector or an expression vector or a gene knockout vector) comprising a nucleic acid molecule of the present invention.

[0025] The present invention further provides a host cell genetically modified to clone, express or overexpress a nucleic acid molecule of the present invention. [0026] The present invention further provides an isolated polypeptide comprising the amino acid sequence of a mammalian PNPGl protein or a fragment thereof. In a specific embodiment, the isolated polypeptide comprises the amino acid sequence of a rat, mouse or human PNPGl protein. More particularly, the rat PNPGl protein comprises the amino acid sequence of SEQ ID NO:2; the mouse PNPGl protein comprises the amino acid sequence of SEQ ID NO: 4; and the human PNPGl protein comprises the amino acid sequence of SEQ ID NO:6.

[0027] The present invention further provides an isolated cellular membrane fraction prepared from a cell, which cell has been genetically modified to express or overexpress a nucleic acid molecule having a nucleotide sequence that encodes a PNPGl protein or a fragment thereof comprising a transmembrane domain, and which cellular membrane fraction comprises the PNPGl protein or a fragment thereof. [0028] The present invention further provides an antibody or antibody fragment that specifically binds to a polypeptide of the present invention.

[0029] The present invention further provides a mammalian cell that has been genetically modified so that its normal expression of a PNPGl -encoding gene has been changed (e.g. , turned on, increased, reduced or eliminated). The present invention further provides genetically modified animals prepared from such cells. The present invention further provides methods for preparing and using such cells and animals.

[0030] The present invention further provides molecules that can specifically inhibit expression of a PNPGl -encoding nucleic acid molecule or a PNPGl protein of the present invention, including without limitation antisense oligonucleotides, ribozymes, triple helix-forming oligonucleotides, and short interfering RNA molecules.

[0031] In conjunction with the nucleic acid molecules disclosed herein, the present invention further provides a method for detecting a pain response in a test cell subjected to a treatment or stimulus, said method comprising: (a) determining the expression level of a nucleic acid molecule encoding a PNPGl protein in the test cell capable of expressing said nucleic acid molecule, which test cell has been subjected to a treatment or stimulus; and

(b) comparing the expression level of the PNPGl -encoding nucleic acid molecule in the test cell to the expression level of the same nucleic acid molecule in a control cell not subjected to the treatment or stimulus;

wherein a detectable change in the expression level of the PNPGl -encoding nucleic acid molecule in the test cell compared to the expression level of the PNPG1- encoding nucleic acid molecule in the control cell indicates that the test cell is exhibiting a pain response. According to the present invention, the detectable change in the expression level is any statistically significant change, and preferably is at least a 1.5-fold change as measured by any available technique such as hybridization or quantitative PCR.

[0032] As disclosed herein, the test cell can be any cell derived from a tissue of an organism experiencing a feeling of pain or associated disorder. Alternatively, the test cell can be any cell grown in vitro under specific conditions. When the test cell is derived from a tissue of an organism experiencing a feeling of pain or associated disorder, it may or may not be known to be located in the region associated with the feeling of pain. The control cell can be any cell which is known to have not been subjected to any treatment or stimulus associated with pain. Preferably, the control cell is otherwise identical to the test cell. For example, when the test cell is derived from a tissue of an animal experiencing a feeling of pain or associated disorder, the control cell can be derived from an identical tissue or body part of a different animal from the same species (preferably closely related) not experiencing a feeling of pain or associated disorder. Alternatively, the control cell can be derived from an identical tissue or body part of the same animal, so long as it can be established that the identical tissue or body part has not been subjected to any treatment or stimulus associated with pain within the timeframe of the experiment. When the test cell is a cell grown in vitro under specific conditions, the control cell can be an identical cell grown in vitro in the absence of such specific conditions. Preferably, the cells used in the method of the invention are neural cells (e.g. , dorsal root ganglia (DRG)). In a specific embodiment, the cells are human cells. In another specific embodiment, the cells are derived from an animal model of pain or associated disorder.

[0033] The present invention further provides a method for identifying a candidate compound useful for modulating the expression of a PNPGl -encoding nucleic acid, said method comprising: (a) contacting a first cell capable of expressing a PNPGl -encoding nucleic acid molecule of the present invention with a test compound under conditions sufficient to allow the cell to respond to said contact with the test compound; (b) determining the expression level of the PNPGl -encoding nucleic acid molecule in the first cell during or after contact with the test compound; and

(c) comparing the expression level of the PNPGl -encoding nucleic acid molecule determined in step (b) to the expression level of the PNPGl -encoding nucleic acid molecule in a second (control) cell capable of expressing the nucleic acid molecule that has not been contacted with the test compound;

wherein a detectable change in the expression level of the PNPGl -encoding nucleic acid molecule in the first cell in response to contact with the test compound compared to the expression level of the PNPGl -encoding nucleic acid molecule in the second (control) cell that has not been contacted with the test compound, indicates that the test compound modulates the expression of the PNPGl -encoding nucleic acid and is a candidate compound. [0034] Further provided herein is a method for monitoring the efficacy of an analgesic treatment in a cell comprising: (a) administering to said cell an analgesic compound under conditions sufficient to allow the cell to respond to said compound; (b) determining in the cell prepared in step (a) the expression level of a PNPGl -encoding nucleic acid molecule; and

(c) comparing the expression level of the PNPGl -encoding nucleic acid molecule determined in step (b) to the expression level of the PNPGl -encoding nucleic acid molecule in a second (control) cell that has not been contacted with the analgesic compound;

wherein a detectable change in the expression level of the PNPGl -encoding nucleic acid molecule in the first cell in response to contact with the analgesic compound compared to the expression level of the PNPGl -encoding nucleic acid molecule in the second (control) cell that has not been contacted with the analgesic compound is indicative of the activity of the analgesic compound.

[0035] The present invention further provides a method for identifying a candidate compound useful for modulating the expression of a PNPGl protein, said method comprising: (a) contacting a first cell capable of expressing a PNPGl protein of the present invention with a test compound under conditions sufficient to allow the cell to respond to said contact with the test compound; (b) determining the expression level of the PNPGl protein in the first cell during or after contact with the test compound; and

(c) comparing the expression level of the PNPGl protein determined in step (b) to the expression level of the PNPGl protein in a second (control) cell capable of expressing the protein that has not been contacted with the test compound;

wherein a detectable change in the expression level of the PNPGl protein in the first cell in response to contact with the test compound compared to the expression level of the PNPGl protein in the second (control) cell that has not been contacted with the test compound, indicates that the test compound modulates the expression of the PNPGl protein and is a candidate compound. In one embodiment, the test compound is an analgesic.

[0036] The present invention further provides a method for identifying a candidate compound capable of binding to a PNPGl protein or peptide fragment thereof, said method comprising: (a) contacting a PNPGl protein or peptide fragment of the present invention with a test compound under conditions that permit binding of the test compound to the PNPGl protein; and

(b) detecting binding of the test compound to the PNPGl protein or peptide fragment. [0037] The present invention further provides a method for identifying a candidate compound capable of modulating the activity of a PNPGl protein, said method comprising:

(a) contacting a PNPGl protein with a test compound under conditions that permit modulation of the activity of the PNPGl protein; and

(b) determining whether the activity of the PNPGl protein is modulated in response to said contact;

wherein a detectable change in the activity of the PNPGl protein in response to contact with the test compound indicates that the test compound modulates the activity of the PNPGl protein and is a candidate compound.

[0038] The present invention further provides a method for treating a condition that can be treated by modulating expression of a PNPGl -encoding nucleic acid molecule or a PNPGl protein, comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound that modulates expression of a PNPGl -encoding nucleic acid molecule or a PNPGl protein. Preferably, the treated condition is a pain or pain-related disorder such as, e.g. , chronic pain, nociceptive pain, neuropathic pain (including all types of hyperalgesia (i.e. , sensation of more pain than the stimulus would warrant) and allodynia (i.e. , a condition in which ordinarily painless stimuli induce the experience of pain)), inflammatory and cancer pain, addiction, seizure (including epilepsy), stroke or ischemia, neurodegenerative disorder (e.g. , Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), or Huntington's chorea), headache (e.g. , general, migraine, cluster or tension), anxiety, depression, asthma, rheumatic disease, osteoarthritis, retinopathy, inflammatory eye disorders, pruritis, ulcer (gastric or duodenal), gastric lesions (e.g. , induced by a necrotizing agent), uncontrollable urination (e.g. , incontinence), an inflammatory or unstable bladder disorders, inflammatory bowel disease (e.g. , Crohn's disease and ulcerative colitis), irritable bowel syndrome (IBS) including irritable bowel disease (IBD), gastroesophageal reflux disease (GERD), functional dyspepsia (e.g. , ulcer-like dyspepsia, dysmotility-like dyspepsia, functional heartburn, and non-ulcer dyspepsia), functional chest pain of presumed esophageal origin, functional dysphagia, non-cardiac chest pain, symptomatic gastroesophageal disease, gastritis, aerophagia, functional constipation, functional diarrhea, burbulence, chronic functional abdominal pain, recurrent abdominal pain (RAP), functional abdominal bloating, functional biliary pain, functional incontinence, functional ano-rectal pain, chronic pelvic pain, pelvic floor dyssenergia, unspecified functional ano-rectal disorder, cholecystalgia, interstitial cystitis, dysmenorrhea, or dyspareunia.

[0039] In a specific embodiment the subject is an animal model of a pain or related disorder. In another specific embodiment, the subject is a mammal such as a human or companion animal (e.g., a dog or cat) suffering from a pain or related disorder.

[0040] In conjunction with the methods of the invention, provided herein are diagnostic kits comprising (i) a nucleic acid molecule of the invention in the form of a hybridization target, primers for quantitative polymerase chain reaction (PCR) analysis, antisense oligonucleotides, ribozymes, RNAi oligonucleotides, etc. and/or (ii) antibodies or antibody fragments that recognize a PNPGl polypeptide of the invention.

4. BRIEF DESCRIPTION OF THE DRAWINGS [0041] Figure 1A represents analysis of expression of the gene corresponding to EST AI228284 in L4 (unligated) and L5 (ligated) dorsal root ganglia (DRG) in the presence or in the absence of gabapentin (GPN) treatment in a rat sciatic nerve ligation (SNL) model of neuropathic pain as compared to expression of the gene corresponding to EST AI228284 in L4 and L5 DRG in naϊve (non-operated) and sham-operated animals. The analysis demonstrates that the expression of the gene corresponding to EST AI228284 in L5 DRG of SNL animals is down-regulated about 3.1 -fold as compared to L5 DRG in naϊve or sham-operated animals. The analysis of EST AI228284 expression was performed using Affymetrix GeneChip^® hybridization (line graph; right axis units expressed as gene expression intensity). Expression down-regulation was confirmed by quantitative real time PCR TaqMan^® (bar graphs; left axis units expressed as relative expression level compared to control gene integrin-linked kinase (ILK)). Further bioinformatic analysis and full-length cloning results indicate that rat PNPGl is the gene corresponding to EST AI228284. [0042] Figure IB represents analysis of expression of the gene corresponding to EST AI228284 in 27 naϊve rat tissues, which demonstrates that EST AI228284 is enriched in DRG. The analysis of EST AI228284 expression was performed using Affymetrix GeneChip^® hybridization (line graph; right axis units expressed as gene expression intensity) and quantitative real time PCR TaqMan^® (bar graphs; left axis units expressed as relative expression level compared to control gene phosphotidylinositol transfer protein, beta (PITPNB)). Further bioinformatic analysis and full-length cloning results indicate that rat PNPGl is the gene corresponding to EST AI228284.

[0043] Figure IC represents analysis of expression of the human ortholog of the gene corresponding to rat EST AI228284 or "human PNPGl" in 23 naϊve human tissues, which demonstrates that PNPGl is expressed in human DRG. The analysis of human PNPGl expression was performed using Affymetrix GeneChip^® hybridization (line graph; right axis units expressed as gene expression intensity) and quantitative real time PCR TaqMan^® (bar graphs; left axis units expressed as relative expression level compared to control gene hypoxanthine phosphoribosyl transferase (HPRT)).

[0044] Figure 2 represents a Northern blot analysis of total mRNA extracted from 10 rat tissues using a probe specific for EST AI228284. The analysis reveals a rat transcript of about 2.3 kb in length. [0045] Figure 3 represents a SMART model (Schultz et al , Proc. Natl.

Acad. Sci. USA 1998; 95: 5857-5864 and Lerunic et al , Nucleic Acids Res. 2002; 30: 242-244) for PNPGl protein which predicts two CTNS domains found in cystinosin, a cystine transporter found on lysosomal membranes. ) [0046] Figure 4 depicts a sequence alignment of rat, mouse and human

PNPGl proteins performed using the ClustalW algorithm alignment tool in Vector NTI software (InforMax™, Invitrogen Life Science Software, Frederick, MD). The three lightest shades of gray indicate identity and similarity in alignment. Shown below is a consensus sequence. Highlighted in dark gray are seven predicted transmembrane domains (TM). Peptides based on the amino acid sequence of rat PNPGl (black highlights, Pep) were synthesized for polyclonal antibody production.

[0047] Figure 5 represents a Western blot analysis of soluble (S) and membrane-bound (M) protein fractions of HEK 293 cells transfected with pcDNA- PNPGl (encoding rat PNPGl fused to V5 epitope-tag) using antibodies directed against the V5 epitope-tag. When the protein extracts were diluted, the predicted 35 kD expressed PNPGl fusion protein was detected in both the soluble (cytosolic) and membrane-bound fractions of pcDNA-PNPGl -transfected HEK 293 cells. The protein was extracted by a 2D sample prep method (Pierce Biotechnology, Rockford, IL) yielding two portions: one containing soluble protein and the other containing membrane-bound protein. SM - size markers; N - negative control (protein extract from untransfected HEK 293 cells); P- positive control (whole cell lysate of HEK 293 cells transfected with pcDNA-PNPGl) .

[0048] Figure 6 represents a confocal microscope image of V5-tagged PNPGl in transiently transfected CHO cells. Arrow indicates observed membrane expression of PNPGl protein.

[0049] Figures 7A and 7B represent brightfield (A) and darkfield (B) images of naϊve rat DRG tissue hybridized in situ with ³⁵S-UTP labeled antisense RNA probe to PNPGl. Arrows point to examples of PNPGl expression in rat DRG neurons.

[0050] Figure 8 depicts vector constructs for epitope-tagged rat PNPGl expression (pcDNA-PNPGl), rat PNPGl siRNA expression (pSi-PNPGl), and Mu opioid receptor siRNA expression (pSi-Mu DNA). Shown are the promoters (PCMV and U6) upstream of the sequence encoding the recombinant protein (rat PNPGl fused to V5+HIS epitope-tag) or siRNA sequence (specific for rat PNPGl or Mu opioid receptor). For the siRNA expression constructs, also shown are cloning sites and RNA Pol III terminator sequence. The vector backbones for pcDNA-PNPGl and pSi-PNPGl were pcDNA3.1D/V5-His-TOPO (Invitrogen, Carlsbad, CA) and pSilencer 2.0-U6 (Ambion Inc., Austin, TX), respectively. [0051] Figures 9A and 9B represent a Western blot analysis confirming

PNPGl siRNA-mediated reduction of recombinant PNPGl protein expression. HEK 293 cells were transfected with pcDNA-PNPGl and vector constructs designed to express either an siRNA targeting PNPGl (pSi-PNPGl) or a negative control siRNA (pSi-Mu, which expresses siRNA specific for the Mu opioid receptor). The total transfected DNA concentration was kept at 200ng/well (on a 24 well plate), but the ratio between pcDNA-PNPGl and pSiPNPGl or pSiMu was changed as indicated. Three days after transfection, 20 μg of total protein was loaded onto an SDS-PAGE gel as indicated. Anti-V5 antibody was used to detect V5-tagged PNPGl (A), and the same membrane was subsequently stripped and re- probed with anti-GAPDH antibody (B) to demonstrate equivalent loading of total protein across samples. The results show that the expression of PNPGl was knocked down when co-expressed with PNPGl siRNA.

[0052] Figures 10A and 10B depict exon-intron organization for the genes encoding rat, mouse and human PNPGl (SEQ ID NOS: 11, 12, and 13, respectively). A. PNPGl gene is encoded by 5, 5, and 6 exons on chromosome 18 of rat, mouse, and human, respectively. Indicated by solid boxes labeled by ORF nt coordinates are the portions of the exons encoding ORF sequence. The coordinates for the UTRs diverged across the three species as indicated by striped boxes. B. Coordinates for the exon-intron boundaries for rat, mouse, and human PNPGl gene are given in the table based on the nucleotide coordinates of rat genomic sequence Accession NW_043157 (GI: 26011374), mouse Mus musculus chromosome 18 genomic contig (GL28525381), and human Homo sapiens chromosome 18 genomic contig (GI: 37545286). Provided for reference are the nt coordinates of the ORF sequence contained within each exon. The rat and human coordinates are reversed indicating that the gene is on the opposite strand with respect to the given reference sequence. [0053] Figure 11 depicts summary experimental timelines for surgery, treatment and behavioral testing.

[0054] Figure 12A depicts the sequence and features of siRNA insert

(oligonucleotide MB0475 annealed to MB0476, SEQ ID NOS: 29 and 30, respectively) that was cloned into pSilencer 2.0-U6 to construct pSi-PNPGl (Figure 8). Critical features include 19 nt PNPGl sense and antisense sequences, a 9 nt loop sequence, RNA Pol III terminator sequence, and 4 nt 5 '-overhangs compatible for annealing and ligation to BamHI and Hindlll restriction enzyme sites.

[0055] Figure 12B shows a schematic representation of a typical Hairpin siRNA produced by expression vector pSilencer 2.0-U6, and the siRNA's relationship to the RNA target sequence. Note that the 19 nt target-specific sequence must occur directly downstream of two adenosines (A) present in the target RNA.

[0056] Figure 13 shows the detection of PNPG1-V5 protein by a purified anti-PNPGl antibody. A Western blot analysis was carried out on whole cell lysates from HEK 293 cells (H) and HEK 293 cells transfected with pcDNA-PNPGl (encoding rat PNPGl fused to a V5 epitope-tag). The same lysates were loaded on right and left panels and detected with anti-V5 antibody (Invitrogen, Cat# R960-25) and purified PNPGl antibody, respectively. 5. DETAILED DESCRIPTION OF THE INVENTION

[0057] The present invention provides nucleic acid molecules having nucleotide sequences encoding a novel protein, designated herein as "PNPGl". These nucleic acid molecules have been identified as corresponding to EST rc_AI228284, which was isolated among 249 known rat genes and 87 rat expressed- sequence tags (ESTs) identified using microarray technology and validated using quantitative real time PCR, representing genes that are differentially expressed in a rat spinal nerve ligation "SNL" model of neuropathic pain.

[0058] The present invention is based on gene expression profiles obtained from a rat spinal nerve ligation "SNL" model of neuropathic pain (Kim and Chung, Pain 1992; 50: 355-363) described in the Example section below. This model is created by tightly ligating the L5 and L6 spinal nerves in the rat. The rat L5/6 inter- vertebral disc is innervated by Ll to L6 dorsal root ganglia (DRG). Symptoms induced by this injury include sensitivity to cold and mechanical stimuli, as well as sensitivity to heat. Behaviors indicative of spontaneous pain, such as sudden licking, gentle biting, or pulling of the nails on the operated side, are also observed. These symptoms are attenuated by drugs commonly used in the clinic to treat neuropathic pain, e.g. , gabapentin.

[0059] Microarrays containing oligonucleotide probe sets representing around 26,000 unique rat genes (GeneChip^®, Rat Genome U34 A, B, and C arrays, Affymetrix, Inc., Santa Clara, CA) were used initially to identify mRNAs which are differentially expressed in SNL rats (with or without gabapentin treatment) as compared to naϊve and sham-operated rats. Two-hundred forty nine known genes and eighty seven ESTs were selected on the basis of their differential expression in both injured (L5 and L6) and non-injured (L4) DRGs. To select from among these differentially expressed genes and ESTs those genes and ESTs likely to be more significant as targets for pain therapeutics, the analysis focused on genes and ESTs the expression of which is co-regulated with the expression of genes known from prior studies to be important as molecular mediators of pain perception and/or restricted in their expression across a panel of 12 normal tissues to the target organ for pain (i.e., the DRG). For the selected set of genes and ESTs, differential expression in the SNL model was validated using quantitative real time PCR (TaqMan^®, Applied Biosystems, Foster City, CA) on mRNA samples isolated from an independent pool of animals. Rat EST rc_AI228284 was selected in all of these steps as one of the differentially expressed ESTs, the expression of which is decreased by about 3 -fold in the DRG of rats subjected to the SNL model, and is closely coupled to the expression of multiple known pain genes. A coding sequence of rat PNPGl was then identified using an EST- walking technique as corresponding to EST rc_AI228284. The rat PNPGl cDNA sequence was used in turn to identify the corresponding rat genomic sequence as well as its human and murine orthologs. 5.1. Definitions

[0060] As used herein, the term "pain" is art recognized and includes a bodily sensation elicited by noxious chemical, mechanical, or thermal stimuli, in a subject, e.g. , a mammal such as a human. The term "pain" includes chronic pain such as lower back pain; pain due to arthritis, e.g. , osteoarthritis; joint pain, e.g. , knee pain or carpal tunnel syndrome; myofascial pain, and neuropathic pain. The term "pain" further includes acute pain, such as pain associated with muscle strains and sprains; tooth pain; headaches; pain associated with surgery; or pain associated with various forms of tissue injury, e.g. , inflammation, infection, and ischemia. [0061] "Neuropathic pain" refers to pain caused by injury or disease of the central or peripheral nervous system. In contrast to the immediate (acute) pain caused by tissue injury, neuropathic pain can develop days or months after a traumatic injury. Neuropathic pain frequently is long-lasting or chronic and is not limited in duration to the period of tissue repair. Neuropathic pain can occur spontaneously or as a result of stimulation that normally is not painful. Neuropathic pain is sustained by aberrant somatosensory processing, and is associated with chronic sensory disturbances, including spontaneous pain, hyperalgesia (i.e. , sensation of more pain than the stimulus would warrant) and allodynia (i.e. , a condition in which ordinarily painless stimuli induce the experience of pain). Neuropathic pain includes but is not limited to pain caused by peripheral nerve trauma, viral infection, diabetes mellitus, causalgia, plexus avulsion, neuroma, limb amputation, vasculitis, nerve damage from chronic alcoholism, hypothyroidism, uremia, and vitamin deficiencies, among other causes. Neuropathic pain is one type of pain associated with cancer. Cancer pain can also be "nociceptive" or "mixed. " [0062] "Chronic pain" can be defined as pain lasting longer than three months (Bonica, Semin. Anesth. 1986, 5:82-99), and may be characterized by unrelenting persistent pain that is not fully amenable to routine pain control methods. Chronic pain includes, but is not limited to, inflammatory pain, postoperative pain, cancer pain, osteoarthritis pain associated with metastatic cancer, trigeminal neuralgia, acute herpetic and post-herpetic neuralgia, diabetic neuropathy, pain due to arthritis, joint pain, myofascial pain, causalgia, brachial plexus avulsion, occipital neuralgia, reflex sympathetic dystrophy, fibromyalgia, gout, phantom limb pain, burn pain, pain associated with spinal cord injury, multiple sclerosis, reflex sympathetic dystrophy and lower back pain and other forms of neuralgia, neuropathic, and idiopathic pain syndromes.

[0063] "Nociceptive pain" is due to activation of pain-sensitive nerve fibers, either somatic or visceral. Nociceptive pain is generally a response to direct tissue damage. The initial trauma causes the release of several chemicals including bradykinin, serotonin, substance P, histamine, and prostaglandin. When somatic nerves are involved, the pain is typically experienced as an aching or pressure-like sensation.

[0064] The "dorsal root ganglion" or "DRG" is the cluster of neurons just outside the spinal cord, made of cell bodies of afferent spinal neurons that comprise the peripheral nervous system (PNS). The cell bodies of sensory nerves that convey somatosensory (sense of touch) information to the brain are found in the DRG. These neurons are unipolar, where the axon splits in two, sending one branch to the sensory receptor and the other to the brain for processing.

[0065] The term "ipsilateral" refers to the side of the animal on which the

L5 and L6 nerves are ligated (wounded) in the rat SNL model of neuropathic pain. The corresponding "ipsilateral" side in a sham-operated animal is the side on which the nerve is exposed but not injured, and the "ipsilateral" side in a naive animal is the side that would have been injured (e.g. , the left side as described in the Section 6 (Example), infra). The term "contralateral" refers to the unligated (unwounded) side of the animal. [0066] In the context of the present invention insofar as it relates to pain, the terms "treat", "treatment", and the like refer to a means to relieve or alleviate the perception of a pain. The terms "treat", "treatment", and the like may mean to relieve or alleviate the intensity and/or duration of a pain (e.g. , burning sensation, tingling, electric-shock-like feelings, etc.) experienced by a subject in response to a given stimulus (e.g. , pressure, tissue injury, cold temperature, etc.). Treatment can occur in a subject (e.g. , a human or companion animal) suffering from a pain condition or having one or more symptoms of another condition that can be treated according to the present invention, or in an animal model of pain, such as the SNL rat model of neuropathic pain described herein, or any other animal model of pain. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pain), the terms "treat", "treatment", and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.

[0067] An "analgesic" refers to any compound (e.g. , small organic or inorganic molecule, polypeptide, nucleic acid molecule, etc.) that is either known or novel, and useful to treat pain. Specific categories of analgesics include but are not limited to opioids (e.g. , morphine, hydromorphone, methadone, levorphanol, fentanyl, oxycodone, oxymorphone), antidepressants (e.g. , fluoxetine (Prozac^®), sertraline (Zoloft^®), amitriptyline), anti-convulsants (e.g., gabapentin, carbamazepine, valproic acid, topiramate, phenytoin), non-steroidal anti- inflammatory drugs (NSAIDs) and anti-pyretics (such as, e.g. , acetaminophen, ibuprofen, fenoprofen, diflusinal, naproxen, aspirin, and other salicylates (e.g. , choline magnesium trisalicylate)), NMDA antagonists (e.g. , ketamine, dextromethorphan), and topical Lidocaine (see also Sindrup et al , Pain 1999; 83: 389-400).

[0068] The term "subject" as used herein refers to a mammal (e.g. , rodent such as a mouse or rat, pig, primate, companion animal (e.g. , dog or cat, etc.). In particular, the term refers to humans.

[0069] "Expression profile" refers to any description or measurement of one or more of the genes that are expressed by a cell, tissue, or organism under or in response to a particular condition. Expression profiles can identify genes that are up-regulated, down-regulated, or unaffected under particular conditions. Gene expression can be detected at the nucleic acid level or at the protein level. The expression profiling at the nucleic acid level can be accomplished using any available technology to measure gene transcript levels. For example, the method could employ in situ hybridization, Northern hybridization or hybridization to a nucleic acid microarray, such as an oligonucleotide microarray, or a cDNA microarray. Alternatively, the method could employ reverse transcriptase- polymerase chain reaction (RT-PCR) such as fluorescent dye-based quantitative real time PCR (TaqMan^® PCR). In the Examples section provided below, nucleic acid expression profiles were obtained by (i) hybridization of labeled cRNA derived from total cellular mRNA to Affymetrix GeneChip^® oligonucleotide microarrays, (ii) TaqMan^® PCR using gene-specific PCR primers, (iii) Northern hybridization, and (iv) in situ hybridization. The expression profiling at the protein level can be accomplished using any available technology to measure protein levels, e.g. , using peptide-specific capture agent arrays (see, e.g., International PCT Publication No. WO 00/04389).

[0070] The term "expressed sequence tag" or "EST" refers to short (usually approximately 200-600 nt) single-pass sequence reads from one or both ends of a cDNA clone. Typically, ESTs are produced in large batches by performing a single, automated, sequencing read of cDNA inserts in a cDNA library using a primer based on the vector sequence. As a result, ESTs often correspond to relatively inaccurate (around 2% error) partial cDNA sequences. Since most ESTs are short, they probably will not contain the entire coding region of a large gene (exceeding 200-600 nt in ORF length). Alternatively, or in addition, ESTs may contain non-coding sequences corresponding to untranslated regions of mRNA. ESTs may provide information about the location, expression, and function of the entire gene they represent. They are useful (e.g. , as hybridization probes and PCR primers) in identifying full-length genomic and coding sequences as well as in mapping exon-intron boundaries, identifying alternatively spliced transcripts, non- translated transcripts, truly unique genes, and extremely short genes. For a review, see Yuan et al, Pharmacology and Therapeutics 2001, 91:115-132. In the present application, the term "EST clone" is used to indicate the entire cloned cDNA segment of which only a portion has been initially end-sequenced to produce the "EST" or "EST sequence" that is stored in public domain sequence databases (e.g., dbEST at NCBI, available on the WorldWideWeb at ncbi.nlm.nih.gov/dbEST/). As with other public domain DNA sequences, these ESTs or EST sequences have accession numbers, and can be analyzed by sequence comparison algorithms such as BLAST, FASTA, DNA Strider, GCG, etc. The Affymetrix GeneChip^® arrays used in the Examples section below include probesets (consisting of 25 nt oligonucleotides) designed to measure mRNA levels of the gene encompassing the EST and are annotated by Affymetrix with the accession number for the relevant EST sequence. Herein, such probesets are referred to by their EST accession number (e.g. , Accession No. AI228284 for EST corresponding to rat PNPGl).

[0071] The terms "array" and "microarray" are used interchangeably and refer generally to any ordered arrangement (e.g., on a surface or substrate) of different molecules, referred to herein as "probes." Each different probe of an array is capable of specifically recognizing and/or binding to a particular molecule, which is referred to herein as its "target," in the context of arrays. Examples of typical target molecules that can be detected using microarrays include mRNA transcripts, cDNA molecules, cRNA molecules, and proteins. As disclosed in the Examples section below, at least one target detectable by the Affymetrix GeneChip^® microarray used as described herein is a PNPGl -encoding nucleic acid (such as an mRNA transcript, or a corresponding cDNA or cRNA molecule).

[0072] Microarrays are useful for simultaneously detecting the presence, absence and quantity of a plurality of different target molecules in a sample (such as an mRNA preparation isolated from a relevant cell, tissue, or organism, or a corresponding cDNA or cRNA preparation). The presence or absence of a probe's target molecule in a sample may be readily determined (and quantified) by analyzing whether (and how much of) a target has bound to a probe at a particular location on the surface or substrate. [0073] In another embodiment, arrays used in the present invention are

"addressable arrays" where each different probe is associated with a particular "address" . For example, in another embodiment where the probes are immobilized on a surface or a substrate, each different probe of the addressable array is immobilized at a particular, known location on the surface or substrate. The presence or absence of that probe's target molecule in a sample may therefore readily be determined by simply detecting whether a target has bound to that particular location on the surface or substrate.

[0074] The arrays according to the present invention are preferably nucleic acid arrays (also referred to herein as "transcript arrays" or "hybridization arrays") that comprise a plurality of nucleic acid probes immobilized on a surface or substrate. The different nucleic acid probes are complementary to, and therefore can hybridize to, different target nucleic acid molecules in a sample. Thus, such probes can be used to simultaneously detect the presence and abundance of a plurality of different nucleic acid molecules in a sample, to determine the expression of a plurality of different genes, e.g. , the presence and abundance of different mRNA molecules, or of nucleic acid molecules derived therefrom (for example, cDNA or cRNA).

[0075] There are two major types of microarray technology; spotted cDNA arrays and manufactured oligonucleotide arrays. The Examples section below describes the use of high density oligonucleotide Affymetrix GeneChip^® arrays.

[0076] The arrays are preferably reproducible, allowing multiple copies of a given array to be produced and the results from each easily compared to each other. Preferably the microarrays are small, usually smaller than 5 cm², and are made from materials that are stable under binding (e.g. , nucleic acid hybridization) conditions. A given binding site or unique set of binding sites in the microarray will specifically bind the target (e.g. , the mRNA of a single gene in the cell). Although there may be more than one physical binding site (hereinafter "site") per specific target, for the sake of clarity the discussion below will assume that there is a single site. It will be appreciated that when cDNA complementary to the RNA of a cell is made and hybridized to a microarray under suitable hybridization conditions, the level or degree of hybridization to the site in the array corresponding to any particular gene will reflect the prevalence in the cell of mRNA transcribed from that gene. For example, when detectably labeled (e.g. , with a fluorophore) cDNA complementary to the total cellular mRNA is hybridized to a microarray, the site on the array corresponding to a gene (i.e. , capable of specifically binding a nucleic acid product of the gene) that is not transcribed in the cell will have little or no signal, and a gene for which the encoded mRNA is highly prevalent will have a relatively strong signal.

[0077] By way of example, GeneChip^® expression analysis (Affymetrix, Santa Clara, CA) generates data for the assessment of gene expression profiles and other biological assays. Oligonucleotide expression arrays simultaneously and quantitatively "interrogate" thousands of mRNA transcripts (genes or ESTs), simplifying large genomic studies. Each transcript can be represented on a probe array by multiple probe pairs to differentiate among closely related members of gene families. Each probe set contains millions of copies of a specific oligonucleotide probe, permitting the accurate and sensitive detection of even low-intensity mRNA hybridization patterns. After hybridization intensity data is captured, e.g. , using optical detection systems (e.g. , a scanner), software can be used to automatically calculate intensity values for each probe cell. Probe cell intensities can be used to calculate an average intensity for each gene, which correlates with mRNA abundance levels. Expression data can be quickly sorted based on any analysis parameter and displayed in a variety of graphical formats for any selected subset of genes. Gene expression detection technologies include, among others, the research products manufactured and sold by Hewlett-Packard, Per kin-Elmer and Gene Logic. [0078] An "antisense" nucleic acid molecule or oligonucleotide is a single stranded nucleic acid molecule, which may be DNA, RNA, a DNA-RNA chimera, or a derivative thereof, which, upon hybridizing under cytoplasmic conditions with complementary bases in an RNA or DNA molecule of interest, inhibits the expression of the corresponding gene by inhibiting, e.g. , mRNA transcription, mRNA splicing, mRNA transport, or mRNA translation or by decreasing mRNA stability. As presently used, "antisense" broadly includes RNA-RNA interactions, RNA-DNA interactions, and RNase-H mediated arrest. Antisense nucleic acid molecules can be encoded by a recombinant gene for expression in a cell (see, e.g., U.S. Patents No. 5,814,500 and 5,811,234), or alternatively they can be prepared synthetically (see, e.g., U.S. Patent No. 5,780,607). According to the present invention, the involvement of PNPGl in regulation of pain may be identified, modulated and studied using antisense nucleic acids derived on the basis of PNPGl - encoding nucleic acid molecules of the invention.

[0079] The term "ribozyme" is used to refer to a catalytic RNA molecule capable of cleaving RNA substrates. Ribozyme specificity is dependent on complementary RNA-RNA interactions (for a review, see Cech and Bass, Annu. Rev. Biochem. 1986; 55: 599-629). Two types of ribozymes, hammerhead and hairpin, have been described. Each has a structurally distinct catalytic center. The present invention contemplates the use of ribozymes designed on the basis of the PNPGl -encoding nucleic acid molecules of the invention to induce catalytic cleavage of the corresponding mRNA, and in this way inhibit expression of the PNPGl gene. Ribozyme technology is described further in Intracellular Ribozyme Applications: Principals and Protocols, Rossi and Couture ed., Horizon Scientific Press, 1999.

[0080] The term "RNA interference" or "RNAi" refers to the ability of double stranded RNA (dsRNA) to suppress the expression of a specific gene of interest in a homology-dependent manner. It is currently believed that RNA interference acts post-transcriptionally by targeting mRNA molecules for degradation. RNA interference commonly involves the use of dsRNAs that are greater than 500 bp; however, it can also be mediated through small interfering RNAs (siRNAs) or small hairpin RNAs (shRNAs), which are typically greater than

18 nucleotides in length. For reviews, see Bosner and Labouesse, Nature Cell Biol.

2000; 2: E31-E36 and Sharp and Zamore, Science 2000; 287: 2431-2433. The present invention exemplifies the use of dsRNAs designed on the basis of PNPGl - encoding nucleic acid molecules of the invention in RNA interference methods to specifically inhibit PNPGl gene expression.

[0081] The term "nucleic acid hybridization" refers to anti-parallel bonding between two nucleic acids, in which A pairs with T (or U if an RNA nucleic acid) and C pairs with G. Nucleic acid molecules are "hybridizable" to each other when at least one strand of one nucleic acid molecule can anneal to a strand of another nucleic acid molecule under defined stringency conditions. Stringency of hybridization is determined, e.g. , by (i) the temperature at which hybridization and/or washing is performed, and (ii) the ionic strength and polarity (e.g. , concentration of formamide) of the hybridization and washing solutions, as well as other parameters. Hybridization requires that the two strands contain substantially complementary sequences. Depending on the stringency of hybridization, however, some degree of mismatches may be tolerated. Under "low stringency" conditions, a number of mismatches are tolerable (i.e. , will not prevent formation of an anti- parallel hybrid). As the stringency increases, hybridization can only occur where there are fewer mismatches. [0082] Typically, hybridization of two strands at high stringency requires that the sequences exhibit a high degree of complementarity over their entire length. Examples of high stringency conditions include: an aqueous solution of 0.5χSSC at 65°C (lxSSC is 0.15 M NaCl, 0.015 M Na citrate); more specifically, for example, hybridization to filter-bound DNA in 0.5 M NaHPO₄, 1% SDS, 1 mM EDTA at 65°C, and washing in O. lx SSC/0.1 % SDS at 68°C or for oligonucleotide molecules washing in 6xSSC/0.5% sodium pyrophosphate at about 37 °C (for 14 nucleotide-long oligos), at about 48°C (for about 17 nucleotide-long oligos), at about 55°C (for 20 nucleotide-long oligos), and at about 60°C (for 23 nucleotide- long oligos)). Accordingly, the term "high stringency hybridization" refers to a combination of solvent and temperature where two strands will pair to form a "hybrid" helix only if their nucleotide sequences are almost perfectly complementary (see Molecular Biology ofthe Cell, Alberts et al , 3^rd ed., New York and London: Garland Publ., 1994, Ch. 7).

[0083] Conditions of intermediate or moderate stringency (such as, for example, an aqueous solution of 2xSSC at 65°C; more specifically, for example, hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.2 x SSC/0.1 % SDS at 42°C) and low stringency (such as, for example, an aqueous solution of 2χSSC at 55°C), require correspondingly less overall complementarity between the hybridizing sequences. Specific temperature and salt conditions for any given stringency hybridization reaction depend on the concentration of the target DNA, length and specific activity of the probe and are normally determined empirically in preliminary experiments, which are routine (see Southern, J. Mol. Biol 1975; 98: 503; Sambrook et al , Molecular Cloning: A Laboratory Manual, 2^nd ed., vol. 2, ch. 9.50, CSH Laboratory Press, 1989; Ausubel et al (eds.), 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).

[0084] As used herein, the term "standard hybridization conditions" refers to hybridization conditions that allow hybridization of sequences having at least 75% sequence identity. According to a specific embodiment, hybridization conditions of higher stringency may be used to allow hybridization of sequences having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity.

[0085] Nucleic acid molecules that "hybridize" to any of the PNPG1- encoding nucleic acids of the present invention may be of any length. In one embodiment, such nucleic acid molecules are at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, and at least 70 nucleotides in length. In another embodiment, nucleic acid molecules that hybridize are, or are about, the same length as the particular PNPGl -encoding nucleic acid.

[0086] The term "homologous" as used in the art commonly refers to the relationship between nucleic acid molecules or proteins that possess a "common evolutionary origin," including nucleic acid molecules or proteins within super families (e.g. , the immunoglobulin superfamily) and homologous nucleic acid molecules or proteins from different species (Reeck et al , Cell 1987; 50: 667). Such nucleic acid molecules or proteins have sequence homology, as reflected by their sequence similarity, whether in terms of substantial percent similarity or the presence of specific residues or motifs at conserved positions.

[0087] The terms "percent (%) sequence similarity", "percent (%) sequence identity", and the like, generally refer to the degree of identity or correspondence between different nucleotide sequences of nucleic acid molecules or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al , supra). Sequence identity can be determined using any of a number of publicly available sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin), etc.

[0088] To determine the percent identity between two amino acid sequences or two nucleic acid molecules, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e. , percent identity = number of identical positions/total number of positions (e.g. , overlapping positions) x 100). In one embodiment, the two sequences are approximately of the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent sequence identity, typically exact matches are counted.

[0089] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl Acad. Sci. USA 1990, 87:2264, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1993, 90:5873- 5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al , J. Mol. Biol. 1990; 215: 403. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to sequences of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to protein sequences of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. 1997, 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationship between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g. , XBLAST and NBLAST) can be used. See ncbi.nlm.nih.gov/BLAST/ on the WorldWideWeb. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS 1988; 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0090] In another embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, J. Mol. Biol 1970, 48:444-453 algorithm which has been incorporated into the GAP program in the GCG software package (Accelrys, Burlington, MA; available at accelrys.com on the WorldWideWeb), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) is using a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. [0091] In addition to the nucleic acid sequences encoding rat, mouse and human PNPGl proteins (as shown in SEQ ID NOS: 1, 3 and 5, respectively), the present invention further provides nucleic acid molecules comprising nucleotide sequences having certain percentage sequence identities to any of the aforementioned sequences. Such sequences preferably hybridize under conditions of moderate or high stringency as described above, and may include species orthologs.

[0092] As used herein, the term "orthologs" refers to genes in different species that apparently evolved from a common ancestral gene by speciation.

Normally, orthologs retain the same function through the course of evolution.

Identification of orthologs can provide reliable prediction of gene function in newly sequenced genomes. Sequence comparison algorithms that can be used to identify orthologs include without limitation BLAST, FASTA, DNA Strider, and the GCG pileup program. Orthologs often have high sequence similarity, as can be seen in the case of rat, mouse and human PNPGl proteins of the present invention. As shown in Figure 4 using ClustalW Vector NTI algorithm (InforMax™, Invitrogen Life Science Software, Frederick, MD), the rat PNPGl protein sequence is 97% identical to the mouse PNPGl protein sequence and is 89% identical to the human PNPGl protein sequence. The present invention encompasses all orthologs of PNPGl. In addition to rat, mouse and human orthologs, particularly useful PNPGl orthologs of the present invention are monkey and porcine orthologs. [0093] As used herein, the term "isolated" means that the material being referred to has been removed from the environment in which it is naturally found, and is characterized to a sufficient degree to establish that it is present in a particular sample. Such characterization can be achieved by any standard technique, such as, e.g. , sequencing, hybridization, immunoassay, functional assay, expression, or the like. Thus, a biological material can be "isolated" if it is free of cellular components, i.e. , components of the cells in which the material is found or produced in nature. For nucleic acid molecules, an "isolated" nucleic acid molecule or an "isolated" oligonucleotide, can be a PCR product, an mRNA transcript, a cDNA molecule, or a restriction fragment. A nucleic acid molecule excised from the chromosome that it is naturally a part of is considered to be isolated. Such a nucleic acid molecule may or may not remain joined to regulatory, or non- regulatory, or non-coding regions, or to other regions located upstream or downstream of the gene when found in the chromosome. Nucleic acid molecules that have been spliced into vectors such as plasmids, cosmids, artificial chromosomes, phages and the like are considered isolated. In a particular embodiment, a PNPGl -encoding nucleic acid spliced into a recombinant vector, and/or transformed into a host cell, is considered to be "isolated".

[0094] Isolated nucleic acid molecules of the present invention do not encompass uncharacterized clones in man-made genomic or cDNA libraries. [0095] A protein that is associated with other proteins and/or nucleic acids with which it is associated in an intact cell, or with cellular membranes if it is a membrane-associated protein, is considered isolated if it has otherwise been removed from the environment in which it is naturally found and is characterized to a sufficient degree to establish that it is present in a particular sample. A protein expressed from a recombinant vector in a host cell, particularly in a cell in which the protein is not naturally expressed, is also regarded as isolated.

[0096] An isolated organelle, cell, or tissue is one that has been removed from the anatomical site (cell, tissue or organism) in which it is found in the source organism. An isolated material may or may not be "purified" .

[0097] The term "purified" as used herein refers to a material (e.g. , a nucleic acid molecule or a protein) that has been isolated under conditions that detectably reduce or eliminate the presence of other contaminating materials. Contaminants may or may not include native materials from which the purified material has been obtained. A purified material preferably contains less than about 90%, less than about 75%, less than about 50%, less than about 25%, less than about 10% , less than about 5%, or less than about 2% by weight of other components with which it was originally associated.

[0098] Methods for purification are well-known in the art. For example, nucleic acids can be purified by precipitation, chromatography (including preparative solid phase chromatography, oligonucleotide hybridization, and triple helix chromatography), ultracentrifugation, and other means. Polypeptides can be purified by various methods including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reverse-phase HPLC, gel filtration, affinity chromatography, ion exchange and partition chromatography, precipitation and salting-out chromatography, extraction, and counter-current distribution. Cells can be purified by various techniques, including centrifugation, matrix separation

(e.g., nylon wool separation), panning and other immunoselection techniques, depletion (e.g., complement depletion of contaminating cells), and cell sorting (e.g., fluorescence activated cell sorting (FACS)). Other purification methods are possible. The term "substantially pure" indicates the highest degree of purity that can be achieved using conventional purification techniques known in the art. In the context of analytical testing of the material, "substantially free" means contaminants, if present, are below the limits of detection, or are detected at levels that are low enough to be acceptable in the relevant art, for example, no more than about 2-5% (w/w). Accordingly, with respect to the purified material, the term "substantially pure" or "substantially free" means that the purified material being referred to is present in a composition where it represents 95% (w/w) or more of the weight of that composition. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, or any other appropriate method known in the art.

[0099] The terms "about" and "approximately" mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, z^'.e. , the limitations of the measurement system. For example, "about" can mean within an acceptable standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to ±20%, preferably up to + 10%, more preferably up to +5% , and more preferably still up to + 1 % of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term "about" is implicit and in this context means within an acceptable error range for the particular value.

[00100] The term "modulator" refers to a compound that differentially affects the expression or activity of a gene or gene product (e.g. , nucleic acid molecule or protein), for example, in response to a stimulus that normally activates or represses the expression or activity of that gene or gene product when compared to the expression or activity of the gene or gene product not contacted with the stimulus. In one embodiment, the gene and gene product the expression or activity of which is being modulated includes a gene, cDNA molecule or mRNA transcript that encodes a mammalian PNPGl protein such as, e.g. , a rat, mouse, companion animal, or human PNPGl protein. Examples of modulators of the PNPGl -encoding nucleic acids of the present invention include without limitation antisense nucleic acids, ribozymes, and RNAi oligonucleotides.

[00101] A "test compound" is a molecule that can be tested for its ability to act as a modulator of a gene or gene product. Test compounds can be selected without limitation from small inorganic and organic molecules (i.e. , those molecules of less than about 2 kD, and more preferably less than about 1 kD in molecular weight), polypeptides (including native ligands, antibodies, antibody fragments, and other immunospecific molecules), oligonucleotides and nucleic acid molecules. In various embodiments of the present invention, a test compound is tested for its ability to modulate the expression of a mammalian PNPGl -encoding nucleic acid or PNPGl protein or bind to a mammalian PNPGl protein. A compound that modulates a nucleic acid or protein of interest is designated herein as a "candidate compound" or "lead compound" suitable for further testing and development. Candidate compounds include, but are not necessarily limited to, the functional categories of agonist and antagonist.

[00102] An "agonist" is defined herein as a compound that interacts with

(e.g. , binds to) a nucleic acid molecule or protein, and promotes, enhances, stimulates or potentiates the biological expression or function of the nucleic acid molecule or protein. The term "partial agonist" is used to refer to an agonist which interacts with a nucleic acid molecule or protein, but promotes only partial function of the nucleic acid molecule or protein. A partial agonist may also inhibit certain functions of the nucleic acid molecule or protein with which it interacts. An "antagonist" interacts with (e.g. , binds to) and inhibits or reduces the biological expression or function of the nucleic acid molecule or protein.

[00103] The phrase "similar or identical expression" (and the like) as used herein refers to an expression level of a PNPGl gene or gene product (i.e. , an mRNA transcript or protein) in a first cell that is + 30%, preferably +20% , and more preferably +10% of a given numerical value of the expression level of the same PNPGl gene or gene product from a second comparator (or control) cell as determined by any quantitative assay known in the art. Preferably, the second cell is either the same type of cell, and preferably from the same type of tissue, as the first cell, or the second cell is from the same cell line as the first cell. This second cell is also referred to herein as a "control cell" or "corresponding cell". The first and second cells are preferably, but need not be, otherwise incubated and treated under the same conditions.

[00104] The term "detectable change" as used herein in relation to an expression level of a gene or gene product (e.g. , PNPGl) means any statistically significant change and preferably at least a 1.5-fold change as measured by any available technique such as hybridization or quantitative PCR.

[00105] As used herein, the term "specific binding" refers to the ability of one molecule, typically an antibody, nucleic acid, polypeptide, or a small molecule ligand to contact and associate with another specific molecule, even in the presence of many other diverse molecules. "Immunospecific binding" refers to the ability of an antibody to specifically bind to (or to be "specifically immunoreactive with") its corresponding antigen.

[00106] "Amplification" of DNA as used herein denotes the use of exponential amplification techniques known in the art such as the polymerase chain reaction (PCR), and non-exponential amplification techniques such as linked linear amplification, that can be used to increase the concentration of a particular DNA sequence present in a mixture of DNA sequences. For a description of PCR, see Saiki et al, Science 1988, 239:487 and U.S. Patent No. 4,683,202. For a description of linked linear amplification, see U.S. Patent Nos. 6,335,184 and 6,027,923; Reyes et al , Clinical Chemistry 2001; 47: 131-40; and Wu et al , Genomics 1989; 4: 560-569.

[00107] As used herein, the phrase "sequence-specific oligonucleotides" refers to oligonucleotides that can be used to detect the presence of a specific nucleic acid molecule, or that can be used to amplify a particular segment of a specific nucleic acid molecule for which a template is present. Such oligonucleotides are also referred to as "primers" or "probes." In a specific embodiment, "probe" is also used to refer to an oligonucleotide, for example about 25 nucleotides in length, attached to a solid support for use on "arrays" and "microarrays" described below.

[00108] The term "host cell" refers to any cell of any organism that is selected, modified, transformed, grown, used or manipulated in any way so as, e.g. , to clone a recombinant vector that has been transformed into that cell, or to express a recombinant protein such as, e.g. , a PNPGl protein of the present invention. Host cells are useful in screening and other assays, as described below.

[00109] As used herein, the terms "transfected cell" and "transformed cell" both refer to a host cell that has been genetically modified to express or over- express a nucleic acid encoding a specific gene product of interest such as, e.g. , a PNPGl protein or a fragment thereof. Any eukaryotic or prokaryotic cell can be used, although eukaryotic cells are preferred, vertebrate cells are more preferred, and mammalian cells are the most preferred. In the case of multi-subunit ion channels, nucleic acids encoding the several subunits are preferably co-expressed by the transfected or transformed cell to form a functional channel. Transfected or transformed cells are suitable to conduct an assay to screen for compounds that modulate the function of the gene product. A typical "assay method" of the present invention makes use of one or more such cells, e.g., in a micro well plate or some other culture system, to screen for such compounds. The effects of a test compound can be determined on a single cell, or on a membrane fraction prepared from one or more cells, or on a collection of intact cells sufficient to allow measurement of activity.

[00110] The term "recombinantly engineered cell" refers to any prokaryotic or eukaryotic cell that has been genetically manipulated to express or over-express a nucleic acid of interest, e.g. , a PNPGl-encoding nucleic acid of the present invention, by any appropriate method, including transfection, transformation or transduction. The term "recombinantly engineered cell" also refers to a cell that has been engineered to activate an endogenous nucleic acid, e.g. , the endogenous PNPGl-encoding gene in a rat, mouse or human cell, which cell would not normally express that gene product or would express the gene product at only a sub- optimal level.

[00111] The terms "vector", "cloning vector" and "expression vector" refer to recombinant constructs including, e.g. , plasmids, cosmids, phages, and the like, with which a nucleic acid molecule (e.g., a PNPGl-encoding nucleic acid or PNPGl siRNA-expressing nucleic acid) can be introduced into a host cell so as to, e.g. , clone the vector or express the introduced nucleic acid molecule. Vectors may further comprise selectable markers.

[00112] The terms "mutant", "mutated" , "mutation" , and the like, refer to any detectable change in genetic material, (e.g., DNA), or any process, mechanism, or result of such a change. Mutations include gene mutations in which the structure (e.g., DNA sequence) of the gene is altered; any DNA or other nucleic acid molecule derived from such a mutation process; and any expression product (e.g., the encoded protein) exhibiting a non-silent modification as a result of the mutation.

[00113] As used herein, the term "genetically modified animal" encompasses all animals into which an exogenous genetic material has been introduced and/or whose endogenous genetic material has been manipulated. Examples of genetically modified animals include without limitation transgenic animals, e.g. , "knock-in" animals with the endogenous gene substituted with a heterologous gene or an ortholog from another species or a mutated gene, "knockout" animals with the endogenous gene partially or completely inactivated, or transgenic animals expressing a mutated gene or overexpressing a wild-type or mutated gene (e.g. , upon targeted or random integration into the genome) and animals containing cells harboring a non-integrated nucleic acid construct (e.g. , viral-based vector, antisense oligonucleotide, shRNA, siRNA, ribozyme, etc.), including animals wherein the expression of an endogenous gene has been modulated (e.g. , increased or decreased) due to the presence of such construct.

[00114] As used herein, a "transgenic animal" is a nonhuman animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include nonhuman primates, sheep, dogs, pigs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a nonhuman animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g. , an embryonic cell of the animal, prior to development of the animal.

[00115] A "knock-in animal" is an animal (e.g., a mammal such as a mouse or a rat) in which an endogenous gene has been substituted in part or in total with a heterologous gene (i.e. , a gene that is not endogenous to the locus in question; see Roamer et al , New Biol. 1991, 3:331). This can be achieved by homologous recombination (see "knockout animal" below), transposition (Westphal and Leder, Curr. Biol. 1997; 7: 530), use of mutated recombination sites (Araki et al , Nucleic Acids Res. 1997; 25: 868), PCR (Zhang and Henderson, Biotechniques 1998; 25: 784), or any other technique known in the art. The heterologous gene may be, e.g. , a reporter gene linked to the appropriate (e.g. , endogenous) promoter, which may be used to evaluate the expression or function of the endogenous gene (see, e.g. , Elegant et al , Proc. Natl Acad. Sci. USA 1998; 95: 11897).

[00116] A "knockout animal" is an animal (e.g., a mammal such as a mouse or a rat) that has had a specific gene in its genome partially or completely inactivated by gene targeting (see, e.g., U.S. Patents Nos. 5,777,195 and 5,616,491). A knockout animal can be a heterozygous knockout (i.e. , with one defective allele and one wild type allele) or a homozygous knockout (i.e. , with both alleles rendered defective). Preparation of a knockout animal typically requires first introducing a nucleic acid construct (a "knockout construct"), that will be used to decrease or eliminate expression of a particular gene, into an undifferentiated cell type termed an embryonic stem (ES) cell. The knockout construct is typically comprised of: (i) DNA from a portion (e.g. , an exon sequence, intron sequence, promoter sequence, or some combination thereof) of a gene to be knocked out; and (ii) a selectable marker sequence used to identify the presence of the knockout construct in the ES cell. The knockout construct is typically introduced (e.g. , electroporated) into ES cells so that it can homologously recombine with the genomic DNA of the cell in a double crossover event. This recombined ES cell can be identified (e.g. , by Southern hybridization or PCR reactions that show the genomic alteration) and is then injected into a mammalian embryo at the blastocyst stage. In another embodiment where the knockout animal is a mammal, a mammalian embryo with integrated ES cells is then implanted into a foster mother for the duration of gestation (see, e.g. , Zhou et al , Genes andDev. 1995; 9: 2623- 34).

[00117] The phrases "disruption of the gene", "gene disruption", and the like, refer to: (i) insertion of a different or defective nucleic acid sequence into an endogenous (naturally occurring) DNA sequence, e.g. , into an exon or promoter region of a gene; or (ii) deletion of a portion of an endogenous DNA sequence of a gene; or (iii) a combination of insertion and deletion, so as to decrease or prevent the expression of that gene in the cell as compared to the expression of the endogenous gene sequence.

[00118] In accordance with the present invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular

Cloning: A Laboratory Manual, 2^nd ed. , Cold Spring Harbor Laboratory Press, Cold

Spring Harbor, New York, 1989 (herein "Sambrook et al, 1989"); DNA Cloning:

A Practical Approach, Volumes I and II (Glover ed. 1985); Oligonucleotide Synthesis (Gait ed. 1984); Nucleic Acid Hybridization (Hames and Higgins eds.

1985); Transcription And Translation (Hames and Higgins eds. 1984); Animal Cell

Culture (Freshney ed. 1986); Immobilized Cells And Enzymes (IRL Press, 1986); B.

Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel et al. eds.,

Current Protocols in Molecular Biology, John Wiley and Sons, Inc. 1994; among others. 5.2. Nucleic Acid Molecules

[00119] The present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a mammalian PNPGl protein. More particularly, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a rat, mouse or human PNPGl protein. In one embodiment, the rat PNPGl protein comprises the amino acid sequence of SEQ ID NO:2. In another embodiment, the mouse PNPGl protein comprises the amino acid sequence of SEQ ID NO:4. In another embodiment, the human PNPGl protein comprises the amino acid sequence of SEQ ID NO:6.

[00120] In one embodiment, the amino acid sequence of the rat PNPGl protein is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: l (cDNA sequence), or a degenerate variant thereof. In another embodiment, the amino acid sequence of the rat PNPGl protein is encoded by a nucleic acid molecule comprising the nucleotide sequence of the rat PNPGl gene (SEQ ID NO: 11), or a PNPGl-encoding portion thereof, or a degenerate variant thereof. The present invention also provides an isolated single-stranded nucleic acid molecule comprising a nucleotide sequence that is the complement of a nucleotide sequence of one strand of any of the aforementioned nucleotide sequences.

[00121] In one embodiment, the amino acid sequence of the mouse PNPGl protein is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 3 (cDNA sequence), or a degenerate variant thereof. In another embodiment, the amino acid sequence of the mouse PNPGl protein is encoded by a nucleic acid molecule comprising the nucleotide sequence of the mouse PNPGl gene (SEQ ID NO: 12), or a PNPGl-encoding portion thereof, or a degenerate variant thereof. The present invention also provides an isolated single-stranded nucleic acid molecule comprising a nucleotide sequence that is the complement of a nucleotide sequence of one strand of any of the aforementioned nucleotide sequences.

[00122] In one embodiment, the amino acid sequence of the human PNPGl protein is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 5 (cDNA sequence), or a degenerate variant thereof. In another embodiment, the amino acid sequence of the human PNPGl protein is encoded by a nucleic acid molecule comprising the nucleotide sequence of the human PNPGl gene (SEQ ID NO: 13) or a PNPGl-encoding portion thereof, or a degenerate variant thereof. The present invention also provides an isolated single-stranded nucleic acid molecule comprising a nucleotide sequence that is the complement of a nucleotide sequence of one strand of any of the aforementioned nucleotide sequences.

[00123] In an alternative embodiment, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a mammalian PNPGl protein, with the proviso that the nucleotide sequence does not comprise the nucleotide sequence consisting of SEQ ID NO: 3 or SEQ ID NO: 5. In another embodiment, the mammalian PNPGl protein encoded by the isolated nucleic acid molecule does not comprise the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO:6.

[00124] The present invention further provides an isolated nucleic acid molecule comprising a nucleotide sequence that hybridizes to the complement of a nucleic acid molecule comprising a nucleotide sequence that encodes the amino acid sequence of the rat, mouse or human PNPGl protein of the present invention, under moderately stringent conditions, such as, for example, an aqueous solution of 2xSSC at 65°C; more specifically, for example, hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.2 x SSC/0.1 % SDS at 42°C (see the Definitions section above). In a another embodiment, the homologous nucleic acid molecule hybridizes to the complement of a nucleic acid molecule comprising a nucleotide sequence that encodes the amino acid sequence of the rat, mouse or human PNPGl protein of the present invention under highly stringent conditions, such as, for example, in an aqueous solution of 0.5xSSC at 65°C; more specifically, for example, hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS 1 mM EDTA at 65°C, and washing in 0.1.x SSC/0.1% SDS at 68°C (see the Definitions Section 5.1., above). In another embodiment, the homologous nucleic acid molecule hybridizes under highly stringent conditions to the complement of a nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO: 3 and SEQ ID NO:5. In another embodiment, the nucleic acid molecule hybridizes under highly stringent conditions to the complement of a nucleic acid molecule consisting of a nucleotide sequence encoding the amino acid of SEQ ID NO:2, and encodes a protein having at least one characteristic of the rat PNPGl protein. In another embodiment, the nucleic acid molecule hybridizes under highly stringent conditions to the complement of a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:l, and encodes a protein having at least one characteristic of the rat PNPGl protein.

[00125] The present invention further provides an isolated nucleic acid molecule comprising a nucleotide sequence that is homologous to the nucleotide sequence of a PNPGl-encoding nucleic acid molecule of the present invention. In another embodiment, such nucleic acid molecule hybridizes under standard conditions to the complement of a nucleic acid molecule comprising a nucleotide sequence that encodes the amino acid sequence of the rat, mouse or human PNPGl protein of the present invention and has at least 75% sequence identity, or at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to the nucleotide sequence of such PNPGl-encoding nucleic acid molecule (e.g. , as determined by a sequence comparison algorithm selected from BLAST, FASTA, DNA Strider, and GCG, and preferably as determined by the BLAST program from the National Center for Biotechnology Information (NCBI-Version 2.2), available on the WorldWideWeb at ncbi.nlm.nih.gov/BLAST/). In another embodiment, the homologous nucleic acid molecule comprises a nucleotide sequence that has at least 95% sequence identity, or at least 98% sequence identity, or at least 99%, sequence identify to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2, and encodes a protein having at least one characteristic of the rat PNPGl protein. In another embodiment, the homologous nucleic acid molecule comprises a nucleotide sequence that has at least 95% sequence identity, or at least 98% sequence identify, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: l, and encodes a protein having at least one characteristic of the rat PNPGl protein. [00126] The present invention further provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to a rat, mouse or human PNPGl protein of the present invention. Preferably, the polypeptides that are homologous to a PNPGl protein of the present invention have the amino acid sequence identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO: 4 or SEQ ID NO: 6, but have one or more amino acid residues conservatively substituted with a different amino acid residue. Conservative amino acid substitutions are well-known in the art. Rules for making such substitutions include those described by Dayhof, 1978, Nat. Biomed. Res. Found. , Washington, D.C , Vol. 5, Sup. 3, among others. More specifically, conservative amino acid substitutions are those that take place within a family of amino acids that are related in acidity, polarity, or bulkiness of their side chains. Genetically encoded amino acids are generally divided into four groups: (1) acidic =aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) non- polar =alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are also jointly classified as aromatic amino acids. One or more replacements within any particular group, e.g. , of a leucine with an isoleucine or valine, or of an aspartate with a glutamate, or of a threonine with a serine, or of any other amino acid residue with a structurally related amino acid residue, e.g. , an amino acid residue with similar acidity, polarity, bulkiness of side chain, or with similarity in some combination thereof, will generally have an insignificant effect on the function or immunogenicity of the polypeptide.

[00127] In one embodiment, the homologous polypeptide has at least about

75%, sequence identify, at least about 80% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, or at least about 99% sequence identity to SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 (e.g. , as determined by a sequence comparison algorithm selected from BLAST, FASTA, DNA Strider, and GCG, and preferably as determined by the BLAST program from the National Center for Biotechnology Information (NCBI-Version 2.2), available at ncbi.nlm.nih.gov/BLAST/ on the WorldWideWeb). In another embodiment, the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 has been conservatively substituted at one, two, three, four or five non-conserved amino acid residue positions. In another embodiment, the homologous polypeptide has at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:2, and exhibits at least one characteristic of the rat PNPGl protein.

[00128] Also encompassed by the present invention are orthologs of the specifically disclosed PNPGl proteins and PNPGl-encoding nucleic acids. Additional PNPGl orthologs can be identified based on the sequences of rat, mouse and human orthologs disclosed herein, using standard sequence comparison algorithms such as BLAST, FASTA, DNA Strider, GCG, etc. In addition to rat, mouse and human orthologs, particularly useful PNPGl orthologs of the present invention are monkey and porcine orthologs.

[00129] The present invention further provides a nucleic acid molecule consisting of a nucleotide sequence that is a substantial portion of the nucleotide sequence of any of the aforementioned PNPGl -related nucleic acid molecules of the present invention, or the complement of such nucleotide sequence. As used herein, a "substantial portion" of a PNPGl-encoding nucleotide sequence means a nucleotide sequence that is less than the nucleotide sequence required to encode a complete PNPGl protein of the present invention, but comprising at least about 5 % , at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50% , at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the contiguous nucleotide sequence of a PNPGl-encoding nucleic acid molecule of the present invention. Such nucleic acid molecules can be used for a variety of purposes including, e.g. , to express a portion of a PNPGl protein of the present invention in an appropriate expression system, or for use in conducting an assay to determine the expression level of a PNPGl gene in a biological sample, or to amplify a PNPGl-encoding nucleic acid molecule. [00130] In addition to the nucleotide sequences of any of the aforementioned

PNPGl -related nucleic acid molecules, _t nucleic acid molecules of the present invention can further comprise, or alternatively may consist of, nucleotide sequences selected from those sequences that naturally flank a PNPGl-encoding nucleotide sequence in the chromosome, including regulatory sequences.

[00131] The nucleic acid molecules encompassed by the present invention exclude uncharacterized clones in man-made genomic or cDNA libraries.

5.3. Oligonucleotides

[00132] The present invention further provides an oligonucleotide molecule that hybridizes to a nucleic acid molecule of the present invention, or that hybridizes to a nucleic acid molecule having a nucleotide sequence that is the complement of a nucleotide sequence of a nucleic acid molecule of the present invention. Such an oligonucleotide molecule: (i) is about 10 nucleotides to about 200 nucleotides in length, preferably from about 15 to about 100 nucleotides in length, and more preferably about 20 to about 50 nucleotides in length, and (ii) hybridizes to one or more of the nucleic acid molecules of the present invention under highly stringent conditions (such as, e.g. , washing in 6x SSC/0.5% sodium pyrophosphate at about

37°C for about 14-base oligos, at about 48°C for about 17-base oligos, at about

55°C for about 20-base oligos, and at about 60°C for about 23-base oligos). In one embodiment, an oligonucleotide molecule of the present invention is 100% complementary to a portion of at least one of the aforementioned nucleic acid molecules of the present invention.

[00133] Specific non-limiting examples of oligonucleotide molecules according to the present invention include oligonucleotide molecules selected from the group consisting of SEQ ID NOS: 14-30 (listed in Tables 2 and 3, below).

[00134] Oligonucleotide molecules of the present invention are useful for a variety of purposes, including as primers in amplification of a PNPGl-encoding nucleic acid molecule for use, e.g. , in differential diagnoses relating to pain conditions, or to encode or act as inhibitory molecules (e.g. , as antisense or short inhibitory (si) RNA molecules) useful in regulating expression of the PNPGl gene product, or to identify PNPGl orthologs in other species. Regarding diagnostics, suitably designed oligonucleotide primers (e.g. , PCR primers) and hybridization probes can be used to detect the presence and quantity of a PNPGl-specific nucleic acid molecule in a biological sample (e.g. , tissue or fluid, such as central nervous system (CNS) or peripheral nervous system (PNS) tissue, lung tissue, placental tissue, blood, cerebrospinal fluid, mucous, urine, amniotic fluid, etc) collected from a subject. According to the methods of the present invention, detection of particular levels of a PNPGl-encoding nucleic acid in a sample can be used for a diagnosis of a pain state in a subject. In addition, such a diagnostic approach can be used to monitor the efficacy of an analgesic treatment or to determine the ability of a compound to modulate expression of the PNPGl-encoding nucleic acid.

[00135] Oligonucleotide molecules can be labeled, e.g., with radioactive labels (e.g. , T^P), biotin, fluorescent labels, etc. In one embodiment, a labeled oligonucleotide molecule can be used as a probe to detect the presence of a nucleic acid. In another embodiment, two oligonucleotide molecules (one or both of which may be labeled) can be used as PCR primers, either for cloning a full-length nucleic acid or a fragment of a nucleic acid encoding a gene product of interest, or to detect the presence of nucleic acids encoding a gene product. Methods for conducting amplifications, such as the polymerase chain reaction (PCR), are described, among other places, in Saiki et al, Science 1988, 239:487 and U.S. Patent No. 4,683,202. Other amplification techniques known in the art, e.g., the ligase chain reaction, can alternatively be used (see, e.g. , U.S. Patent Nos. 6,335,184 and 6,027,923; Reyes et al , Clinical Chemistry 2001; 47: 131-40; and Wu et al , Genomics 1989; 4: 560- 569).

[00136] In a further embodiment, an oligonucleotide molecule of the present invention can form a triple helix with a PNPGl-encoding nucleic acid molecule, thereby inhibiting PNPGl expression. In still another embodiment, an oligonucleotide molecule can be a short interfering or small hairpin RNA, or an antisense oligonucleotide, useful to inhibit expression of the PNPGl gene. Generally, oligonucleotide molecules are prepared synthetically, preferably on a nucleic acid synthesizer, and may be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, where appropriate. 5.4. Recombinant Expression Systems 5.4.1. Cloning and Expression Vectors

[00137] The present invention further provides compositions and constructs for cloning and expressing any of the nucleic acid molecules of the present invention, including cloning vectors, expression vectors, transformed host cells comprising any of said vectors, and novel strains or cell lines derived therefrom. In one embodiment, the present invention provides a recombinant vector comprising a nucleic acid molecule having a nucleotide sequence encoding a mammalian PNPGl protein. In one embodiment, the mammalian PNPGl protein is a rat, mouse or human PNPGl protein. In a specific embodiment, the rat PNPGl protein comprises the amino acid sequence of SEQ ID NO:2. In another specific embodiment, the mouse PNPGl protein comprises the amino acid sequence of SEQ ID NO:4. In another specific embodiment, the human PNPGl protein comprises an amino acid sequence of SEQ ID NO:6.

[00138] In one specific non-limiting embodiment, the present invention provides plasmid pPNPGl (ATCC Accession No.PTA-5617; deposited with the American Type Culture Collection (ATCC) at 10801 University Boulevard, Manassas, VA 20110-2209, USA on October 24, 2003), which comprises a nucleic acid molecule having a nucleotide sequence encoding the rat PNPGl protein. The rat PNPGl sequence was obtained by fully sequencing Invitrogen Rat UI EST clone UI-R-BJ2-bor-e-06-0-UI (Invitrogen Cat.#99002). This EST clone came out of government-sponsored research: the I.M.A.G.E. Consortium at Washington University (see http://image.llnl.gov/). It was this same clone that was deposited with the ATCC (the backbone vector is pT7T3-Pac, Invitrogen).

[00139] Recombinant vectors of the present invention, particularly expression vectors, are preferably constructed so that the coding sequence for the nucleic acid molecule of the present invention is in operative association with one or more regulatory elements necessary for transcription and translation of the coding sequence to produce a polypeptide. As used herein, the term "regulatory element" includes but is not limited to nucleotide sequences that encode inducible and non- inducible promoters, enhancers, operators and other elements known in the art that serve to drive and/or regulate expression of nucleic acid coding sequences. Also, as used herein, the coding sequence is in operative association with one or more regulatory elements where the regulatory elements effectively regulate and allow for the transcription of the coding sequence or the translation of its mRNA, or both.

[00140] Methods are known in the art for constructing recombinant vectors containing particular coding sequences in operative association with appropriate regulatory elements, and these can be used to practice the present invention. These methods include in vitro recombinant techniques, synthetic techniques, and in vivo genetic recombination. See, e.g. , the techniques described in Ausubel et al , 1989, above; Sambrook et al. , 1989, above; Saiki et al , 1988, above; Reyes et al , 2001, above; Wu et al , 1989, above; U.S. Patent Nos. 4,683,202; 6,335,184 and 6,027,923.

[00141] A variety of expression vectors are known in the art that can be utilized to express a nucleic acid molecule of the present invention, including recombinant bacteriophage DNA, plasmid DNA, and cosmid DNA expression vectors containing the particular coding sequences. Typical prokaryotic expression vector plasmids that can be engineered to contain a nucleic acid molecule of the present invention include pUC8, pUC9, pBR322 and pBR329 (Biorad Laboratories, Richmond, CA), pPL and pKK223 (Pharmacia, Piscataway, NJ), pQE50 (Qiagen, Chatsworth, CA), and pGEM-T EASY (Promega, Madison, WI), among many others. Typical eukaryotic expression vectors that can be engineered to contain a nucleic acid molecule of the present invention include an ecdysone-inducible mammalian expression system (Invitrogen, Carlsbad, CA), cytomegalovirus promoter-enhancer-based systems (Promega, Madison, WI; Stratagene, La Jolla, CA; Invitrogen), and baculovirus-based expression systems (Promega), among many others. [00142] The regulatory elements of these and other vectors can vary in then- strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements can be used. For instance, when cloning in mammalian cell systems, promoters isolated from the genome of mammalian cells, e.g. , mouse metallothionein promoter, or from viruses that grow in these cells, e.g. , vaccinia virus 7.5 K promoter or Maloney murine sarcoma virus long terminal repeat, can be used. Promoters obtained by recombinant DNA or synthetic techniques can also be used to provide for transcription of the inserted sequence. In addition, expression from certain promoters can be elevated in the presence of particular inducers, e.g. , zinc and cadmium ions for metallothionein promoters. Non-limiting examples of transcriptional regulatory regions or promoters include for bacteria, the β-gal promoter, the T7 promoter, the TAC promoter, λ left and right promoters, trp and lac promoters, trp-lac fusion promoters, etc.; for yeast, glycolytic enzyme promoters, such as ADH-I and -II promoters, GPK promoter, PGI promoter, TRP promoter, etc.; and for mammalian cells, SV40 early and late promoters, and adenovirus major late promoters, among others.

[00143] Specific initiation signals are also required for sufficient translation of inserted coding sequences. These signals typically include an ATG initiation codon and adjacent sequences. In cases where the nucleic acid molecule of the present invention, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translation control signals may be needed. However, in cases where only a portion of a coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, may be required. These exogenous translational control signals and initiation codons can be obtained from a variety of sources, both natural and synthetic. Furthermore, the initiation codon must be in-phase with the reading frame of the coding regions to ensure in-frame translation of the entire insert.

[00144] Expression vectors can also be constructed that will express a fusion protein comprising a protein or polypeptide of the present invention. Such fusion proteins can be used, e.g. , to raise anti-sera against a PNPGl protein, to study the biochemical properties of the PNPGl protein, to engineer a variant of a PNPGl protein exhibiting different immunological or functional properties, or to aid in the identification or purification, or to improve the stability, of a recombinantly- expressed PNPGl protein. Possible fusion protein expression vectors include but are not limited to vectors incorporating sequences that encode β-galactosidase and trpE fusions, maltose-binding protein fusions, glutathione-S-transferase fusions and polyhistidine fusions (carrier regions). Methods known in the art can be used to construct expression vectors encoding these and other fusion proteins.

[00145] The fusion protein can be useful to aid in purification of the expressed protein. In non-limiting embodiments, e.g. , a PNPGl-maltose-binding fusion protein can be purified using amylose resin; a PNPGl -glutathione-S- transferase fusion protein can be purified using glutathione-agarose beads; and a PNPGl -polyhistidine fusion protein can be purified using divalent nickel resin. Alternatively, antibodies against a carrier protein or peptide can be used for affinity chromatography purification of the fusion protein. For example, a nucleotide sequence coding for the target epitope of a monoclonal antibody can be engineered into the expression vector in operative association with the regulatory elements and situated so that the expressed epitope is fused to a PNPGl protein of the present invention. In a non-limiting embodiment, a nucleotide sequence coding for the FLAG™ epitope tag (International Biotechnologies Inc.), which is a hydrophilic marker peptide, can be inserted by standard techniques into the expression vector at a point corresponding, e.g. , to the amino or carboxyl terminus of the PNPGl protein. The expressed PNPGl protein-FLAG™ epitope fusion product can then be detected and affinity-purified using commercially available anti-FLAG™ antibodies. The expression vector can also be engineered to contain polylinker sequences that encode specific protease cleavage sites so that the expressed PNPGl protein can be released from a carrier region or fusion partner by treatment with a specific protease. For example, the fusion protein vector can include a nucleotide sequence encoding a thrombin or factor Xa cleavage site, among others.

[00146] A signal sequence upstream from, and in reading frame with, the

PNPGl coding sequence can be engineered into the expression vector by known methods to direct the trafficking and secretion of the expressed protein. Non- limiting examples of signal sequences include those from α-factor, immunoglobulins, outer membrane proteins, penicillinase, and T-cell receptors, among others.

[00147] To aid in the selection of host cells transformed or transfected with a recombinant vector of the present invention, the vector can be engineered to further comprise a coding sequence for a reporter gene product or other selectable marker. Such a coding sequence is preferably in operative association with the regulatory elements, as described above. Reporter genes that are useful in practicing the invention are known in the art, and include those encoding chloramphenicol acetyltransferase (CAT), green fluorescent protein, firefly luciferase, and human growth hormone, among others. Nucleotide sequences encoding selectable markers are known in the art, and include those that encode gene products conferring resistance to antibiotics or anti-metabolites, or that supply an auxotrophic requirement. Examples of such sequences include those that encode thymidine kinase activity, or resistance to metliotrexate, ampicillin, kanamycin, chloramphenicol, zeocin, pyrimethamine, aminoglycosides, or hygromycin, among others. 5.4.2. Transformation of Host Cells

[00148] The present invention further provides a transformed host cell comprising a nucleic acid molecule or recombinant vector of the present invention, and a cell line derived therefrom. Such host cells are useful for cloning and/or expressing a nucleic acid molecule of the present invention. Such transformed host cells include but are not limited to microorganisms, such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA vectors, or yeast transformed with a recombinant vector, or animal cells, such as insect cells infected with a recombinant virus vector, e.g. , baculovirus, or mammalian cells infected with a recombinant virus vector, e.g. , adenovirus or vaccinia virus, among others. For example, a strain of E. coli can be used such as, e.g. , the DH5α strain available from the ATCC, Manassas, VA, USA (Accession No. 31343), or from Stratagene (La Jolla, CA). Eukaryotic host cells include yeast cells, although mammalian cells, e.g. , from a mouse, rat, hamster, cow, monkey, or human cell line, among others, can also be utilized effectively. Examples of eukaryotic host cells that can be used to express a recombinant protein of the invention include Chinese hamster ovary (CHO) cells (e.g. , ATCC Accession No. CCL-61), NIH Swiss mouse embryo cells NIH/3T3 (e.g. , ATCC Accession No. CRL-1658), human epithelial kidney cells HEK 293 (e.g. , ATCC Accession No. CRL-1573), and Madin-Darby bovine kidney (MDBK) cells (ATCC Accession No. CCL-22).

[00149] The recombinant vector of the invention is preferably transformed or transfected into one or more host cells of a substantially homogeneous culture of cells. The vector is generally introduced into host cells in accordance with known techniques, such as, e.g. , by protoplast transformation, calcium phosphate precipitation, calcium chloride treatment, microinjection, electroporation, transfection by contact with a recombined virus, liposome-mediated transfection, DEAE-dextran transfection, transduction, conjugation, or microprojectile bombardment, among others. Selection of transformants can be conducted by standard procedures, such as by selecting for cells expressing a selectable marker, e.g. , antibiotic resistance, associated with the recombinant expression vector.

[00150] Once an expression vector is introduced into the host cell, the presence of the nucleic acid molecule of the present invention, either integrated into the host cell genome or maintained episomally, can be confirmed by standard techniques, e.g. , by DNA-DNA, DNA-RNA, or RNA-antisense RNA hybridization analysis, restriction enzyme analysis, PCR analysis including reverse transcriptase PCR (RT-PCR), detecting the presence of a "marker" gene function, or by immunological or functional assay to detect the expected protein product. 5.4.3. Expression and Purification of Recombinant Polypeptides

[00151] Once a nucleic acid molecule of the present invention has been stably introduced into an appropriate host cell, the transformed host cell is clonally propagated, and the resulting cells can be grown under conditions conducive to the efficient production (i.e. , expression or overexpression) of the encoded polypeptide. Where the expression vector comprises an inducible promoter, appropriate induction conditions such as, e.g. , temperature shift, exhaustion of nutrients, addition of gratuitous inducers (e.g. , analogs of carbohydrates, such as isopropyl-β-D- thiogalactopyranoside (IPTG)), accumulation of excess metabolic by-products, or the like, are employed as needed to induce expression. If necessary and desired, a signal sequence that matches with a host cell can be added to the N-terminus of the polypeptide. Examples of the signal sequences that can be used are PhoA signal sequence, OmpA signal sequence, etc. , in the case of using bacteria of the genus Escherichia as the host; α-amylase signal sequence, subtilisin signal sequence, etc., in the case of using bacteria of the genus Bacillus as the host; MFα signal sequence, SUC2 signal sequence, etc. , in the case of using yeast as the host; and insulin signal sequence, c-interferon signal sequence, antibody molecule signal sequence, etc. , in the case of using animal cells as the host, respectively.

[00152] Where the polypeptide is retained inside the host cells or contained in a cell membrane, the cells are harvested and lysed, and the product is substantially purified or isolated from the lysate or membrane fraction under extraction conditions known in the art to minimize protein degradation such as, e.g. , at 4°C, or in the presence of protease inhibitors, or both. Where the polypeptide is secreted from the host cells, the exhausted nutrient medium can simply be collected and the polypeptide substantially purified or isolated therefrom.

[00153] The polypeptide can be substantially purified or isolated from cell lysates, membrane fractions, or culture medium, as necessary, using standard methods, including but not limited to one or more of the following methods: ammonium sulfate precipitation, size fractionation, ion exchange chromatography, HPLC, density centrifugation, affinity chromatography, ethanol precipitation, and chromatofocusing. During purification, the polypeptide can be detected based, e.g. , on size, or reactivity with a polypeptide-specific antibody, or by detecting the presence of a fusion tag.

[00154] According to the present invention, the recombinantly expressed full- length PNPGl protein is most likely to be associated with the cellular membrane as a transmembrane protein. Such protein can be isolated from membrane fractions of host cells. The cell membrane fraction refers to a fraction abundant in cell membrane obtained by cell disruption and subsequent fractionation by any of the known methods. Useful cell disruption methods include, e.g. , cell squashing using a Potter-Elvehjem homogenizer, disruption using a Waring blender or Polytron (manufactured by Kinematica Inc.), disruption by ultrasonication, and disruption by cell spraying through thin nozzles under an increased pressure using a French press or the like. Cell membrane fractionation is effected mainly by fractionation using a centrifugal force, such as centrifugation for fractionation and density gradient centrifugation. For example, cell disruption fluid can be centrifuged at a low speed (500 rpm to 3,000 rpm) for a short period of time (normally about 1 to about 10 minutes), the resulting supernatant is then centrifuged at a higher speed (15,000 rpm to 30,000 rpm) normally for 30 minutes to 2 hours. The precipitate thus obtained can be used as the membrane fraction. The membrane fraction is rich in membrane components such as cell-derived phospholipids and transmembrane and membrane- associated proteins. In yet other embodiments, the membrane fraction may be further solubilized with a detergent. Detergents that may be used with the present invention include without limitation Triton X-100, (3-octyl glucoside, and CHAPS (see also Langridge et al , Biochim. Biophys. Acts. 1983; 751: 318).

[00155] Upon isolation of the membrane fraction, the peripheral proteins of these membranes can be removed by extraction with high salt concentrations, high pH or chaotropic agents such as lithium diiodosalicylate. The integral proteins

(including the PNPGl protein) can then be solubilized using a detergent such as

Triton X-100, /3-octyl glucoside, CHAPS, or other compounds of similar action

(see, e.g. , Beros et al , J. Biol Chem. 1987; 262: 10613). A combination of several standard chromatographic steps (e.g. , ion exchange chromatography, gel permeation chromatography, adsorption chromatography or isoelectric focusing) and/or a single purification step involving immuno-affinity chromatography using immobilized antibodies (or antibody fragments) to the PNPGl protein and/or preparative polyacrylamide gel electrophoresis using instrumentation such as the Applied Biosystems "230A EPEC System" can be then used to purify the PNPGl protein and remove it from other integral proteins of the detergent-stabilized mixture. It is recognized that the hydrophobic nature of the transmembrane protein may necessitate the inclusion of amphiphilic compounds such as detergents and other surfactants (see bud Kar and Maloney, J. Biol Chem. 1986; 261: 10079) during handling.

[00156] For use in practicing the present invention, the polypeptide can be in an unpurified state as secreted into the culture fluid or as present in a cell lysate or membrane fraction. Alternatively, the polypeptide may be purified therefrom. Once a polypeptide of the present invention of sufficient purity has been obtained, it can be characterized by standard methods, including by SDS-PAGE, size exclusion chromatography, amino acid sequence analysis, immunological activity, biological activity, etc. The polypeptide can be further characterized using hydrophilicity analysis (see, e.g. , Hopp and Woods, Proc. Natl. Acad. Sci. USA 1981; 78: 3824), or analogous software algorithms, to identify hydrophobic and hydrophilic regions. Structural analysis can be carried out to identify regions of the polypeptide that assume specific secondary structures. Biophysical methods such as X-ray crystallography (Engstrom, Biochem. Exp. Biol 1914; 11: 7-13), computer modeling (Fletterick and Zoller eds., In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1986), and nuclear magnetic resonance (NMR) can be used to map and study potential sites of interaction between the polypeptide and other putative interacting proteins/receptors/molecules. Information obtained from these studies can be used to design deletion mutants, and to design or select therapeutic compounds that can specifically modulate the biological function of the PNPGl protein in vivo. 5.5. Polypeptides

[00157] The present invention provides an isolated polypeptide encoded by a nucleic acid of the present invention, which polypeptide may or may not also be purified.

[00158] In one embodiment, the isolated polypeptide is a rat PNPGl protein comprising the amino acid sequence of SEQ ID NO:2. In another embodiment, the isolated polypeptide is a mouse PNPGl protein comprising the amino acid sequence of SEQ ID NO:4. In another embodiment, the isolated polypeptide is a human PNPGl protein comprising the amino acid sequence of SEQ ID NO:6.

[00159] The present invention further provides a polypeptide that is homologous to a rat, mouse or human PNPGl protein of the present invention, as the term "homologous" is defined above for polypeptides.

[00160] The present invention further provides a polypeptide consisting of a substantial portion of a rat, mouse or human PNPGl protein of the present invention. As used herein, a "substantial portion" (also referred to as a "peptide fragment") of such a protein refers to a polypeptide consisting of less than the complete amino acid sequence of the corresponding full-length protein, but comprising at least about 10%, at least about 20%, at least about 30%, at least about 40% , at least about 50%, at least about 60%, at least about 70% , at least about 80%, at least about 90%, at least about 95% or at least about 99% of the contiguous amino acid sequence of the full-length protein. Such peptide fragments are useful if they either exhibit PNPGl activity or are immunogenic, i.e. , capable of inducing an immune response resulting in the production of antibodies that react specifically against the corresponding full-length PNPGl polypeptide.

[00161] The present invention further provides fusion proteins comprising any of the aforementioned polypeptides (proteins or peptide fragments) fused to a carrier or fusion partner, as known in the art.

[00162] Polypeptides of the present invention are useful for a variety of purposes, including for use in cell-based or non-cell-based assays to study the biological function of PNPGl, to identify molecules that interact with PNPGl in vivo, to screen for compounds that bind to PNPGl and modulate its expression and/or activity, or as antigens to raise polyclonal or monoclonal antibodies, as described below. Such antibodies can be used as therapeutic agents to modulate PNPGl activity, or as diagnostic reagents, e.g. , using standard techniques such as Western blot assays, to screen for PNPGl protein expression levels in cell, tissue or fluid samples collected from a subject. [00163] A polypeptide of the present invention can be modified at the protein level to improve or otherwise alter its biological or immunological characteristics. One or more chemical modifications of the polypeptide can be carried out using known techniques to prepare analogs therefrom, including but not limited to any of the following: substitution of one or more L-amino acids of the polypeptide with corresponding D-amino acids, amino acid analogs, or amino acid mimics, so as to produce, e.g. , carbazates or tertiary centers; or specific chemical modification, such as, e.g. , proteolytic cleavage with trypsin, chymotrypsin, papain or V8 protease, or treatment with NaBHt or cyanogen bromide, or acetylation, formylation, oxidation or reduction, etc. Alternatively or additionally, a polypeptide of the present invention can be modified by genetic recombination techniques.

[00164] A polypeptide of the present invention can be derivatized by conjugation thereto of one or more chemical groups, including but not limited to acetyl groups, sulfur bridging groups, glycosyl groups, lipids, and phosphates, and/or by conjugation to a second polypeptide of the present invention, or to another protein, such as, e.g. , serum albumin, keyhole limpet hemocyanin, or commercially available BSA, or to a polyamino acid (e.g. , polylysine), or to a polysaccharide, (e.g. , sepharose, agarose, or modified or unmodified celluloses), among others. Such conjugation is preferably by covalent linkage at amino acid side chains and/or at the N-terminus or C-terminus of the polypeptide. Methods for carrying out such conjugation reactions are known in the field of protein chemistry.

[00165] Derivatives useful in practicing the claimed invention also include those in which a water-soluble polymer such as, e.g. , polyethylene glycol, is conjugated to a polypeptide of the present invention, or to an analog or derivative thereof, thereby providing additional desirable properties while retaining, at least in part, the immunogenicity of the polypeptide. These additional desirable properties include, e.g. , increased solubility in aqueous solutions, increased stability in storage, increased resistance to proteolytic degradation, and increased in vivo half- life. Water-soluble polymers suitable for conjugation to a polypeptide of the present invention include but are not limited to polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol, wherein said homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group, poly oxy ethylated polyols, polyvinyl alcohol, polysaccharides, polyvinyl ethyl ethers, and α, β -poly [2-hydroxy ethyl] -DL- aspartamide. Polyethylene glycol is particularly preferred. Methods for making water-soluble polymer conjugates of polypeptides are known in the art and are described, among other places, in U.S. Patent Nos. 3,788,948; 3,960,830; 4,002,531; 4,055,635; 4,179,337; 4,261,973; 4,412,989; 4,414,147; 4,415,665; 4,609,546; 4,732,863; and 4,745,180; European Patent (EP) 152,847; EP 98,110; and Japanese Patent 5,792,435; which patents are incorporated herein by reference. 5.6. Antibodies

[00166] As used herein, "antibody" refers to a human, nonhuman, or chimeric (e.g., humanized) immunoglobulin, or binding fragment thereof, that specifically binds to an antigen, e.g., a PNPGl protein. Suitable antibodies may be polyclonal (e.g., sera or affinity purified preparations), monoclonal, or recombinant. Examples of useful fragments include separate heavy chains, light chains, Fab, F(ab')₂, Fabc, and Fv fragments. Fragments can be produced by enzymatic or chemical separation of intact immunoglobulins or by recombinant DNA techniques. Fragments may be expressed in the form of phage-coat fusion proteins (see, e.g., International PCT Publication Nos. WO 91/17271, WO 92/01047, and WO 92/06204). Typically, the antibodies, fragments, or similar binding agents bind a specific antigen with an affinity of at least IO⁷, 10⁸, IO⁹, or 10¹⁰ M^"1.

[00167] The present invention provides an isolated antibody directed against a polypeptide of the present invention. In a specific embodiment, antibodies can be raised against a PNPGl protein of the invention using known methods in view of this disclosure. Various host animals selected, e.g. , from pigs, cows, horses, rabbits, goats, sheep, rats, or mice, can be immunized with a partially or substantially purified PNPGl protein, or with a peptide homolog, fusion protein, peptide fragment, analog or derivative thereof, as described above. An adjuvant can be used to enhance antibody production. [00168] Polyclonal antibodies can be obtained and isolated from the serum of an immunized animal and tested for specificity against the antigen using standard techniques. Alternatively, monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, Nature 1975; 256: 495-497; the human B-cell hybridoma technique (Kosbor et al , Immunology Today 1983; 4: 72; Cote et al , Proc. Natl. Acad. Sci. USA 1983; 80: 2026-2030); and the EBV-hybridoma technique (Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985, pp. 77-96). Alternatively, techniques described for the production of single chain antibodies (see, e.g. , U.S. Patent No. 4,946,778) can be adapted to produce PNPGl -specific single chain antibodies.

[00169] Antibody fragments that contain specific binding sites for a polypeptide of the present invention are also encompassed within the present invention, and can be generated by known techniques. Such fragments include but are not limited to F(ab')₂ fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed (Huse et al , Science 1989; 246: 1275-1281) to allow rapid identification of Fab fragments having the desired specificity to the particular PNPGl protein.

[00170] Techniques for the production and isolation of monoclonal antibodies and antibody fragments are known in the art, and are generally described, among other places, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, and in Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, London, 1986.

[00171] Antibodies or antibody fragments can be used in methods known in the art relating to the localization and activity of PNPGl, e.g. , in Western blotting, in situ imaging, measuring levels thereof in appropriate physiological samples, etc. Immunoassay techniques using antibodies include radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassay s, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using, e.g. , colloidal gold, enzyme or radioisotope labels), precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. Antibodies can also be used in microarrays (see, e.g., International PCT Publication No. WO 00/04389). Furthermore, antibodies can be used as therapeutics to inhibit the activity of a PNPGl protein.

[00172] Recent advances in antibody engineering have allowed the genes encoding antibodies to be manipulated, so that antigen-binding molecules can be expressed within mammalian cells. Application of gene technologies to antibody engineering has enabled the synthesis of single-chain fragment variable (scFv) antibodies that combine within a single polypeptide chain the light and heavy chain variable domains of an antibody molecule covalently joined by a pre-designed peptide linker. Intracellular antibody (or "intrabody") strategy serves to target molecules involved in essential cellular pathways for modification or ablation of protein function. Antibody genes for intracellular expression can be derived, e.g. , either from murine or human monoclonal antibodies or from phage display libraries. For intracellular expression, small recombinant antibody fragments containing the antigen recognizing and binding regions can be used. Intrabodies can be directed to different intracellular compartments by targeting sequences attached to the antibody fragments.

[00173] Various methods have been developed to produce intrabodies. Techniques described for the production of single chain antibodies (see, e.g. , U.S. Patents No. 5,476,786; 5,132,405; and 4,946,778) can be adapted to produce polypeptide-specific single chain antibodies. Another method called intracellular antibody capture (IAC), is based on a genetic screening approach (Tanaka et al. , Nucleic Acids Res. 2003; 31: e23). Using this technique, consensus immunoglobulin variable frameworks are identified that can form the basis of intrabody libraries for direct screening. The procedure comprises in vitro production of a single antibody gene fragment from oligonucleotides and diversification of CDRs of the immunoglobulin variable domain by mutagenic PCR to generate intrabody libraries. This method obviates the need for in vitro production of antigen for pre-selection of antibody fragments, and also yields intrabodies with enhanced intracellular stability.

[00174] Intrabodies can be used to modulate cellular physiology and metabolism through a variety of mechanisms, including blocking, stabilizing, or mimicking protein-protein interactions, by altering enzyme function, or by diverting proteins from their usual intracellular compartments. Intrabodies can be directed to the relevant cellular compartments by modifying the genes that encode them to specify N- or C-terminal polypeptide extensions for providing intracellular- trafficking signals. 5.7. Targeted Mutation of the PNPGl Gene

[00175] Based on the present disclosure of nucleic acid molecules, genetic constructs can be prepared for use in disabling or otherwise mutating a mammalian

PNPGl gene. For example, the rat, mouse or human PNPGl gene can be mutated using an appropriately designed genetic construct in combination with genetic techniques currently known or to be developed in the future. For example, a rat, mouse or human PNPGl gene can be mutated using a genetic construct that functions to: (a) delete all or a portion of the coding sequence or regulatory sequence of the PNPGl gene; or (b) replace all or a portion of the coding sequence or regulatory sequence of the PNPGl gene with a different nucleotide sequence; or

(c) insert into the coding sequence or regulatory sequence of the PNPGl gene one or more nucleotides, or an oligonucleotide molecule, or nucleic acid molecule, which can comprise a nucleotide sequence from the same species or from a heterologous source; or (d) carry out some combination of (a), (b) and (c).

[00176] Cells, tissues and animals in which the PNPGl gene has been mutated are useful, among others, in studying the biological function of PNPGl, identifying molecules that interact with PNPGl in vivo, as well as in conducting screens to identify therapeutic compounds that selectively modulate PNPGl expression and/or activity. In another embodiment, the mutation serves to partially or completely disable the PNPGl gene, or partially or completely disable the protein encoded by the PNPGl gene. In this context, a PNPGl gene or protein is considered to be partially or completely disabled if either no protein product is made (for example, where the gene is deleted), or a protein product is made that can no longer carry out its normal biological function or can no longer be transported to its normal cellular location,, or a protein product is made that carries out its normal biological function but at a significantly reduced level.

[00177] In a non-limiting embodiment, a genetic construct of the present invention is used to mutate a wild-type PNPGl gene by replacement of at least a portion of the coding or regulatory sequence of the wild-type gene with a different nucleotide sequence such as, e.g. , a mutated coding sequence or mutated regulatory region, or portion thereof. A mutated PNPGl gene sequence for use in such a genetic construct can be produced by any of a variety of known methods, including by use of error-prone PCR, or by cassette mutagenesis. For example, oligonucleotide-directed mutagenesis can be employed to alter the coding or regulatory sequence of a wild-type PNPGl gene in a defined way, e.g. , to introduce a frame-shift or a termination codon at a specific point within the sequence. A mutated nucleotide sequence for use in the genetic construct of the present invention can be prepared by insertion into the coding or regulatory (e.g. , promoter) sequence of one or more nucleotides, oligonucleotide molecules or nucleic acid molecules, or by replacement of a portion of the coding sequence or regulatory sequence with one or more different nucleotides, oligonucleotide molecules or nucleic acid molecules. Such oligonucleotide molecules or nucleic acid molecules can be obtained from any naturally occurring source or can be synthetic. The inserted sequence can serve simply to disrupt the reading frame of the PNPGl gene, or can further encode a heterologous gene product such as a selectable marker.

[00178] Mutations to produce modified cells, tissues and animals that are useful in practicing the present invention can occur anywhere in the PNPGl gene, including in the open reading frame, or in the promoter or other regulatory region, or in any other portion of the sequence that naturally comprises the gene or ORF. Such cells include mutants in which a modified form of the PNPGl protein normally encoded by the PNPGl gene is produced, or in which no protein normally encoded by the PNPGl gene is produced. Such cells can be null, conditional or leaky mutants.

[00179] Alternatively, a genetic construct can comprise nucleotide sequences that naturally flank the PNPGl gene or ORF in situ, with only a portion or no nucleotide sequences from the actual coding region of the gene itself. Such a genetic construct can be useful to delete the entire PNPGl gene or ORF.

[00180] Methods for carrying out homologous gene replacement are known in the art. For targeted gene mutation through homologous recombination, the genetic construct is preferably a plasmid, either circular or linearized, comprising a mutated nucleotide sequence as described above. In a non-limiting embodiment, at least about 200 nucleotides of the mutated sequence are used to specifically direct the genetic construct of the present invention to the particular targeted PNPGl gene for homologous recombination, although shorter lengths of nucleotides may also be effective. In addition, the plasmid preferably comprises an additional nucleotide sequence encoding a reporter gene product or other selectable marker constructed so that it will insert into the genome in operative association with the regulatory element sequences of the native PNPGl gene to be disrupted. Reporter genes that can be used in practicing the invention are known in the art, and include those encoding CAT, green fluorescent protein, and β-galactosidase, among others. Nucleotide sequences encoding selectable markers are also known in the art, and include those that encode gene products conferring resistance to antibiotics or anti- metabolites, or that supply an auxotrophic requirement.

[00181] In view of the present disclosure, methods that can be used for creating the genetic constructs of the present invention will be apparent, and can include in vitro recombinant techniques, synthetic techniques, and in vivo genetic recombination, as described, among other places, in Ausubel et al , 1989, above; Sambrook et al , 1989, above; Innis et al , 1995, above; and Erlich, 1992, above. [00182] Mammalian cells can be transformed with a genetic construct of the present invention in accordance with known techniques, such as, e.g. , by electroporation. Selection of transformants can be carried out using standard techniques, such as by selecting for cells expressing a selectable marker associated with the construct. Identification of transformants in which a successful recombination event has occurred and the particular target gene has been disabled can be carried out by genetic analysis, such as by Southern blot analysis, or by Northern analysis to detect a lack of mRNA transcripts encoding the particular protein, or by the appearance of cells lacking the particular protein, as determined, e.g. , by immunological analysis, or some combination thereof.

[00183] The present invention thus provides modified mammalian cells in which the native PNPGl gene has been mutated. The present invention further provides modified animals in which the PNPGl gene has been mutated. 5.8. Genetically Modified Animals

[00184] Genetically modified animals can be prepared for studying the biological function of PNPGl in vivo and for screening and/or testing candidate compounds for their ability to affect the expression and/or activity of PNPGl as potential therapeutics for treating pain and related disorders such as chronic pain, neuropathic pain, inflammatory and cancer pain, addiction, seizure (including epilepsy), stroke or ischemia, neurodegenerative disorder (e.g. , Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), or Huntington's chorea), headache (e.g. , general, migraine, cluster or tension), anxiety, depression, asthma, rheumatic disease, retinopathy, inflammatory eye disorders, pruritis, ulcer (gastric or duodenal), gastric lesions induced by a necrotizing agent, uncontrollable urination (e.g. , incontinence), inflammatory or unstable bladder disorders, and inflammatory bowel disease.

[00185] To investigate the function of PNPGl in vivo in animals, PNPGl- encoding nucleic acid molecules or PNPGl -inhibiting antisense nucleic acid molecules, shRNAs, siRNAs, or ribozymes can be introduced into test animals, such as mice or rats, using, e.g. , viral vectors or naked nucleic acid molecules. Alternatively, transgenic animals can be produced. Specifically, "knock-in" animals with the endogenous PNPGl gene substituted with a heterologous gene or an ortholog from another species or a mutated PNPGl gene, "knockout" animals with PNPGl gene partially or completely inactivated, or transgenic animals expressing or overexpressing a wild-type or mutated PNPGl gene (e.g. , upon targeted or random integration into the genome) can be generated.

[00186] PNPGl-encoding nucleic acid molecules can be introduced into animals using viral delivery systems. Exemplary viruses for production of delivery vectors include without limitation adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV). See, e.g. , Becker et al , Meth. Cell Biol 1994; 43: 161-89; Douglas and Curiel, Science & Medicine 1997; 4: 44-53; Yeh and Perricaudet, FASEB J. 1997; 11: 615-623; Kuo et al , Blood 1993; 82: 845; Markowitz et al , J. Virol. 1988; 62: 1120; Mann et al , Cell 1983; 33: 153; U.S. Patents No. 5,399,346; 4,650,764; 4,980,289; 5,124,263; and International Publication No. WO 95/07358.

[00187] In an alternative method, a PNPGl-encoding nucleic acid molecule can be introduced by liposome-mediated transfection, a technique that provides certain practical advantages, including the molecular targeting of liposomes to specific cells. Directing transfection to particular cell types (also possible with viral vectors) is particularly advantageous in a tissue with cellular heterogeneity, such as the brain, pancreas, liver, and kidney. Lipids may be chemically coupled to other molecules for the purpose of targeting. Targeted peptides (e.g. , hormones or neurotransmitters), proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.

[00188] In another embodiment, target cells can be removed from an animal, and a nucleic acid molecule can be introduced as a naked construct. The transformed cells can be then re-implanted into the body of the animal. Naked nucleic acid constructs can be introduced into the desired host cells by methods known in the art, e.g. , transfection, electroporation, microinj ection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g. , Wu et al , J. Biol Chem. 1992; 267: 963-7; Wu et al , J. Biol. Chem. 1988; 263: 14621-4.

[00189] As specified above, transgenic animals can also be generated.

Methods of making transgenic animals are well-known in the art (for transgenic mice see Gene Targeting: A Practical Approach, 2^nd Ed., Joyner ed., IRL Press at Oxford University Press, New York, 2000; Manipulating the Mouse Embryo: A Laboratory Manual, Nagy et al. eds., Cold Spring Harbor Press, New York, 2003; Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson ed. , IRL Press at Oxford University Press, 1987; Transgenic Animal Technology: A Laboratory Handbook, Pinkert ed., Academic Press, New York, 1994; Hogan, Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1986; Brinster et al , Proc. Nat. Acad. Sci. USA 1985; 82: 4438- 4442; Capecchi, Science 1989; 244: 1288-1292; Joyner et al , Nature 1989; 338: 153-156; U.S. Patents No. 4,736,866; 4,870,009; 4,873,191; for particle bombardment see U.S. Patent No. 4, 945,050; for transgenic rats see, e.g. , Hammer et al , Cell 1990; 63: 1099-1112; for non-rodent transgenic mammals and other animals see, e.g. , Pursel et al , Science 1989; 244: 1281-1288 and Simms et al , Bio /Technology 1988; 6: 179- 183; and for culturing of embryonic stem (ES) cells and the subsequent production of transgenic animals by the introduction of DNA into ES cells using methods such as electroporation, calcium phosphate/DNA precipitation and direct injection see, e.g. , Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, Robertson ed., IRL Press, 1987). Clones of the nonhuman transgenic animals can be produced according to available methods (see e.g. , Wilmut et al , Nature 1997; 385: 810-813 and International Publications No. WO 97/07668 and WO 97/07669).

[00190] In one embodiment, the transgenic animal is a "knockout" animal having a heterozygous or homozygous alteration in the sequence of an endogenous PNPGl gene that results in a decrease of PNPGl function, preferably such that PNPGl expression is undetectable or insignificant. Knockout animals are typically generated by homologous recombination with a vector comprising a transgene having at least a portion of the gene to be knocked out. Typically a deletion, addition or substitution has been introduced into the transgene to functionally disrupt it.

[00191] Knockout animals can be prepared by any method known in the art

(see, e.g. , Snouwaert et al , Science 1992; 257: 1083; Lowell et al , Nature 1993; 366: 740-42; Capecchi, Science 1989; 244: 1288-1292; Palmiter et al , Ann. Rev. Genet. 1986; 20: 465-499; Bradley, Current Opinion in Bio/Technology 1991; 2: 823-829; and International Publications No. WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169). Preparation of a knockout animal typically requires first introducing a nucleic acid construct (a "knockout construct"), that will be used to decrease or eliminate expression of a particular gene, into an undifferentiated cell type termed an embryonic stem (ES) cell. The knockout construct is typically comprised of: (i) DNA from a portion (e.g. , an exon sequence, intron sequence, promoter sequence, or some combination thereof) of a gene to be knocked out; and (ii) a selectable marker sequence used to identify the presence of the knockout construct in the ES cell. The knockout construct is typically introduced (e.g. , electroporated or microinj ected) into ES cells so that it can homologously recombine with the genomic DNA of the cell in a double crossover event. This recombined ES cell can be identified (e.g. , by Southern hybridization or PCR reactions that show the genomic alteration) and is then injected into a mammalian embryo at the blastocyst stage. In another embodiment where the knockout animal is a mammal, a mammalian embryo with integrated ES cells is then implanted into a foster mother for the duration of gestation (see, e.g. , Zhou et al. , Genes and Dev. 1995; 9: 2623- 34).

[00192] In a specific embodiment, the knockout vector is designed such that, upon homologous recombination, the endogenous PNPGl-related gene is functionally disrupted (i.e. , no longer encodes a functional protein). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous PNPGl-related gene is mutated or otherwise altered but still encodes functional protein (e.g. , the upstream regulatory region can be altered to thereby alter the expression of the PNPGl-related polypeptide). In the homologous recombination vector, the altered portion of PNPGl-related gene is preferably flanked at its 5' and 3' ends by additional nucleic acid of the PNPGl-related gene to allow for homologous recombination to occur between the exogenous PNPGl- related gene carried by the vector and an endogenous PNPGl-related gene in an embryonic stem cell. The additional flanking PNPGl-related nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (at both the 5' and 3' ends) are included in the vector (see, e.g. , Thomas and Capecchi, Cell 1987; 51: 503). The vector is introduced into an ES cell line (e.g. , by electroporation), and cells in which the introduced PNPGl-related gene has homologously recombined with the endogenous PNPGl-related gene are selected (see, e.g. , Li et al , Cell 1992; 69: 915). The selected cells are then injected into a blastocyst of an animal (e.g. , a mouse) to form aggregation chimeras (see, e g. , Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson ed., IRL, Oxford, 1987, pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.

[00193] The phenotype of knockout animals can be predictive of the in vivo function of the gene and of the effects or noneffect of its antagonists or agonists. Knockout animals can also be used to study the effects of the PNPGl protein in models of disease, including, pain and pain-related disorders. In a specific embodiment, knockout animals, such as mice harboring the PNPGl gene knockout, may be used to produce antibodies against the heterologous PNPGl protein (e.g. , human PNPGl) (Claesson et al., Scan. J. Immunol. 1994; 0: 257-264; Declerck et al. , J. Biol Chem. 1995; 270: 8397-400).

[00194] Genetically modified animals expressing or harboring PNPG1- specific antisense nucleic acid molecules, shRNA, siRNA, or ribozymes can be used analogously to knockout animals described above. [00195] In another embodiment of the invention, the transgenic animal is an animal having an alteration in its genome that results in altered expression (e.g. , increased or decreased expression) of the PNPGl gene, e.g. , by introduction of additional copies of PNPGl gene in various parts of the genome, or by operatively inserting a regulatory sequence that provides for altered expression of an endogenous copy of the PNPGl gene. Such regulatory sequences include inducible, tissue-specific, and constitutive promoters and enhancer elements. Suitable promoters include metallothionein, albumin (Pinkert et al , Genes Dev. 1987; 1: 268-76), and K-14 keratinocyte (Vassar et al, Proc. Natl Acad. Sci. USA 1989; 86: 1563-1567) gene promoters. Overexpression of the wild-type PNPGl polypeptide, polypeptide fragment or a mutated version thereof may alter normal cellular processes, resulting in a phenotype that identifies a tissue in which PNPGl expression is functionally relevant and may indicate a therapeutic target for the PNPGl, its agonists or antagonists. For example, a transgenic test animal can be engineered to overexpress a full-length PNPGl sequence, which may result in a phenotype that shows similarity with human diseases.

[00196] Transgenic animals can also be produced that allow for regulated

(e.g. , tissue-specific) expression of the transgene. One example of such a system that may be produced is the Cre-Lox recombinase system of bacteriophage PI (Lakso et al , Proc. Natl. Acad. Sci. USA 1992; 89: 6232-6236; U.S. Patents No. 4,959,317 and 5,801,030). If the Cre-Lox recombinase system is used to regulate expression of a transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g. , by mating two transgenic or gene-targeted animals, one containing a transgene encoding a selected protein or containing a targeted allele (e.g., a loxP flanked exon), and the other containing a transgene encoding a recombinase (e.g., a tissue-specific expression of Cre recombinase). Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O' Gorman et al , Science 1991; 251: 1351-1355; U.S. Patent No. 5,654,182). In another embodiment, both Cre- Lox and Flp-Frt are used in the same system to regulate expression of the transgene, and for sequential deletion of vector sequences in the same cell (Sun et al. , Nat. Genet. 2000; 25: 83-6). Regulated transgenic animals can be also prepared using the tet-repressor system (see, e.g. , U.S. Patent No. 5,654,168).

[00197] The in vivo function of PNPGl can also be investigated through making "knock-in" animals. In such animals the endogenous PNPGl gene can be replaced, e.g. , by a heterologous gene, by a PNPGl ortholog or by a mutated PNPGl gene. See, for example, Wang et al , Development 1997; 124: 2507-2513; Zhuang et al , Mol Cell Biol. 1998; 18: 3340-3349; Geng et al , Cell 1999; 97: 767-777; Baudoin et al , Genes Dev. 1998; 12: 1202-1216. Thus, a non-human transgenic animal can be created in which: (i) a human ortholog of the non-human animal PNPGl gene has been stably inserted into the genome of the animal; and/or (ii) the endogenous non-human animal PNPGl gene has been replaced with its human counterpart (see, e.g., Coffman, Semin. Nephrol. 1997; 17: 404; Esther et al , Lab. Invest. 1996; 74: 953; Murakami et al , Blood Press. Suppl 1996; 2: 36). In one aspect of this embodiment, a human PNPGl gene inserted into the transgenic animal is the wild-type human PNPGl gene. In another aspect, the PNPGl gene inserted into the transgenic animal is a mutated form or a variant of the human PNPGl gene.

[00198] Included within the scope of the present invention are transgenic animals, preferably mammals (e.g., mice) in which, in addition to the PNPGl gene, one or more additional genes (preferably, associated with pain or related disorders) have been knocked out, or knocked in, or overexpressed. Such animals can be generated by repeating the procedures set forth herein for generating each construct, or by breeding two animals of the same species (each with a single gene manipulated) to each other, and screening for those progeny animals having the desired genotype. 5.9. Use of a Nucleic Acid Molecule to Modulate PNPGl Gene Expression

[00199] As specified above, the PNPGl-encoding nucleic acid molecules of the invention or the nucleic acid molecules comprising sequences that hybridize to them under standard hybridization conditions (including their homologs/orthologs, complementary sequences and various oligonucleotide probes and primers derived from them) can be used to inhibit the expression of PNPGl genes (e.g. , by inhibiting transcription, splicing, transport, or translation or by promoting degradation of corresponding mRNAs). Specifically, the nucleic acid molecules of the invention can be used to "knock down" or "knock out" the expression of the PNPGl genes in a cell or tissue (e.g., in an animal model or in cultured cells) by using their sequences to design antisense oligonucleotides, RNA interference (RNAi) molecules, ribozymes, nucleic acid molecules to be used in triplex helix formation, etc. Preferred methods to inhibit gene expression are described below. 5.9.1. RNA Interference (RNAi)

[00200] RNA interference (RNAi) is a process of sequence-specific post- transcriptional gene silencing by which double stranded RNA (dsRNA) homologous to a target locus can specifically inactivate gene function in plants, fungi, invertebrates, and vertebrates, including mammals (Hammond et al , Nature Genet. 2001; 2: 110-119; Sharp, Genes Dev. 1999;13: 139-141). This dsRNA-induced gene silencing is mediated by short double-stranded small interfering RNAs (siRNAs) generated from longer dsRNAs by ribonuclease III cleavage (Bernstein et al , Nature 2001; 409: 363-366 and Elbashir et al, Genes Dev. 2001; 15: 188-200). RNAi-mediated gene silencing is thought to occur via sequence-specific mRNA degradation, where sequence specificity is determined by the interaction of an siRNA with its complementary sequence within a target mRNA (see, e.g. , Tuschl, Chem. Biochem. 2001; 2: 239-245).

[00201] For mammalian systems, RNAi commonly involves the use of dsRNAs that are greater than 500 bp; however, it can also be activated by introduction of either siRNAs (Elbashir, et al , Nature 2001; 411: 494-498) or short hairpin RNAs (shRNAs) bearing a fold back stem-loop structure (Paddison et al, Genes Dev. 2002; 16: 948-958; Sui et al, Proc. Natl Acad. Sci. USA 2002; 99: 5515-5520; Brummelkamp et al, Science 2002; 296: 550-553; Paul et al , Nature Biotechnol 2002; 20: 505-508).

[00202] The siRNAs to be used in the methods of the present invention are preferably short double stranded nucleic acid duplexes comprising annealed complementary single stranded nucleic acid molecules. In other embodiments, the siRNAs are short dsRNAs comprising annealed complementary single strand RNAs. However, the invention also encompasses embodiments in which the siRNAs comprise an annealed RNA:DNA duplex, wherein the sense strand of the duplex is a DNA molecule and the antisense strand of the duplex is a RNA molecule.

[00203] Preferably, each single stranded nucleic acid molecule of the siRNA duplex is of from about 19 nucleotides to about 27 nucleotides in length. In other embodiments, duplexed siRNAs have a 2 or 3 nucleotide 3' overhang on each strand of the duplex. In other embodiments, siRNAs have 5 '-phosphate and 3 '-hydroxyl groups.

[00204] The RNAi molecules to be used in the methods of the present invention comprise nucleic acid sequences that are complementary to the nucleic acid sequence of a portion of the target locus. In certain embodiments, the portion of the target locus to which the RNAi probe is complementary is at least about 15 nucleotides in length. In other embodiments, the portion of the target locus to which the RNAi probe is complementary is at least about 19 nucleotides in length. The target locus to which an RNAi probe is complementary may represent a transcribed portion of the PNPGl gene or an untranscribed portion of the PNPGl gene (e.g. , intergenic regions, repeat elements, etc.).

[00205] The RNAi molecules may include one or more modifications, either to the phosphate-sugar backbone or to the nucleoside. For example, the phosphodiester linkages of natural RNA may be modified to include at least one heteroatom other than oxygen, such as nitrogen or sulfur. In this case, for example, the phosphodiester linkage may be replaced by a phosphothioester linkage. Similarly, bases may be modified to block the activity of adenosine deaminase. Where the RNAi molecule is produced synthetically, or by in vitro transcription, a modified ribonucleoside may be introduced during synthesis or transcription.

[00206] According to the present invention, siRNAs may be introduced to a target cell as an annealed duplex siRNA, or as single stranded sense and anti-sense nucleic acid sequences that, once within the target cell, anneal to form the siRNA duplex. Alternatively, the sense and anti-sense strands of the siRNA may be encoded on an expression construct that is introduced to the target cell (e.g. , as in construct pSi-PNPGl disclosed in Figures 8 and 12A and in Section 6.6., below). Upon expression within the target cell, the transcribed sense and antisense strands may anneal to reconstitute the siRNA.

[00207] The shRNAs to be used in the methods of the present invention comprise a single stranded "loop" region connecting complementary inverted repeat sequences that anneal to form a double stranded "stem" region (see Figure 12B). Structural considerations for shRNA design are discussed, for example, in McManus et al, RNA 2002; 8: 842-850. In certain embodiments the shRNA may be a portion of a larger RNA molecule, e.g. , as part of a larger RNA that also contains U6 RNA sequences (Paul et al, supra).

[00208] In other embodiments, the loop of the shRNA is from about 1 to about 9 nucleotides in length. In other embodiments the double stranded stem of the shRNA is from about 19 to about 33 base pairs in length. In other embodiments, the 3' end of the shRNA stem has a 3' overhang. In other embodiments, the 3' overhang of the shRNA stem is from 1 to about 4 nucleotides in length. In other embodiments, shRNAs have 5 '-phosphate and 3 '-hydroxyl groups.

[00209] Although the RNAi molecules useful according to the invention preferably contain nucleotide sequences that are fully complementary to a portion of the target locus, 100% sequence complementarity between the RNAi probe and the target locus is not required to practice the invention.

[00210] RNA molecules useful for RNAi may be chemically synthesized, for example using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. RNAs produced by such methodologies tend to be highly pure and to anneal efficiently to form siRNA duplexes or shRNA hairpin stem-loop structures. Following chemical synthesis, single stranded RNA molecules are deprotected, annealed to form siRNAs or shRNAs, and purified (e.g. , by gel electrophoresis or HPLC). [00211] Alternatively, standard procedures may used for in vitro transcription of RNA from DNA templates carrying RNA polymerase promoter sequences (e.g. , T7 or SP6 RNA polymerase promoter sequences). Efficient in vitro protocols for preparation of siRNAs using T7 RNA polymerase have been described (Donze and Picard, Nucleic Acids Res. 2002; 30: e46; and Yu et al, Proc. Natl. Acad. Sci. USA 2002; 99: 6047-6052). Similarly, an efficient in vitro protocol for preparation of shRNAs using T7 RNA polymerase has been described (Yu et al, supra). The sense and antisense transcripts may be synthesized in two independent reactions and annealed later, or may be synthesized simultaneously in a single reaction.

[00212] RNAi molecules may be formed within a cell by transcription of

RNA from an expression construct introduced into the cell. For example, both a protocol and an expression construct for in vivo expression of siRNAs are described in Yu et al, supra. Similarly, protocols and expression constructs for in vivo expression of shRNAs have been described (Brummelkamp et al, supra; Sui et al, supra; Yu et al, supra; McManus et al, supra; Paul et al, supra).

[00213] The expression constructs for in vivo production of RNAi molecules comprise RNAi encoding sequences operably linked to elements necessary for the proper transcription of the RNAi encoding sequence(s), including promoter elements and transcription termination signals. Preferred promoters for use in such expression constructs include the polymerase-III HI-RNA promoter (see, e.g. , Brummelkamp et al, supra) and the U6 polymerase-III promoter (see, e.g., Sui et al, supra; Paul, et al. supra; and Yu et al, supra). The RNAi expression constructs can further comprise vector sequences that facilitate the cloning of the expression constructs. Standard vectors that maybe used in practicing the current invention are known in the art (e.g. , pSilencer 2.0-U6 vector, Ambion Inc., Austin, TX).

[00214] Examples of two PNPGl -specific oligonucleotides useful for the production of PNPGl -specific siRNA are shown in Figure 12A (oligonucleotides MB0475 and MB0476, SEQ ID NOS: 29 and 30, respectively, see also Table 3, below). 5.9.2. Antisense Nucleic Acids

[00215] In a specific embodiment, to achieve inhibition of expression of a

PNPGl gene, the nucleic acid molecules of the invention can be used to design antisense oligonucleotides. An antisense oligonucleotide is typically 18 to 25 bases in length (but can be as short as 13 bases in length) and is designed to bind to a selected PNPGl mRNA. This binding prevents translation of that specific PNPGl mRNA, inhibiting production of the corresponding PNPGl protein. The antisense oligonucleotides of the invention comprise at least 6 nucleotides and preferably comprise from 6 to about 50 nucleotides. In specific aspects, the antisense oligonucleotides comprise at least 10 nucleotides, at least 15 nucleotides, at least 25, at least 30, at least 100 nucleotides, or at least 200 nucleotides.

[00216] The antisense nucleic acid oligonucleotides of the invention comprise sequences complementary to at least a portion of the corresponding PNPGl mRNA. However, 100% sequence complementarity is not required so long as formation of a stable duplex (for single stranded antisense oligonucleotides) or triplex (for double stranded antisense oligonucleotides) can be achieved. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense oligonucleotides. Generally, the longer the antisense oligonucleotide, the more base mismatches with the corresponding mRNA can be tolerated. One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

[00217] The antisense oligonucleotides can be DNA or RNA or chimeric mixtures, or derivatives or modified versions thereof, and can be single-stranded or double-stranded. The antisense oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, or a combination thereof. For example, a PNPGl-specific antisense oligonucleotide can comprise at least one modified base moiety selected from a group including but not limited to 5-fluorouracil, 5- bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5-methoxycarboxymethyluracil, 5-methoxyuracil, 2- methylthio-N6-isopentenyladenine, uracil-5 -oxy acetic acid (v), pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5- methyluracil, uracil-5-oxyacetic acid methylester, 5-methyl-2-thiouracil, 3-(3-amino- 3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

[00218] In another embodiment, the PNPGl -specific antisense oligonucleotide comprises at least one modified sugar moiety, e.g. , a sugar moiety selected from arabinose, 2-fluoroarabinose, xylulose, and hexose.

[00219] In yet another embodiment, the PNPGl -specific antisense oligonucleotide comprises at least one modified phosphate backbone selected from a phosphor othioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

[00220] The antisense oligonucleotide can include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, Proc. Natl Acad. Sci. USA 1989; 86: 6553-6556; Lemaitre et al, Proc. Natl. Acad. Sci. USA 1987; 84: 648-652; PCT Publication No. WO

88/09810) or blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134), hybridization-triggered cleavage agents (see, e.g., Krol et al , BioTechniques 1988; 6: 958-976), intercalating agents (see, e.g., Zon, Pharm. Res. 1988; 5: 539-549), etc.

[00221] In another embodiment, the antisense oligonucleotide can include a- anomeric oligonucleotides. An c-anomeric oligonucleotide forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gautier et al , Nucl. Acids Res. 1987; 15: 6625-6641). [00222] In yet another embodiment, the antisense oligonucleotide can be a morpholino antisense oligonucleotide (t.e. , an oligonucleotide in which the bases are linked to 6-membered morpholine rings, which are connected to other morpholine- linked bases via non-ionic phosphorodiamidate intersubunit linkages). Morpholino oligonucleotides are resistant to nucleases and act by sterically blocking transcription of the target mRNA.

[00223] Similar to the above-described RNAi molecules, the antisense oligonucleotides of the invention can be synthesized by standard methods known in the art, e.g., by use of an automated synthesizer. Antisense nucleic acid oligonucleotides of the invention can also be produced intracellularly by transcription from an exogenous sequence. For example, a vector can be introduced in vivo such that it is taken up by a cell within which the vector or a portion thereof is transcribed to produce an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, so long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. In another embodiment, "naked" antisense nucleic acid molecules can be delivered to adherent cells via "scrape delivery", whereby the antisense oligonucleotide is added to a culture of adherent cells in a culture vessel, the cells are scraped from the walls of the culture vessel, and the scraped cells are transferred to another plate where they are allowed to re-adhere. Scraping the cells from the culture vessel walls serves to pull adhesion plaques from the cell membrane, generating small holes that allow the antisense oligonucleotides to enter the cytosol.

[00224] The present invention thus provides a method for inhibiting the expression of a PNPGl gene in a eukaryotic, preferably mammalian, and more preferably rat, mouse or human, cell, comprising providing the cell with an effective amount of a PNPGl -inhibiting antisense oligonucleotide. [00225] PNPGl oligonucleotides that may be useful as antisense reagents include those comprising the nucleotide sequence of SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9, or a fragment or a derivative thereof. 5.9.3. Ribozyme Inhibition

[00226] In another embodiment, the expression of PNPGl genes of the present invention can be inhibited by ribozymes designed based on the nucleotide sequence thereof.

[00227] Ribozyme molecules catalytically cleave mRNA transcripts and can be used to prevent translation of mRNA and, therefore, expression of the gene product. Ribozymes are enzymatic RNA molecules capable of catalyzing the sequence-specific cleavage of RNA (for a review, see Rossi, Current Biology 1994; 4: 469-471). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage event. The composition of ribozyme molecules must include: (i) one or more sequences complementary to the target gene mRNA; and (ii) a catalytic sequence responsible for mRNA cleavage (see, e.g., U.S. Patent No. 5,093,246).

[00228] According to the present invention, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA has the following sequence of two bases: 5'-UG-3'. The construction of hammerhead ribozymes is known in the art, and described more fully in Myers, Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, New York, 1995 (see especially Figure 4, page 833) and in Haseloff and Gerlach, Nature 1988; 334: 585-591.

[00229] Preferably, the ribozymes of the present invention are engineered so that the cleavage recognition site is located near the 5' end of the corresponding mRNA, i.e. , to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts. [00230] As in the case of RNAi and antisense oligonucleotides, ribozymes of the invention can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.). These can be delivered to mammalian cells, and preferably mouse, rat, or human cells, which express the target PNPGl protein in vivo. A method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous mRNA encoding the protein and inhibit translation. Because ribozymes, unlike antisense molecules, are catalytic, a lower intracellular concentration may be required to achieve an adequate level of efficacy.

[00231] Ribozymes can be prepared by any method known in the art for the synthesis of DNA and RNA molecules, as discussed above. Ribozyme technology is described further in Intracellular Ribozyme Applications: Principals and Protocols, Rossi and Couture eds., Horizon Scientific Press, 1999. 5.9.4. Triple Helix Formation

[00232] Nucleic acid molecules useful to inhibit PNPGl gene expression via triple helix formation are preferably composed of deoxynucleotides. The base composition of these oligonucleotides is typically designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex. Nucleotide sequences may be pyrimidine-based, resulting in TAT and CGC triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, nucleic acid molecules may be chosen that are purine-rich, e.g. , those containing a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex. [00233] Alternatively, sequences can be targeted for triple helix formation by creating a so-called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

[00234] Similarly to PNPGl -specific RNAi, antisense oligonucleotides, and ribozymes, triple helix molecules of the invention can be prepared by any method known in the art. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides such as, e.g. , solid phase phosphor amidite chemical synthesis. Alternatively, RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences "encoding" the particular RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. 5.10. Diagnostic Methods

[00235] A variety of methods can be employed for the diagnostic evaluation of pain, and for the identification and evaluation of subjects experiencing pain and related disorders. These methods may utilize reagents such as the nucleic acid molecules and oligonucleotides of the present invention. The methods may alternatively utilize an antibody or antibody fragment that binds specifically to a PNPGl protein. Such reagents can be used for: (i) the detection of either an over- or an under-expression of the PNPGl gene relative to its expression in an unaffected state (e.g. , in a subject or individual not having pain or a related disorder); (ii) the detection of either an increase or a decrease in the level of the PNPGl protein relative to its level in an unaffected state; and (iii) the detection of an aberrant PNPGl gene product activity relative to the unaffected state.

[00236] In another embodiment, a diagnostic method of the present invention utilizes quantitative hybridization (e.g. , quantitative in situ hybridization, Northern blot analysis or microarray hybridization) or quantitative PCR (e.g. , TaqMan^®) using a PNPGl -specific nucleic acid molecule of the invention as a hybridization probe and PCR primers, respectively.

[00237] The present invention provides a method for detecting a pain response in a test cell subjected to a treatment or stimulus, said method comprising: (a) determining the expression level of a nucleic acid molecule encoding a PNPGl protein in the test cell capable of expressing said nucleic acid molecule, which test cell has been subjected to a treatment or stimulus; and

(b) comparing the expression level of the PNPGl-encoding nucleic acid molecule in the test cell to the expression level of the same nucleic acid molecule in a control cell not subjected to the treatment or stimulus;

wherein a detectable change in the expression level of the PNPGl-encoding nucleic acid molecule in the test cell compared to the expression level of the PNPG1- encoding nucleic acid molecule in the control cell indicates that the test cell is exhibiting a pain response.

[00238] According to the present invention, the detectable change in the expression level is any statistically significant change and preferably at least a 1.5- fold change as measured by any available technique such as hybridization or quantitative PCR (see the Definitions Section, above).

[00239] The test and control cells are preferably the same type of cells from the same species and tissue, and can be any cells useful for conducting this type of assay where a meaningful result can be obtained. Any cell type in which a PNPGl- encoding nucleic acid molecule is ordinarily expressed, or in which a PNPG1- encoding nucleic acid molecule is expressed in connection with pain or a related treatment or stimulus, may be used. For example, the test cell can be any cell derived from a tissue of an organism experiencing a feeling of pain or associated disorder. Alternatively, the test cell can be any cell grown in vitro under specific conditions. When the test cell is derived from a tissue of an organism experiencing a feeling of pain or associated disorder, it may or may not be known to be located in the region associated with the feeling of pain.

[00240] In one embodiment, the test and control cells are cells from the central nervous system (CNS) or peripheral nervous system (PNS). Preferably, the test and control cells are neuronal cells from the dorsal root ganglia (DRG). The test and control cells can be derived from any appropriate organism, but are preferably human, rat or mouse cells. In a specific embodiment, the test and control cells are from an animal model of pain (e.g. , rat SNL model of neuropathic pain) or any related disorder, and may or may not be isolated from that animal model. In another embodiment, the first cell is from a subject, such as a human or companion animal, for which the test is being conducted to determine the pain state of that subject, and the second cell is an appropriate control cell. The first cell may or may not be isolated from the subject being tested.

[00241] The control cell can be any cell which is known to have not been subjected to any treatment or stimulus associated with pain. Preferably, the control cell is otherwise identical to the test cell. For example, when the test cell is derived from a tissue of an animal experiencing a feeling of pain or associated disorder, the control cell can be derived from an identical tissue or body part of a different animal from the same species (preferably closely related) not experiencing a feeling of pain or associated disorder. Alternatively, the control cell can be derived from an identical tissue or body part of the same animal, wherein it can be established that said identical tissue or body part has not been subjected to any treatment or stimulus associated with pain within the timeframe of the experiment. When the test cell is a cell grown in vitro under specific conditions, the control cell can be an identical cell grown in vitro in the absence of such specific conditions.

[00242] In one embodiment, the test cell has been exposed to a treatment or stimulus that is, or that simulates or mimics, a pain condition prior to determining the expression level of the nucleic acid molecule encoding the PNPGl protein, and the control cell is useful as an appropriate comparator cell to allow a determination of whether or not the test cell is exhibiting a pain response. For example, where the test cell has been exposed to a treatment or stimulus that is, or that simulates or mimics, a pain condition, the control cell has not been exposed to such a treatment or stimulus. In another embodiment, the test cell has been exposed to a compound that is being tested to determine whether it simulates or mimics a pain condition.

[00243] In one embodiment, the nucleic acid molecule the expression of which is being determined according to this method encodes a mammalian PNPGl protein. In one embodiment, the nucleic acid molecule encodes a rat, mouse or human PNPGl protein. In one embodiment, the rat PNPGl protein comprises the amino acid sequence of SEQ ID NO:2. In another embodiment, the mouse PNPGl protein comprises the amino acid sequence of SEQ ID NO:4. In another embodiment, the human PNPGl protein comprises the amino acid sequence of SEQ ID NO:6.

[00244] In one embodiment, the expression level of the nucleic acid molecule in each of the test and control cells is determined by quantifying the amount of PNPGl-encoding mRNA present in the two cells. In another embodiment, the expression level of the nucleic acid molecule in each of the test and control cells is determined by quantifying the amount of PNPGl protein present in each of the two cells. In another embodiment, the expression level of the nucleic acid molecule in each of the test and control cells is determined by quantifying the amount of PNPGl activity present in each of the two cells. Where the test cell has a detectable change in the expression level of the PNPGl-encoding nucleic acid molecule compared to the expression level of the PNPGl-encoding nucleic acid molecule in the control cell, a pain response in the test cell has been detected.

[00245] To assay levels of a PNPGl-encoding nucleic acid in a sample, a variety of standard nucleic acid isolation and quantification methods can be employed. As specified above, in another embodiment, a diagnostic method of the present invention utilizes quantitative hybridization (e.g. , quantitative in situ hybridization, Northern blot analysis or microarray hybridization) or quantitative PCR (e.g. , TaqMan^®) using PNPGl-specific nucleic acid molecules of the invention as hybridization probes and PCR primers, respectively. [00246] In PCR-based assays, gene expression can be measured after extraction of cellular mRNA and preparation of cDNA by reverse transcription (RT). A sequence within the cDNA can then be used as a template for a nucleic acid amplification reaction. Nucleic acid molecules of the present invention can be used to design PNPGl-specific RT and PCR oligonucleotide primers (such as, e.g. , SEQ ID NOS: 14, 15, 17, 18, 20, 21, and 23-28, see Table 2, below). Preferably, the oligonucleotide primers are at least about 9 to about 30 nucleotides in length. The amplification can be performed using, e.g. , radioactively labeled or fluorescently-labeled nucleotides, for detection. Alternatively, enough amplified product may be made such that the product can be visualized simply by standard ethidium bromide or other staining methods.

[00247] A preferred PCR-based detection method of the present invention is quantitative real time PCR (e.g. , TaqMan^® technology, Applied Biosystems, Foster City, CA). This method is based on the observation that there is a quantitative relationship between the amount of the starting target molecule and the amount of PCR product produced at any given cycle number. Real time PCR detects the accumulation of amplified product during the reaction by detecting a fluorescent signal produced proportionally during the amplification of a PCR product. The method takes advantage of the properties of Taq DNA polymerases having 5' exo- nuclease activity (e.g. , AmpliTaq^®) and Fluorescent Resonant Energy Transfer (FRET) method for detection in real time. The 5' exo-nuclease activity of the Taq DNA polymerase acts upon the surface of the template to remove obstacles downstream of the growing amplicon that may interfere with its generation. FRET is based on the observation that when a high-energy dye is in close proximity to a low-energy dye, a transfer of energy from high to low will typically occur. The real time PCR probe is designed with a high-energy dye termed a "reporter" at the 5' end, and a low-energy molecule termed a "quencher" at the 3' end. When this probe is intact and excited by a light source, the reporter dye's emission is suppressed by the quencher dye as a result of the close proximity of the dyes. When the probe is cleaved by the 5' nuclease activity of the Taq enzyme, the distance between the reporter and the quencher increases, causing the transfer of energy to stop, resulting in an increase of fluorescent emissions of the reporter, and a decrease in the fluorescent emissions of the quencher. The increase in reporter signal is captured by the Sequence Detection instrument and displayed. The amount of reporter signal increase is proportional to the amount of product being produced for a given sample. According to this method, the data is preferably measured at the exponential phase of the PCR reaction.

[00248] Specifically, a fluorogenic probe complementary to the target sequence is designed to anneal to the target sequence between the traditional forward and reverse primers. The probe is labeled at the 5' end with a reporter fluorochrome (e.g. , 6-carboxyfluorescein (6-FAM)). A quencher fluorochrome (e.g. , 6-carboxy-tetramethyl-rhodamine (TAMRA)) is added at any T position or at the 3' end. The probe is designed to have a higher melting temperature (Tm) than the primers, and during the extension phase, the probe must be 100% hybridized for success of the assay. As long as both fluorochromes are on the probe, the quencher molecule stops all fluorescence by the reporter. However, as Taq polymerase extends the primer, the intrinsic 5' nuclease activity of Taq degrades the probe, releasing the reporter fluorochrome which results in an increase in the fluorescence intensity of the reporter dye. The amount of fluorescence released during the amplification cycle is proportional to the amount of product generated in each cycle. This process occurs in every cycle and does not interfere with the accumulation of PCR product.

[00249] In a high throughput setting, to induce fluorescence during PCR, laser light is distributed to 96 sample wells via a multiplexed array of optical fibers. The resulting fluorescent emission returns via the fibers and is directed to a spectrograph with a charge-coupled device (CCD) camera. Emissions sent through the fiber to the CCD camera are analyzed by the software's algorithms. Collected data are subsequently sent to the computer. Emissions are measured, e.g. , every 7 seconds. The sensitivity of detection allows acquisition of data when PCR amplification is still in the exponential phase and makes real time PCR more reliable than end-point measurements of accumulated PCR products used by traditional PCR methods. [00250] Some of the preferred parameters of the quantitative real time PCR reactions of the present invention include: (i) designing the probe so that its Tm is 10°C higher than for the PCR primers, (ii) having primer Tm>s between 58°C and 60 °C, (iii) having amplicon sizes between 50 and 150 bases, and (iv) avoiding 5' Gs. However, other parameters can be used (e.g. , determined using Primer Express^® software, Applied Biosystems, Foster City, CA). For example, the best design for primers and probes to use for the quantitation of mRNA expression involves positioning of a primer or probe over an intron (exemplified by the primers/probes SEQ ID NOS: 14-22 disclosed in Table 2, below).

[00251] For more details on quantitative real time PCR, see Gibson et al ,

Genome Res. 1996; 6: 995-1001; Heid et α/. , Genome Res. 1996; 6: 986-994; Livak et al , PCR Methods Appl 1995; 4: 357-362; Holland et al , Proc. Natl Acad. Sci. USA 1991; 88: 7276-7280.

[00252] SYBR Green Dye PCR (Molecular Probes, Inc. , Eugene, OR), competitive PCR as well as other quantitative PCR techniques can also be used to quantify PNPGl gene expression according to the present invention.

[00253] PNPGl gene expression detection assays of the invention can also be performed in situ (e.g. , directly upon sections of fixed or frozen tissue collected from a subject, thereby eliminating the need for nucleic acid purification). Nucleic acid molecules of the invention or portions thereof can be used as labeled probes or primers for such in situ procedures (see, e.g. , Figures 7A-B and section 6.5., below; see also, e.g. , Nuovo, PCR in situ Hybridization: Protocols And Application, Raven Press, New York, 1992). Alternatively, if a sufficient quantity of the appropriate cells can be obtained, standard quantitative Northern analysis can be performed to determine the level of gene expression using the nucleic acid molecules of the invention or portions thereof as labeled probes (see, e.g. , Figure 2 and section 6.4. , below).

[00254] For in vitro cell cultures or in vivo animal models, the diagnostic reagents of the invention can be used in screening assays as surrogates for the pain state to find compounds that affect expression of the PNPGl gene. For example, probes for the human PNPGl gene can be used for diagnosing individuals experiencing a pain or a related condition, and also for monitoring the effectiveness of a pain therapy.

[00255] Various techniques can be used to measure the levels of PNPGl protein in a sample, including the use of anti-PNPGl antibodies or antibody fragments. Antibodies and various immunoassay methods also have important applications for assessing the efficacy of pain treatments. For example, anti-PNPGl antibodies or antibody fragments can be used in a method to screen test compounds to identify those compounds that can modulate PNPGl protein production. For example, anti-PNPGl antibodies or antibody fragments can be used to detect the presence of the PNPGl protein by, e.g. , immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric or fluorimetric detection methods. Such techniques are particularly preferred for detecting the presence of the PNPGl protein on the surface of cells. In addition, protein isolation methods such as those described by Harlow and Lane (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988) can also be employed to measure the levels of PNPGl protein in a sample.

[00256] Antibodies or antigen-binding fragments thereof may also be employed histologically, e.g. , in immunofluorescence or immunoelectron microscopy techniques, for in situ detection of the PNPGl protein. In situ detection may be accomplished by, e.g. , removing a tissue sample from a patient and applying to the tissue sample a labeled antibody or antibody fragment of the present invention. This procedure can be used to detect both the presence of the PNPGl protein and its distribution in the tissue. 5.11. Screening Methods

[00257] The present invention further provides a method for identifying a candidate compound useful for modulating the expression of a PNPGl-encoding nucleic acid, said method comprising: (a) contacting a first cell capable of expressing a PNPGl-encoding nucleic acid molecule of the present invention with a test compound under conditions sufficient to allow the cell to respond to said contact with the test compound; (b) determining the expression level of the PNPGl-encoding nucleic acid molecule in the first cell during or after contact with the test compound; and

(c) comparing the expression level of the PNPGl-encoding nucleic acid molecule determined in step (b) to the expression level of the PNPGl-encoding nucleic acid molecule in a second (control) cell capable of expressing the nucleic acid molecule, which second cell has not been contacted with the test compound;

wherein a detectable change in the expression level of the PNPGl-encoding nucleic acid molecule in the first cell in response to contact with the test compound compared to the expression level of the PNPGl-encoding nucleic acid molecule in the second (control) cell that has not been contacted with the test compound, indicates that the test compound modulates the expression of the PNPGl-encoding nucleic acid.

[00258] In one embodiment, the candidate compound decreases the expression of the PNPGl-encoding nucleic acid molecule. In another embodiment, the candidate compound increases the expression of a PNPGl-encoding nucleic acid molecule. In another embodiment, the first and second cells are incubated under conditions that induce the expression of a PNPGl-encoding nucleic acid molecule, but the test compound is tested for its ability to inhibit or reduce the induction of such expression in the first cell. In another embodiment, the first and second cells are incubated under conditions that induce the expression of a PNPGl-encoding nucleic acid molecule, but the test compound is tested for its ability to potentiate the induction of such expression in the first cell. [00259] This method of the present invention can be used to identify a candidate compound useful to treat a condition that can be treated by modulating the expression of a PNPGl-encoding nucleic acid of the present invention.

[00260] The test compound can be, without limitation, a small organic or inorganic molecule, a polypeptide (including an antibody, antibody fragment, or other immunospecific molecule), an oligonucleotide molecule, a nucleic acid molecule, or a chimera or derivative thereof.

[00261] The first and second cells are preferably the same types of cells, and can be any cells useful for conducting this type of assay where a meaningful result can be obtained. Such cells can be prokaryotic, but are preferably eukaryotic. Such eukaryotic cells are preferably mammalian cells, and more preferably rat, mouse or human cells. In one non-limiting embodiment, the first and second cells are cells that have been genetically modified to express or over-express a PNPGl nucleic acid molecule. In another non-limiting embodiment, the first and second cells are cells that express a PNPGl nucleic acid molecule, either naturally (e.g. , DRG cells) or in response to an appropriate stimulus. In one embodiment, the first and second cells have been exposed to a condition or stimulus that is, or that simulates or mimics, a pain condition prior to, or at the same time as, exposing the cells to the test compound to determine the effect of the test compound on the expression level of a nucleic acid molecule encoding a PNPGl protein.

[00262] In one embodiment, the first and second cells are from an animal model of pain (e.g. , rat SNL model of neuropathic pain or other animal models described below), and may or may not be isolated from that animal model. In another embodiment, the first cell is from a subject, such as a human or companion animal, and the second cell is an appropriate control cell. The first cell may or may not be isolated from the subject being tested.

[00263] In one embodiment, the nucleic acid molecule the expression of which is being determined according to this method encodes a mammalian PNPGl protein. In a further embodiment, the nucleic acid molecule encodes a rat, mouse or human PNPGl protein. In one embodiment, the rat PNPGl protein comprises the amino acid sequence of SEQ ID NO:2. In another embodiment, the mouse PNPGl protein comprises the amino acid sequence of SEQ ID NO:4. In another embodiment, the human PNPGl protein comprises the amino acid sequence of SEQ ID NO:6.

[00264] The expression level of the nucleic acid molecule in each of the first and second cells can be determined by quantifying the amount of PNPGl-encoding mRNA present in each of the first and second cells. Alternatively, the expression level of the nucleic acid molecule in each of the first and second cells can be determined by quantifying and comparing the amount of PNPGl protem present in the first and second cells. Where the first cell has a detectable change in the expression level of the nucleic acid encoding a PNPGl protein compared to the expression level of the nucleic acid encoding the PNPGl protein in the second cell, the test compound is identified as a candidate compound useful for modulating the expression of a PNPGl-encoding nucleic acid.

[00265] The present invention further provides a method for monitoring the efficacy of an analgesic treatment in a cell comprising: (a) administering to said cell an analgesic compound under conditions sufficient to allow the cell to respond to said compound; (b) determining in the cell prepared in step (a) the expression level of a PNPGl-encoding nucleic acid molecule; and

(c) comparing the expression level of the PNPGl-encoding nucleic acid molecule determined in step (b) to the expression level of the PNPGl-encoding nucleic acid molecule in a second (control) cell that has not been contacted with the analgesic compound;

wherein a detectable change in the expression level of the PNPGl-encoding nucleic acid molecule in the first cell in response to contact with the analgesic compound compared to the expression level of the PNPGl-encoding nucleic acid molecule in the second (control) cell that has not been contacted with the analgesic compound is indicative of the activity of the analgesic compound.

[00266] Also provided herein is a method for identifying a candidate compound useful for modulating the expression of a PNPGl protein, said method comprising: (a) contacting a first cell capable of expressing a PNPGl protein of the present invention with a test compound under conditions sufficient to allow the cell to respond to said contact with the test compound; (b) determining the expression level of the PNPGl protein in the first cell during or after contact with the test compound; and

(c) comparing the expression level of the PNPGl protein determined in step (b) to the expression level of the PNPGl protein in a second (control) cell capable of expressing the protein that has not been contacted with the test compound;

wherein a detectable change in the expression level of the PNPGl protein in the first cell in response to contact with the test compound compared to the expression level of the PNPGl protein in the second (control) cell that has not been contacted with the test compound, indicates that the test compound modulates the expression of the PNPGl protein and is a candidate compound.

[00267] The present invention further provides a method for identifying a candidate compound capable of binding to a PNPGl protein, said method comprising: (a) contacting a PNPGl protein or peptide fragment of the present invention with a test compound under conditions that permit binding of the test compound to the PNPGl protein or peptide fragment; and

(b) detecting binding of the test compound to the PNPGl protem or peptide fragment. [00268] The present invention further provides a method for identifying a candidate compound capable of modulating the activity of a PNPGl protein, said method comprising:

(a) contacting a PNPGl protein with a test compound under conditions that permit modulation of the activity of the PNPGl protein; and

(b) determining whether the activity of the PNPGl protein is modulated in response to said contact;

wherein a detectable change in the activity of the PNPGl protein in response to contact with the test compound indicates that the test compound modulates the activity of the PNPGl protein and is a candidate compound. In a specific embodiment, the activity of a PNPGl protein is a transporter activity.

[00269] The above-identified screening methods can be used to identify a candidate compound that can be used to treat a condition that can be treated by modulating the expression and/or activity of a PNPGl protein of the present invention.

[00270] Appropriate controls can be included in the binding assay to ensure that the binding of the compound to the PNPGl protein or peptide variant is specific.

[00271] The test compound can be, without limitation, a small organic or inorganic molecule, a polypeptide (including an antibody, antibody fragment, or other immunospecific molecule), an oligonucleotide molecule, a nucleic acid molecule, or a chimera or derivative thereof.

[00272] Test compounds that specifically bind to a PNPGl-encoding nucleic acid molecule or to a PNPGl protein of the present invention can be identified, for example, by high-throughput screening (HTS) assays, including cell-based and cell- free assays, directed against individual protein targets. Several methods of automated assays that have been developed in recent years enable the screening of tens of thousands of compounds in a short period of time (see, e.g., U.S. Patent Nos. 5,585,277, 5,679,582, and 6,020,141). Such HTS methods are particularly preferred.

[00273] In conjunction with the screening methods of the present invention, also provided herein are the following features:

(1) a method for identifying a ligand or binding partner to the PNPGl protein of the present invention, which comprises bringing a labeled test compound in contact with the PNPGl protein or a fragment thereof and measuring the amount of the labeled test compound specifically bound to the PNPGl protein or to the fragment thereof;

(2) a method for identifying a ligand or binding partner to the PNPGl protein of the present invention, which comprises bringing a labeled test compound in contact with cells or a cell membrane fraction containing the PNPGl protein, and measuring the amount of the labeled test compound specifically bound to the cells or the membrane fraction; and,

(3) a method for determining a ligand or binding partner to the PNPGl protein of the present invention, which comprises culturing a transfected cell containing the DNA encoding the PNPGl protein under conditions that permit or induce expression of the PNPGl protein, bringing a labeled test compound in contact with the PNPGl protein expressed on a membrane of said cell, and measuring the amount of the labeled test compound specifically bound to the PNPGl protein.

[00274] For example, the ligand or binding partner of the PNPGl protein of the present invention can be determined by the following procedures. First, a standard PNPGl preparation can be prepared by suspending cells containing the PNPGl protein, or the membrane fraction of such cells, in a buffer appropriate for use in the determination method. Any buffer can be used so long as it does not inhibit the ligand-PNPGl binding. Such buffers include, e.g. , a phosphate buffer or a Tris-HCI buffer having pH of 4 to 10 (preferably pH of 6 to 8). For the purpose of minimizing non-specific binding, a surfactant such as CHAPS, Tween-80™ (manufactured by Kao-Atlas Inc.), digitonin or deoxycholate, and various proteins such as bovine serum albumin or gelatin, may optionally be added to the buffer. For the purpose of suppressing degradation of the PNPGl or ligand by proteases, a protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Institute, Inc.) and pepstatin can be added. A given amount (e.g. , 5,000 to 500,000 cpm) of the test compound labeled with [Η], [¹²⁵I], [¹⁴C], [³⁵S] or the like can be added to about 0.01 ml to 10 ml of the solution containing PNPGl. To determine the amount of non-specific binding (NSB), a reaction tube containing an unlabeled test compound in a large excess is also prepared. The reaction is carried out at approximately 0 to 50°C, preferably about 4 to 37°C for about 20 minutes to about 24 hours, preferably about 30 minutes to about 3 hours. After completion of the reaction, the cells or membranes containing any bound ligand are separated, e.g. , the reaction mixture is filtered through glass fiber filter paper and washed with an appropriate volume of the same buffer. The residual radioactivity on the glass fiber filter paper can be measured by means of a liquid scintillation counter, a β-counter, or a γ-counter. A test compound exceeding 0 cpm obtained by subtracting NSB from the total binding (B) (B minus NSB) may be selected as a ligand or binding partner of the PNPGl protein of the present invention.

[00275] The present invention further provides a method for studying additional biological activities of the PNPGl protein. The biological activity of the PNPGl protein can be studied using: (i) intact cells that express the PNPGl protein (either constitutively or as a result of a specific stimulus or treatment); (ii) membrane fractions comprising the PNPGl protein; or (iii) the isolated PNPGl protein (e.g. as soluble PNPGl protein, peptide fragments or PNPGl fusion proteins). For example, a biological activity of the PNPGl protein can be studied by measuring in a cell that expresses the PNPGl protein: (i) an activity that promotes or suppresses the production of a particular "index substance" (e.g. , arachidonic acid release, acetylcholine release, intracellular Ca²⁺ release, intracellular cAMP production, intracellular cGMP production, or inositol phosphate production); (ii) a change in cell membrane potential; (iii) phosphorylation of an intracellular protein; (iv) activation of c-fos; (v) pH reduction, etc. [00276] PNPGl -mediated activities can be determined by any known method.

For example, cells containing the PNPGl protein can first be cultured on a multi- well plate, etc. Prior to the activity determination, the medium can be replaced with fresh medium or with an appropriate non-cytotoxic buffer, followed by incubation for a given period of time in the presence of a test compound, etc. Subsequently, the cells can be extracted or the supernatant can be recovered and the resulting product can be quantified by appropriate procedures. Where it is difficult to detect the production of the "index substance" for the cell-stimulating activity due to a degrading enzyme contained in the cells, an inhibitor against such a degrading enzyme may be added prior to the assay. For detecting activities such as the cAMP production suppression activity, the baseline production in the cells can be increased by forskolin or the like and the suppressing effect on the increased baseline production may then be detected.

[00277] In one embodiment, the PNPGl protein is a mammalian PNPGl protein. In one embodiment, the PNPGl protein is a rat, mouse or human PNPGl protein. In one embodiment, the rat PNPGl protein comprises the amino acid sequence of SEQ ID NO:2. In another embodiment, the mouse PNPGl protein comprises the amino acid sequence of SEQ ID NO:4. In another embodiment, the human PNPGl protein comprises the amino acid sequence of SEQ ID NO: 6. 5.12. Methods of Treatment

[00278] The present invention provides a method for treating a condition that can be treated by modulating expression of a PNPGl-encoding nucleic acid molecule or a PNPGl protein, comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound that modulates expression of a PNPGl-encoding nucleic acid molecule or a PNPGl protein.

[00279] Conditions that can be treated using the method disclosed herein include a pain condition or a pain-related disorder selected without limitation from chronic pain, nociceptive pain, neuropathic pain (including all types of hyperalgesia (i.e. , sensation of more pain than the stimulus would warrant) and allodynia (i.e. , a condition in which ordinarily painless stimuli induce the experience of pain)), inflammatory and cancer pain, addiction, seizure (including epilepsy), stroke or ischemia, neurodegenerative disorder (e.g. , Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), or Huntington's chorea), headache (e.g. , general, migraine, cluster or tension), anxiety, depression, asthma, rheumatic disease, osteoarthritis, retinopathy, inflammatory eye disorders, pruritis, ulcer (gastric or duodenal), gastric lesions (e.g. , induced by a necrotizing agent), uncontrollable urination (e.g. , incontinence), an inflammatory or unstable bladder disorders, inflammatory bowel disease (e.g. , Crohn's disease and ulcerative colitis), irritable bowel syndrome (IBS) including irritable bowel disease (IBD), gastroesophageal reflux disease (GERD), functional dyspepsia (e.g. , ulcer-like dyspepsia, dysmotility-like dyspepsia, functional heartburn, and non-ulcer dyspepsia), functional chest pain of presumed esophageal origin, functional dysphagia, non-cardiac chest pain, symptomatic gastroesophageal disease, gastritis, aerophagia, functional constipation, functional diarrhea, burbulence, chronic functional abdominal pain, recurrent abdominal pain (RAP), functional abdominal bloating, functional biliary pain, functional incontinence, functional ano-rectal pain, chronic pelvic pain, pelvic floor dyssenergia, unspecified functional ano-rectal disorder, cholecystalgia, interstitial cystitis, dysmenorrhea, and dyspareunia.

[00280] In another embodiment, the condition treated by the method of the present invention is neuropathic pain. In another embodiment, the condition treated by the method of the present invention is chronic pain.

[00281] The term "therapeutically effective amount" is used here to refer to an amount or dose of a compound sufficient to: (i) detectably change the level of expression of a PNPGl-encoding nucleic acid or PNPGl protein in a subject; or (ii) detectably change the level of activity of a PNPGl protein in a subject; or (iii) cause a detectable improvement in a clinically significant symptom or condition (e.g. , amelioration of pain) in a subject.

[00282] A candidate compound useful in conducting a therapeutic method of the present invention is advantageously formulated in a pharmaceutical composition with a pharmaceutically acceptable carrier. The candidate compound may be designated as an active ingredient or therapeutic agent for the treatment of pain or other indication.

[00283] The concentration of the active ingredient depends on the desired dosage and administration regimen, as discussed below. Suitable doses ranges of the active ingredient are from about 0.01 mg/kg to about 1500 mg/kg of body weight per day.

[00284] Therapeutically effective compounds can be provided to the patient in standard formulations, and may include any pharmaceutically acceptable additives, such as excipients, lubricants, diluents, flavor ants, colorants, buffers, and disintegrants. The formulation may be produced in useful dosage units for administration by oral, parenteral, transmucosal, intranasal, rectal, vaginal, or transdermal routes or locally by topical administration. Parental routes include intravenous, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration.

[00285] The pharmaceutical composition may also include other biologically active substances in combination with the candidate compound. Such substances include but are not limited to opioids, non-steroidal anti-inflammatory drugs

(NSAIDs), and other analgesics. *

[00286] The pharmaceutical composition can be added to a retained physiological fluid such as blood or synovial fluid. For CNS administration, a variety of techniques are available for promoting transfer of the therapeutic agent across the blood brain barrier, including disruption by surgery or injection, co- administration of a drug that transiently opens adhesion contacts between CNS vasculature endothelial cells, and co-administration of a substance that facilitates translocation tlirough such cells.

[00287] In another embodiment, the active ingredient can be delivered in a vesicle, particularly a liposome.

[00288] In another embodiment, the therapeutic agent can be delivered in a controlled release manner. For example, a therapeutic agent can be administered using intravenous infusion with a continuous pump, in a polymer matrix such as poly-lactic/glutamic acid (PLGA), in a pellet containing a mixture of cholesterol and the active ingredient (SilasticR™; Dow Corning, Midland, MI; see U.S. Patent No. 5,554,601), by subcutaneous implantation, or by transdermal patch. 5.13. Neuronal Cell Cultures

[00289] DRG neuronal cell cultures are useful in practicing various aspects of the present invention, and can be prepared using ordinary techniques known in the art. The screening methods and biological activity assays of the present invention can use cultured cells or cell lines to screen for candidate compounds useful as therapeutic agents. The cells are preferably neurons or other cells present in CNS or PNS.

[00290] Cultured post-mitotic or neuronal precursors can be obtained using various methods. As one example, primary neurons or neural progenitor cells are isolated and cultured according to methods known in the art (see, e.g., U.S. Patent No. 5,654,189). Examples of neurons useful for carrying out the methods of the present invention include brain or spinal cord neurons collected from mammals, and neuronal cell lines grown in the presence of growth factors such as NGF (nerve growth factor), IGF (insulin-like growth factor), etc.

[00291] For example, DRG neurons from rats can be dissociated following the procedure of Caldero et al , J. Neurosci. 1998; 18: 356-370. Following dissociation, neurons can be placed in tissue culture dishes or micro-wells coated, e.g., with ornithine-laminin, in medium supplemented with glutamine, fetal bovine serum (FBS), putrescine, sodium selenite, progesterone and antibiotics (see, e.g. , Baudet et al , Development 2000; 127: 4335-4344). Growth factors such as NGF, FGF (fibroblast growth factor), EGF (epidermal growth factor), interieukin 6 (Ann. Rev. Pharmacol. Toxicol. 1991; 31: 205-228), IGF (J. Cell Biol. 1986; 102: 1949- 1954), and those described in Cell Culture in the Neurosciences , New York: Plenum Press, 1955, pp. 95-123, can also be included.

[00292] In another embodiment, transformed neuronal cell lines, such as those created with tetracarcinoma cell lines, can also be used. [00293] In another embodiment, clonal cell lines can be isolated from a conditionally immortalized neural precursor cell line (see, e.g., U.S. Patent No. 6,255,122). A skilled artisan will readily appreciate that cells or cell cultures used in the methods of this invention should be carefully controlled for parameters such as cell passage number, cell density, the methods by which the cells are dispensed, and growth time after dispensing. 5.14. Animal Models of Pain

[00294] As specified above, the diagnostic and screening methods of the present invention can be conducted in (i) any cell derived from a tissue of an organism experiencing a feeling of pain or a pain-related condition or (ii) any cell grown in vitro in tissue culture under specific conditions that mimic some aspect of tissue conditions in an organism experiencing a feeling of pain (e.g. , nerve injury, inflammation, viral infection, etc.). Particularly useful for the diagnostic and screening methods of the present invention are cells (especially neural cells) derived from animal models of pain and related disorders. The PNPGl gene of the invention was identified using a rat spinal nerve ligation (SNL) model of neuropathic pain (Kim and Chung, Pain 1992; 50: 355-363), which is a particularly useful source of cells in the methods of the present invention. Some of the additional useful models are described below. 5.14.1. FCA Injection Model

[00295] A chronic pain condition can be reproduced in mice or rats by the injection of Freund's complete adjuvant (FCA) containing heat-killed Mycobacterium into the base of the tail or into the hind footpads (Colpaert et al. , Life Sci. 1980; 27: 921-928; De Castro Costa et al , Pain 1981; 10: 173-185; Larson et al. , Pharmacol. Biochem Behav. 1986; 24: 9-53).

[00296] For example, a chronic pain condition can be induced by intradermal injection of 50 μl of 50% FCA into one hindpaw, wherem undiluted FCA consists of 1 mg/ml heat-killed and dried Mycobacterium, each ml of vehicle contains 0.85 ml paraffin oil + 0.15 ml mannide monooleate (Sigma, St. Louis, MO), and FCA is diluted in a ratio of 1: 1 (vol: vol) with 0.9% saline. Intradermal injection can be performed under isoflurane/02 inhalation anesthesia. The treated and control (e.g. , given an intradermal injection of 0.9% saline) animals can be tested between 24 and 72 hours following FCA injection.

[00297] FCA injection causes an inflammation (in the case of injection into the base of the tail, wide-spread joint inflammation mimicking rheumatoid arthritis) that lasts for several days, and is evidenced by the classical signs of inflammation (erythema, edema, heat), as well as hyperalgesia (e.g. , to thermal and mechanical stimuli) and allodynia (Fundytus et al , Pharmacol Biochem & Behav 2002; 73: 401-410; Binder et al , Anesthesiology 2001; 94: 1034-1044). The pain sensitivity (i.e. , alterations in nociceptive thresholds) can then be measured in the injected and neighboring regions by decreases in response latency (compared to control animals injected with either the same adjuvant lacking heat-killed Mycobacterium or 0.9% saline). For example, thermal hyperalgesia can be assessed by applying focused radiant heat to the plantar surface of the hindpaw and measuring the latency for the animal to withdraw its paw from the stimulus (Hargreaves et al , Pain 1988; 32: 77- 88; D'Amour and Smith, J. Pharmacol. Exp. Ther. 1941; 72: 74-79; see also the hot-plate assay described by Eddy and Leimbach, J. Pharmacol. Exp. Ther. 1953; 107: 385-393). A decrease in the paw withdrawal latency following FCA injection indicates thermal hyperalgesia. Mechanical hyperalgesia can be assessed with the paw pressure test, where the paw is placed on a small platform, and weight is applied in a graded manner until the paw is completely withdrawn (Stein, Biochemistry & Behavior 1988; 31: 451-455, see also the Examples section, below). Mechanical allodynia can be also assessed by applying thin filaments (von Frey hairs) to the plantar surface of the hindpaw and determining the response threshold for paw withdrawal (see Dixon, J. Am Stat. Assoc. 1965; 60: 967-978). 5.14.2. Sciatic Nerve Injury Models

[00298] The first animal model of neuropathic pain to be developed was the simple cutting of the sciatic nerve, termed "axotomy" (Wall et al , Pain 1979; 7: 103-111). Following axotomy, neuromas form at the ends of the cut nerve. With this type of injury, self-mutilation of the injured foot, termed "autotomy", is often observed. [00299] In this model, a unilateral nerve injury is induced by exposing and cutting one sciatic nerve. The ends of the cut sciatic nerve are then ligated to prevent re-growth. Surgery is performed under isoflurane/O₂ anesthesia. The wound is closed with 4-0 Vicryl, dusted with antibiotic powder, and the animals are allowed to recover on a warm heating pad before being returned to their home cages. Sham-operated animals are used as a control. Sham-operation consists of exposing but not injuring one sciatic nerve. Animals are observed for up to two weeks to assess pain behaviors. Animals are tested with the thermal and mechanical tests as described above.

[00300] One of the most commonly used experimental animal models for neuropathic pain is the chronic constriction injury (CCI) where four loose ligatures are tied around the sciatic nerve (Bennett and Xie, Pain 1988; 33: 87-107). One disadvantage of this model is the introduction of foreign material into the wound, which causes a local inflammatory reaction, whereas hyperalgesia does not have to be associated with inflammation. Thus, the distinction between the neuropathic and the inflammatory component of pain is difficult in this model. In order to produce a pure nerve injury model without an epineurial inflammatory component due to foreign material, Lindenlaub and Sommer (Pain 2000; 89: 97-106) have recently performed a partial sciatic nerve transection (PST) in rats. These rats developed thermal hyperalgesia and mechanical allodynia comparable to the CCI model. In both models, animals' thermal withdrawal thresholds are commonly assessed by response to radiant heat on the plantar surface of the hindpaw (Hargreaves et al , Pain 1988; 32: 77-88). Mechanical hypersensitivity is commonly determined by measuring the withdrawal thresholds to von Frey hairs (Dixon, J. Am Stat. Assoc. 1965; 60: 967-978).

[00301] Decosterd and Woolf have recently developed a variant of partial denervation, the spared nerve injury model (Decosterd and Woolf, Pain 2000, 87: 149-58). This model involves a lesion of two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve intact. The spared nerve injury model differs from the SNL, CCI and PST models in that the co-mingling of distal intact axons with degenerating axons is restricted, and it permits behavioral testing of the non-injured skin territories adjacent to the denervated areas. The spared nerve injury model results in early (less than 24 hours), prolonged (greater than 6 months), robust (all animals are responders) behavioral modifications. The mechanical sensitivity (as determined, e.g. , by sensitivity to von Frey hairs and pinprick test) and thermal (hot and cold) responsiveness is increased in the ipsilateral sural and to a lesser extent saphenous territories, without any change in heat thermal thresholds. 5.14.3. Cancer Pain Models

[00302] The models of neuropathic pain described above involve acute or sub- acute insult of the peripheral nerve, and do not necessarily reflect gradual but progressive insult of the nerve, which is expected to occur in such common neuropathic pain conditions as neuropathic cancer pain. Neuropathic cancer pain can, however, be, reproduced by inoculating Meth A sarcoma cells to the immediate proximity of the sciatic nerve in BALB/c mice (Shimoyama et al. , Pain 2002; 99: 167-174). The tumor grows predictably with time and gradually compresses the nerve, thereby causing thermal hyperalgesia (as determined, e.g. , by paw withdrawal latencies to radiant heat stimulation), mechanical allodynia (as determined, e.g. , by sensitivity of paws to von Frey hairs), and signs of spontaneous pain (as detected, e.g. , by spontaneous lifting of the paw).

[00303] A rat model of bone cancer pain was also recently reported

(Medhurst et al , Pain 2002; 96: 129-40). In this model, Sprague-Dawley rats receive intra-tibial injections of 3 x 10³ or 3 x IO⁴ syngeneic MRMT-1 rat mammary gland carcinoma cells, which produce rapidly expanding tumors within the boundaries of the tibia, causing severe remodeling of the bone. Rats receiving intra- tibial injections of MRMT-1 cells develop behavioral signs indicative of pain, including the gradual development of mechanical allodynia and mechanical hyperalgesia/reduced weight bearing on the affected limb, beginning on day 12-14 or 10-12 following injection of 3 x 10³ or 3 x 10⁴ cells, respectively. These symptoms are not observed in rats receiving heat-killed cells or vehicle. Acute treatment with morphine produces a dose-dependent reduction in the response frequency of hind paw withdrawal to von Frey hairs as well as reduction in the difference in hind limb weight bearing. 5.14.4. Incisional Model of Post-Operative Pain

[00304] Brennan and colleagues have developed an animal model of post- operative pain (Brennan et al, Pain 1996; 64: 493-501), which involves making a surgical incision on the plantar aspect of the rat hindpaw. The mechanical hyperalgesia that is observed in this rat model parallels the time course of pain in post-operative patients, and is alleviated by systemic and intrathecal (i.t.) morphine (Zahn et al , Anesthesiology 1997; 86: 1066-1077).

[00305] Specifically, a 1 cm incision is made in the plantar surface of one hindpaw under isoflurane/O₂ inhalation anesthesia. The incision is closed with two sutures using 4-0 Vicryl. Rats are allowed to recover in their home cages. Naϊve rats are used as control animals. Mechanical and thermal sensitivity is measured 24 hours after injury, e.g. , as described above. 5.15. Use of Arrays

[00306] . In one embodiment, the differentially expressed gene of the present invention, i.e. , the PNPGl gene, will be used in screening methods comprising microarrays. 5.15.1. Cell-Based Arrays

[00307] Cell-based arrays combine the technique of cell culture in conjunction with the use of fluidic devices for measurement of cell response to test compounds in a sample of interest, screening of samples for identifying molecules that induce a desired effect in cultured cells, and selection and identification of cell populations with novel and desired characteristics. High-throughput screens (HTS) can be performed on fixed cells using fluorescent-labeled antibodies, biological ligands and/or nucleic acid hybridization probes, or on live cells using multicolor fluorescent indicators and biosensors. The choice of fixed or live cell screens depends on the specific cell-based assay required. [00308] There are numerous single- and multi-cell-based array techniques known in the art. Recently developed techniques such as micro-patterned arrays (described, e.g. , in International PCT Publications WO 97/45730 and WO 98/38490) and microfluidic arrays provide valuable tools for comparative cell-based analysis. Transfected cell microarrays are a complementary technique in which array features comprise clusters of cells overexpressing defined cDNAs. Complementary DNAs cloned in expression vectors are printed on microscope slides, which become living arrays after the addition of a lipid transfection reagent and adherent mammalian cells (Bailey et al , Drug Discov. Today 2002; 7(18 Suppl): S113-8). Cell-based arrays are described in detail in, e.g., Beske, Drug Discov. Today 2002; 7(18 Suppl): S131-5; Sundberg et al , Curr. Opin. Biotechnol. 2000; 11: 47-53; Johnston et al , Drug Discov. Today 2002; 7: 353-63; U.S. Patents No. 6,406,840 and 6,103,479, and U.S. published patent application No. 2002/0197656. For cell-based assays specifically used to screen for modulators of ligand-gated ion channels, see Mattheakis et al. , Curr. Opin. Drug Discov. Devel. 2001; 1: 124-34; and Baxter et al , J. Biomol. Screen. 2002; 7: 79-85.

5.15.2. Protein Arrays

[00309] Protein arrays are solid-phase, ligand binding assay systems using immobilized proteins on surfaces that are selected from glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles. The ligand binding assays using these arrays are highly parallel and often miniaturized. Their advantages are that they are rapid, can be automated, are capable of high sensitivity, are economical in their use of reagents, and provide an abundance of data from a single experiment.

[00310] Automated multi-well formats are the best-developed HTS systems.

Automated 96-well plate-based screening systems are the most widely used. The current trend in plate based screening systems is to reduce the volume of the reaction wells further, thereby increasing the density of the wells per plate (96 wells to 384 wells, and 1,536 wells per plate). The reduction in reaction volumes results in increased throughput, dramatically decreased bioreagent costs, and a decrease in the number of plates that need to be managed by automation. For a description of protein arrays that can be used for HTS, see, e.g. , U.S. Patents No. 6,475,809; 6,406,921; and 6,197,599; and International Publications No. WO 00/04389 and WO 00/07024.

[00311] For construction of arrays, sources of proteins include cell-based expression systems for recombinant proteins, purification from natural sources, production in vitro by cell-free translation systems, and synthetic methods for peptides. For capture arrays and protein function analysis, it is important that proteins are correctly folded and functional. This is not always the case, e.g., where recombinant proteins are extracted from bacteria under denaturing conditions, whereas other methods (isolation of natural proteins, cell free synthesis) generally retain functionality. However, arrays of denatured proteins can still be useful in screening antibodies for cross-reactivity, identifying auto-antibodies, and selecting ligand binding proteins.

[00312] The immobilization method used should be reproducible, applicable to proteins of different properties (size, hydrophilic, hydrophobic), amenable to high throughput and automation, and compatible with retention of fully functional protein activity. Both covalent and non-covalent methods of protein immobilization can be used. Substrates for covalent attachment include, e.g. , glass slides coated with amino- or aldehyde-containing silane reagents (Telechem). In the Versalinx™ system (Prolinx), reversible covalent coupling is achieved by interaction between the protein derivatized with phenyldiboronic acid, and salicylhydroxamic acid immobilized on the support surface. Covalent coupling methods providing a stable linkage can be applied to a range of proteins. Non-covalent binding of unmodified protein occurs within porous structures such as HydroGel™ (PerkinElmer), based on a 3-dimensional polyacrylamide gel. 5.15.3. Detection

[00313] For detection of molecules using screening assays, a molecule (e.g., an antibody or nucleic acid probe) can be detectably labeled with an atom (e.g., radionuclide), detectable molecule (e.g., fluorescein), or complex that, due to its physical or chemical property, serves to indicate the presence of the molecule. A molecule can also be detectably labeled when it is covalently bound to a "reporter" molecule (e.g., a biomolecule such as an enzyme) that acts on a substrate to produce a detectable product. Detectable labels suitable for use in the present invention include any composition detectable by specfroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the present invention include, but are not limited to, biotin for staining with labeled avidin or streptavidin conjugate, magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein, fluorescein-isothiocyanate (FITC), Texas red, rhodamine, green fluorescent protein, enhanced green fluorescent protein, lissamine, phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX [Amersham], SyBR Green I & II [Molecular Probes], and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., hydrolases, particularly phosphatases such as alkaline phosphatase, esterases and glycosidases, or oxidoreductases, particularly peroxidases such as horse radish peroxidase, and the like), substrates, cofactors, inhibitors, chemiluminescent groups, chromogenic agents, and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Examples of patents describing the use of such labels include U.S. Patents No. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

[00314] Means of detecting such labels are known to those of skill in the art.

For example, radiolabels and chemiluminescent labels can be detected using photographic film or scintillation counters; fluorescent markers can be detected using a photo-detector to detect emitted light (e.g., as in fluorescence-activated cell sorting); and enzymatic labels can be detected by providing the enzyme with a substrate and detecting, e.g. , a colored reaction product produced by the action of the enzyme on the substrate.

[00315] The present invention is now described, in detail, by way of the following particular examples. However, the use of such examples is illustrative only and is not intended to limit the scope or meaning of this invention or of any exemplified term. Nor is the invention limited to any particular preferred embodiment(s) described herein. Indeed, many modifications and variations of the invention will be apparent to those skilled in the art upon reading this specification, and such "equivalents" can be made without departing from the invention in spirit or scope. The invention is therefore limited only by the terms of the appended claims, along with the full scope of equivalents to which the claims are entitled. 6. EXAMPLE: PNPGl Gene Expression is Modulated in a Neuropathic Pain Model

[00316] The present inventors have identified a novel gene PNPGl. The change in PNPGl gene expression in the SNL model of neuropathic pain parallels change in expression of several genes known to be molecular mediators of pain, thereby linking PNPGl to a role in mediating or responding to pain. 6.1. Preparation of Neuropathic Pain Model

[00317] Rats having the L5-L6 spinal nerves ligated (SNL) according to the method of Kim and Chung, Pain 1992; 50:355-63 were used in this experiment. Briefly, nerve injury was induced by tight ligation of the left L5 and L6 spinal nerves, producing symptoms of neuropathic pain as described below. The advantage of this model is that it allows the investigation of dorsal root ganglia that are injured (L5 and L6) versus dorsal root ganglia that are not injured (L4). Thus, it is possible to see changes in gene expression specifically in response to nerve injury. Surgery was performed under isoflurane/O2 inhalation anesthesia. Following induction of anesthesia, a 3 cm incision was made just lateral to the spinal vertebrae. The left paraspinal muscles were separated from the spinous process at the L4-S2 levels. The L6 transverse process was carefully removed with a pair of small rongeurs to visually identify the L4-L6 spinal nerves. The left L5 and L6 spinal nerves were isolated and tightly ligated with 7-0 silk suture. A complete hemostasis was confirmed, and the wound was sutured using non- absorbable sutures, such as 4-0 Vicryl.

[00318] Both naϊve and sham-operated animals were used as controls. Sham- operation consisted of exposing the spinal nerves without ligation or manipulation. After surgery, animals were weighed and administered a subcutaneous (s.c.) injection of Ringers lactate solution. Following injection, the wound area was dusted with antibiotic powder and the animals were kept on a warm pad until recovery from anesthesia. Animals were then returned to their home cages until behavioral testing. The naϊve control group consisted of rats that were not operated on (naϊve). Eight to twelve rats in each group were evaluated.

[00319] Some rats from the SNL and naϊve groups were also treated with gabapentin (GPN) as described below. Gabapentin (GPN), an anti-convulsant, has been shown in the clinic to be effective for treating neuropathic pain (Mellegers et al , Clin. J. Pain 2001; 17: 284-295; Rose and Kam, Anaesthesia 2002; 57: 451- 462).

[00320] L4, L5 and L6 DRGs from the SNL model of neuropathic pain were used to identify genes involved in mediating and responding to pain (including genes affected by GPN treatment) by using expression profiling, which is based on identifying probes on a "genome-scale" microarray that are differentially expressed in SNL DRGs as compared to DRGs of naϊve and sham-operated animals.

Table 1 summarizes five experimental groups consisting of sham surgery, naϊve or SNL surgery with or without GPN treatment:

6.2. Behavioral Testing

[00321] Mechanical sensitivity was assessed using the paw pressure test. This test measures mechanical hyperalgesia. Hind paw withdrawal thresholds ("PWT") (measured in grams) in response to a noxious mechanical stimulus were determined using an analgesymeter (Model 7200, commercially available from Ugo Basile of Italy), as described in Stein, Biochemistry & Behavior 1988; 31: 451-455. The rat's paw was placed on a small platform, and weight was applied in a graded manner up to a maximum of 250 grams. The endpoint was taken as the weight at which the paw was completely withdrawn. PWT was determined once for each rat at each time point, and only the injured ipsilateral paw (i.e. , the hind paw on the same side of the animal as the ligation in SNL animals, or the side of the animal where the nerve was exposed but not injured in sham-operated animals) was used in the test. For naϊve animals, the left paw or the side that "would have been" subjected to surgery (herein also referred to as "ipsilateral") was used for the test.

[00322] Rats were tested prior to injury (SNL or sham surgery; naϊve rats were tested at the same time) to determine a baseline, or normal, PWT. To verify that the surgical procedure was successful, rats were again tested at 12-14 days after surgery. At this time, rats with an SNL injury should exhibit a significantly reduced PWT compared to their baseline PWT, while sham-operated and naϊve rats should have PWT that is not significantly different from their baseline PWT. Only rats that met these criteria were included in further behavioral testing and the gene expression study.

[00323] Rats that met the behavior criteria were divided into the treatment, groups (described above): 1) naϊve + vehicle; 2) naϊve + GPN; 3) sham + vehicle; 4) SNL + vehicle; 5) SNL + GPN (Table 1). Vehicle (0.9% saline) and GPN (dissolved in 0.9% saline) were administered intraperitoneally (i.p.) in a volume of 2 ml/kg. The dose of GPN was 100 mg/kg. The rats in the above treatment groups were treated each day for 7 days (with either vehicle or GPN as per their group), and on the last (7^th) treatment day (corresponding to 19-21 days post surgery), rats were again assessed for mechanical sensitivity using the paw pressure test described above, in particular to confirm the reversal of neuropathic pain with GPN treatment. Following testing, tissues were collected as described below. See Figure 11 for a summary of the experimental timelines for surgery, treatment, and testing. 6.3. Determining Gene Expression Profiles in the SNL Model 6.3.1. Tissue Collection and RNA Preparation

[00324] Eight to twelve rats meeting behavioral criteria for the five experimental groups described above were sacrificed, and the following tissues were collected separately: ipsilateral and contralateral dorsal root ganglia (DRG) for L4, L5 and L6. Samples were rapidly frozen on dry ice. Next, for each experimental group and tissue (5 groups x 6 tissues = 30 total), the samples were separated into two pools (Pool 1 and Pool 2), consisting of half or 4-6 animals each.

[00325] Total RNA from each tissue sample pool was prepared using Tri- Reagent (Sigma, St. Louis, MO). Total RNA was quantified by measuring absorption at 260 nm. RNA quality was assessed by measuring absorption at 260 nm/280 nm and by capillary electrophoresis on an RNA Lab-on-chip using Bioanalyzer 2100 (Agilent, Palo Alto, CA) to ensure that the ratio of 260 nm/280 nm exceeded 2.0, and that the ratio of 28S rRNA to 18S rRNA exceeded 1.0 for each sample. Pool 1 total RNA was used for the Affymetrix microarray hybridization, and Pool 2 total RNA was used for validation of gene expression profiles by TaqMan^® analysis.

[00326] Total RNA was also prepared for Affymetrix GeneChip^® and

TaqMan^® analysis from 27 rat organ tissues dissected from naϊve rats. These included duodenum, lung, ovary, esophagus, diaphragm, skin, heart, colon, optic nerve, thyroid, thymus, trachea, superior cervical ganglion, prostate, dorsal root ganglia, sciatic nerve, spinal cord, brain, adrenal, aorta, fetal brain, kidney, liver, quadriceps muscle, spleen, submaxillary gland, and testis.

[00327] Total RNA for Affymetrix GeneChip^® and TaqMan^® analysis of 23 human tissues was obtained from Clontech (Palo Alto, CA). These included adrenal, bladder, bone marrow, cerebellum, colon, dorsal root ganglion, fetal brain, heart, kidney, liver, lung, ovary, pancreas, prostate, salivary gland, skeletal muscle, smooth muscle, spinal cord, testis, thymus, thyroid, uterus, and whole brain. 6.3.2. Microarray Analysis

[00328] GeneChip^® (Affymetrix, Santa Clara, CA) technology allows comparative analysis of the relative expression of thousands of known genes annotated in the public domain (herein, referred to as simply "known genes"), and genes encompassing ESTs (herein, referred to as simply "ESTs"), under multiple experimental conditions. Each gene is represented by a "probeset" consisting of multiple pairs of oligonucleotides (25 nt in length) with sequence complementary to the gene sequence or EST sequence of interest, and the same oligonucleotide sequence with a one base-pair mismatch. These probeset pairs allow for the detection of gene-specific nucleic acid hybridization signals as described below. The Affymetrix Rat U34 A, B and C arrays used for the described analysis contain probesets representing approximately 26,000 genes including 1200 genes of known relevance to the field of neurobiology. For example, these arrays include probesets specific for detecting the mRNA for kinases, cell surface receptors, cytokines, growth factors and oncogenes.

[00329] Hybridization probes were prepared according to the Affymetrix

Technical Manual (available on the WorldWideWeb at affymetrix . com/support/technical/manual/expression_manual . affx) . First-strand cDNA synthesis was primed for each total RNA sample (10 μg), using 5 mM of oligonucleotide primer encoding the T7 RNA polymerase promoter linked to oligo- dT₂₄ primer. cDNA synthesis reactions were carried out at 42 °C using Superscript II - reverse transcriptase (Invitrogen, Carlsbad, CA). Second-strand cDNA synthesis was carried out using DNA polymerase I and T4 DNA ligase. Each double-stranded cDNA sample was purified by sequential Phase Lock Gels (Brinkman Instrument, Westbury, NY) and extracted with a 1:1 mixture of phenol to chloroform (Ambion Inc., Austin, TX). Half of each cDNA sample was transcribed in vitro into copy RNA (cRNA) labeled with biotin-UTP and biotin-CTP using the Bio Array High Yield RNA Transcript Labeling Kit (Enzo Biochemicals, New York, NY). These cRNA transcripts were purified using RNeasy™ columns (Qiagen, Hilden Germany), and quantified by measuring absorption at

260nm/280nm. Aliquots (15 μg) of each cRNA sample were fragmented at 95°C for 35 min in 40 mM Tris-acetate, pH 8.0, 100 mM KOAc, and 30 mM MgOAc to a mean size of about 50 to 150 nucleotides. Hybridization buffer (0.1 M MES, pH 6.7, IM NaCl, 0.01 % Triton, 0.5 mg/ml BSA, 0.1 mg/ml H. sperm DNA, 50 pM control oligo B2, and lx eukaryotic hybridization control (Affymetrix, Santa Clara, CA)) was added to each sample.

[00330] Samples were then hybridized to RG-U34 A, B, and C microarrays

(Affymetrix) at 45°C for 16 h. Microarrays were washed and sequentially incubated with streptavidin phycoerythrin (Molecular Probes, Inc. , Eugene, OR), biotinylated anti-streptavidin antibody (Vector Laboratories, Inc. , Burlingame, CA), and streptavidin phycoerythrin on the Affymetrix Fluidic Station. Finally, the microarrays were scanned with a gene array scanner (Hewlett Packard Instruments, TX) to capture the fluorescence image of each hybridization. Microarray Suite 5.0 software (Affymetrix) was used to extract gene expression intensity signal from the scanned array images for each probeset under each experimental condition. 6.3.3. Statistical Criteria

[00331] Based on cumulative historical statistical analysis of replicate sample data (not shown), it was determined that the reproducibility of GeneChip^® data is dependent on the intensity of the signal. For intensities above 130, the reproducibility exhibits a coefficient of variation (CV; standard deviation divided by the average intensity) of 0.2 or better. Below 130, the reproducibility quickly falls off to CVs approaching infinity. Therefore, for genes having a gene expression intensity greater than 130, there is a high confidence of greater than two standard deviations for apparent fold-changes of three-fold or more.

[00332] As has been observed by others (Wang et al , Neuroscience 2002; 114: 529-546), the apparent gene regulation in L5 and L6 was much more robust than in L4 (data not shown). In order to optimize filtering criteria to reduce the approximately 26,000 rat genes represented on the GeneChip^® to those most relevant for pain, multiple filtering criteria were applied based on different threshold detection limits, and fold-regulation in various tissues and conditions. The best criteria that captured the most genes known to be molecular substrates of pain, and most likely to be reproducibly regulated by the SNL model in L4, L5 or L6, are listed below.

[00333] For L4, it was required that: 1. The maximum value between L4 sham (ipsilateral), SNL (ipsilateral), and SNL (contralateral) be at least 130, AND

2. that the L4 SNL (ipsilateral) compared to L4 sham (ipsilateral) exhibit at least three-fold regulation, AND

3. that the L4 SNL (ipsilateral) compared to L4 SNL (contralateral) exhibit at least three-fold regulation.

[00334] For L5 and L6, it was required that: 1. The maximum value between L5 sham (ipsilateral), L5 SNL (ipsilateral), L6 sham (ipsilateral), and L6 SNL (ipsilateral) be at least 130, AND

2. that the L5 SNL (ipsilateral) compared to L5 sham (ipsilateral) exhibit at least three-fold regulation, AND

3. that the L6 SNL (ipsilateral) compared to L6 sham (ipsilateral) exhibit at least three-fold regulation.

[00335] Probesets representing 249 known genes and 87 ESTs were selected based on the above criteria. Thirteen genes known to be molecular mediators of pain captured by the filtering criteria included the vanilloid receptor (VR-1), voltage-gated sodium channels NaN and SNS/PN3/Navl.8, serotonin receptor (5HT3), glutamate receptor (iGluR5), regulator of G protein signaling (RGS4), nicotinic acetylcholine receptor alpha 3 subunit, transcription factor DREAM, galanin receptor type 2, somatostatin, galanin, vasoactive intestinal peptide, and neuropeptide Y. Included among the ESTs was a DNA sequence annotated by Affymetrix as GenBank Accession #AI228284 (SEQ ID NO: 10). [00336] In order to further characterize the 336 genes (249 known plus 87

ESTs) regulated by SNL according to the stringent criteria described above, hierarchical clustering algorithms were used with a standard correlation distance measure available in GeneSpring software (Silicon Genetics, Redwood City, CA) to order the 336 genes based on their gene expression profiles. The experimental samples used for the hierarchical clustering analysis included: L4 naϊve ipsi, L4 naϊve contra, L4 sham ipsi, L4 SNL ipsi, L4 SNL contra, L4 GPN ipsi, L5 naϊve ipsi, L5 sham ipsi, L5 SNL ipsi, L5 SNL contra, L5 SNLGPN ipsi, L6 naϊve ipsi, L6 sham ipsi, L6 SNL ipsi, L6 SNL contra, L6 SNLGPN ipsi, sciatic nerve, spinal cord, brain, adrenal, aorta, fetal brain, kidney, liver, quadriceps muscle, spleen, submaxillary gland, and testis. Using the results of hierarchical clustering and determining the functional annotations of grouped genes, nine transcript regulation classes were determined and designated as: (1) known and novel DRG-specific pain targets; (2) neuronal cellular signal transduction proteins; (3) neuronal markers; (4) cellular signal transduction proteins; (5) known and novel neuropeptides or secreted molecules; (6) inflammatory response genes A; (7) inflammatory response genes B; (8) markers of muscle tissue; and (9) unknown. The gene expression profile of EST AI228284 (SEQ ID NO: 10) fell into transcript class (1), consisting of probesets representing ten ESTs and twenty-two different known genes, including eight known pain genes (VR-1, NaN, SNS/PN3/ Navl.8, 5HT3, iGluR5, RGS4, nicotinic acetylcholine receptor, and DREAM). The tight coupling of EST AI228284 gene expression profile to that of multiple genes known to be molecular mediators of pain suggests that the full-length gene sequence encompassing EST AI228284 encodes a protein whose temporal and spatial expression and function is involved in pain. 6.3.4. TaqMan^® Quantitative Real-Time PCR

[00337] The expression profiles across 27 rat naϊve tissue samples

(duodenum, lung, ovary, esophagus, diaphragm, skin, heart, colon, optic nerve, thyroid, thymus, trachea, superior cervical ganglion, prostate, dorsal root ganglia, sciatic nerve, spinal cord, brain, adrenal, aorta, fetal brain, kidney, liver, quadriceps muscle, spleen, submaxillary gland, and testis) and across 10 SNL samples (L4 naϊve ipsi, L4 naϊve contra, L4 sham ipsi, L4 SNL ipsi, L4 SNL contra, L4 GPN ipsi, L5 naϊve ipsi, L5 sham ipsi, L5 SNL ipsi, and L5 SNL GPN ipsi) for 18 known genes in transcript class 1 and for EST AI228284 (Figures 1A-B) were confirmed by TaqMan^® analysis as described below.

[00338] Once the complete rat open reading frame encompassing EST AI228284 was determined (described below) and the human ortholog identified (described below), the human expression profile across 23 naϊve tissues (adrenal, bladder, bone marrow, cerebellum, colon, dorsal root ganglion, fetal brain, heart, kidney, liver, lung, ovary, pancreas, prostate, salivary gland, skeletal muscle, smooth muscle, spinal cord, testis, thymus, thyroid, uterus, and whole brain) was also determined by TaqMan^® and Affymetrix GeneChip^® analysis ("human PNPGl" , Figure IC) as described below.

[00339] Total RNA (10 ng) was used to synthesize cDNA with random hexamers using a TaqMan^® Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Real-time PCR analysis was performed on an Applied Biosystems ABI Prism 7700 Sequence Detection System. Matching primers and fluorescence probes were designed for the gene or EST sequences using Primer Express software from Applied Biosystems. Primers and probe sequences used for rat EST AI228284 and for the human ortholog are listed in Table 2. Table 2. List of primer sequences (with nucleotide sequences shown from 5' to 3'):

[00340] Both forward and reverse primers were used at 200 nM. In all cases, the final probe concentration was 200 nM. The real-time PCR reaction was performed in a final volume of 25 μl using TaqMan^® Universal PCR Master Mix containing AmpliTaq Gold DNA Polymerase, AmpErase UNG, dNTPs (with dUTP), Passive Reference 1, optimized buffer components (Applied Biosystems, Foster City, CA) and 5 μl of cDNA template. Three replicates of reverse transcription and real-time PCR for each RNA sample were performed on the same reaction plate. A control lacking a DNA template, and controls using reference genes with stable expressions in all samples in the SNL/GPN study, were included on the same plate to minimize the reaction variability.

[00341] By Affymetrix GeneChip^® analysis and TaqMan^®, expression of EST AI228284 is decreased by about 3-fold in injured SNL L5 and L6 DRGs and is also decreased in uninjured SNL L4 DRGs (which is also known to experience pain). By TaqMan^® analysis, in rat, EST AI228284 is highly enriched in DRGs, but is also found in kidney, liver, lung, diaphragm, and testes. The human ortholog is expressed more ubiquitously, including in DRG, but is particularly highly expressed in liver (Figure IC). 6.4. Identification of PNPGl

[00342] Northern Blot Analysis. As a first step to identify the full-length rat gene encompassing EST AI228284, Northern blot analysis was performed. Using two EST sequence-specific primers, MB0284 and MB0285 (Table 2, SEQ ID NOS: 23 and 24, respectively), a 397 bp DNA fragment was amplified by PCR for probe synthesis. ³²P-dATP labeled probe prepared by using a random primed labeling kit (Catalog# 300385; Stratagene, La Jolla, CA) was then hybridized to a rat Multiple Tissue Northern Blot (Blot-1; Sigma, Saint Louis, MO). Hybridization was carried out with ExpressHyb Solution (BD BioSciences, Palo Alto, CA) supplemented with 0.1 mg/ml sheared salmon sperm DNA at 65 °C for 1 hour and imaged by exposure to Kodak BioMax MS scientific imaging film overnight. Figure 2 shows that the full-length of the rat transcript detected was approximately 2.3 kb.

[00343] Determination of the sequence for the full-length open reading frame of the gene encompassing EST AI228284. By BLAST analysis, two publicly available EST clones were identified that overlapped and shared nearly 100% identity with the known sequence of EST AI228284: UI-R-BJ2-bor-e-06-0-UI (about 2 kb in length), and UI-R-A0-bi-b-04-0-UI (about 0.7 kb in length). These two clones were obtained from Invitrogen (Catalog# 99002; Carlsbad, CA) and for both, the sequence for the entire EST (beyond the publicly available end-reads) was finished by dideoxy-sequencing. The DNA sequencing reaction was carried out using DTCS Quick Start Kit and sequenced on a CEQ 8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA). The sequence reads (which included overlapping reads from both strands) for the two clones were aligned in Sequencher™ (Gene Codes Corporation, Ann Arbor, MI). The consensus for the contiguous sequence determined included a complete open reading frame (ORF) and was designated as rat PNPGl sequence (SEQ ID NO: 1). The gene encoding the identified open reading frame was named "PNPGl".

[00344] The finished nucleotide sequence of rat PNPGl (SEQ ID NO: 1) is

1889 nt in length and contains an ORF found to share 93% and 85% identity with the ORF of mouse sequence AK018759 (SEQ ID NO: 3), and human sequence NM_025078 (SEQ ID NO: 5), herein referred to as mouse and human PNPGl, respectively. The encoded protein of rat PNPGl (SEQ ID NO: 2) shares 97% and 89% amino acid sequence identity with mouse PNPGl (SEQ ID NO: 4) and human PNPGl (also annotated as hypothetical protein FLJ22378; SEQ ID NO: 6), respectively (see the alignment of all three sequences in Figure 4; the alignment was obtained using ClustalW, Vector NTI software (Invitrogen Life Science Software, Frederick, MD)). No proteins were found that have the same or similar sequence in the public rat DNA or protein databases. In addition, no annotated function for either the mouse sequence or the human hypothetical protein was found in the published literature.

[00345] Summary. Since the full-length gene encompassing EST AI228284 is

PNPGl, PNPGl also falls within transcript class 1. Transcript class 1, designated as "known and novel DRG-specific pain targets", distinguishes itself from the others, mostly due to higher expression in DRG in the naϊve conditions, and reduced expression in other neuronal tissues such as brain and spinal cord. In addition, known transcript class 1 genes encode proteins previously described in the literature to be involved in pain (described above). Since these known genes, such as the vanilloid receptor and the voltage-gated sodium channel, are known drug targets for therapeutics, the present inventors conclude that PNPGl is also useful as a drug target for pain and related disease states. 6.5. Further Characterization of PNPGl

[00346] Bioinformatic analysis. Analysis of the PNPGl protein structure was performed by two methods. The Simple Modular Architecture Research Tool, SMART (Schultz et al , Proc. Natl Acad. Sci. USA 1998; 95: 5857-5864; and Letunic et al. , Nucleic Acids Res. 2002; 30: 242-244) indicated that PNPGl protein contains CTNS domains (Figure 3) found in cystinosin, a cystine transporter with seven transmembrane domains found on lysosomal membranes (Anikster et al , Hum. Mutat. 1999; 14: 454-8). A different algorithm, PredictProtein (Rost, Meth. in Enzym. 1996; 266: 525-539; available on WorldWideWeb at cubic.bioc.columbia.edu), predicted seven transmembrane domains (Figure 4).

[00347] Determining the gene structure for rat, mouse, and human PNPGl.

In order to determine the gene structure of PNPGl, the rat, mouse, and human PNPGl cDNA sequences (SEQ ID NOS: 1, 3, and 5) were used to BLAST public domain genomic sequences for rat, mouse, and human, respectively. After hand aligning the cDNA sequence to the genomic region determined by BLAST (using Sequencher™, Gene Codes Corporation, Ann Arbor, MI), it was observed that the exon-intron structure of PNPGl gene is quite consistent across the three species (Figure 10). All three orthologs are encoded on each species' chromosome 18 and the exon-intron boundaries within the ORF regions are conserved. The UTR boundaries diverge across the three species. In fact, the human 5'-UTR spans an additional exon labeled " la" in Figure 10. Coordinates for the exon-intron boundaries for rat, mouse, and human PNPGl gene are summarized in Figure 10 based on the nucleotide coordinates of rat genomic sequence Accession NW_043157 (GL26011374), mouse chromosome 18 genomic contig (G 28525381), and human chromosome 18 genomic contig (GI: 37545286), respectively. SEQ ID NOS: 11, 12, and 13 contain the sequences for rat, mouse, and human PNPGl genes, respectively, including 2 kb of regulatory region upstream of the 5 '-most and downstream of the 3'-most exons. Strings of Ns in SEQ ID NOS: 11, 12, and 13 indicate regions of a contig discontinuity. These strings do not have a precisely defined length. In other words, N is variable both as to the identity and number of bases. [00348] In the analysis of the rat PNPGl gene sequence (SEQ ID NO: 11), derived from coordinates 612338-652647 of Rattus norvegicus chromosome 18 WGS supercontig (GI: 26011374), the present inventors found a "GC" not present between nt 94-95 of the rat PNPGl cDNA sequence (SEQ ID NO: 1). This discrepancy is in the 5' UTR and would not be expected to affect rat PNPGl protein sequence. The present inventors also found a "T" in the rat gene sequence (SEQ ID NO: 11) which is a "C" at nt 550 in the rat PNPGl cDNA sequence (SEQ ID NO: 1). This is a silent mutation that does not alter the coded amino acid (Tyr¹⁴² of the rat PNPGl protein; SEQ ID NO: 2), and likely represents a polymorphic allele. Another polymorphic allele, "C" was found at nt 1835 in the 3' UTR of the rat PNPGl cDNA sequence (SEQ ID NO: 1) which is a "T" in the rat gene sequence (SEQ ID NO: 11).

[00349] The mouse PNPGl gene sequence (SEQ ID NO: 12) comes from coordinates 2331137-2372020 of Mus musculus chromosome 18 genomic contig, strain C57BL/6J (GI: 28525381). Coordinates 37919-38018 of the mouse genomic contig (GI: 28525381) are represented by a string of N's indicating a contig discontinuity in the most recent assembly of the mouse genome. By aligning the mouse PNPGl cDNA sequence (SEQ ID NO: 3) to GI: 28525381, a predicted exon spans this region from upstream of the discontinuity to downstream indicating that it should be replaced by a 29 nt sequence. Accordingly, SEQ ID NO: 12 differs from the public domain sequence (GI: 28525381) in 29 nt.

[00350] The human gene sequence comes from coordinates 1888667-1941797 of Homo sapiens chromosome 18 genomic contig (GI: 37545286).

[00351] Vector Construction. In order to confirm protein expression and membrane localization of rat PNPGl, two primers, MB0473 and MB0474 (SEQ ID NOS: 25 and 26, respectively) were designed to specifically amplify the rat PNPGl ORF using the UI-R-BJ2-bor-e-06-0-UI EST clone as template. The resulting PCR product was cloned into an expression vector, pcDNA3.1D/V5-His-TOPO vector (Cat. No. K4900-01, Invitrogen, Carlsbad, CA). The construct (Figure 8) was named pcDNA-PNPGl and can be used to express a recombinant rat PNPGl protein fused to an epitope tag (V5+HIS) for Western blot, and immunohistochemical detection (using anti-V5 or anti-HIS antibodies).

[00352] Detection of PNPGl Protein Expression. Membrane fractionation and

Western blot analysis. HEK-293 cells were transfected with pcDNA-PNPGl and the recombinant protein was detected with antibody directed against the V5 epitope- tag (Cat. No. R960-25, Invitrogen, Carlsbad, CA). The full length fusion protein detected by Western blot analysis is an approximately 35 kD protein as predicted by 271 amino acids for the rat PNPGl protein and 45 amino acids for the combined V5/HIS-epitope tag (Figure 5). Both soluble and membrane-bound fractions were prepared by a 2D sample prep method (Pierce Biotechnology, Rockford, IL). When the isolated membrane-bound extract was loaded without dilution, a V5 positive band was detected in a high molecular weight band at the top of the gel (Figure 5). As this extract was diluted (1:5 and 1:20), an additional band was detected at a molecular weight of about 35 kD, corresponding to the expected size for the V5- tagged PNPGl protein (Figure 5). These results suggest that PNPGl is associated with high-order protein complexes found in the membrane-bound fraction. Upon dilution, these high-order protein complexes are sufficiently reduced, releasing PNPGl monomers.

[00353] PNPGl-pep2 peptide (SEQ ID NO:31), corresponding to rat PNPGl amino acid position 111 to 125, was used to immunize rabbits. To purify the antibody serum, the peptide PNPGl-pep2 was conjugated on PIERCE beads according to the PIERCE EZ antibody production and purification kit (Cat#77627).

The rabbit antibody (#921-1) was bound on the peptide beads, washed and then eluted from the beads with the PIERCE ImmunoPure Binding/Elution Buffer system (Cat# 21001). Fractions 3 through 6 were combined, concentrated and dialyzed in a

PIERCE 3.5 K cut off dialysis cassette (Cat # 66330 ) in PBS overnight. The purified antibody was used to detect PNPGl protein by Western blot (Figure 13).

Anti-V5 antibody was used as a positive control to detect the PNPG1-V5 fusion protein resulting in a strong signal for pcDNA-PNPGl-V5 transfected cell lysate (Figure 13, right panel). The purified PNPGl antibody (#921-1) was also able to detect a strong signal in the pcDNA-PNPGl-V5 transfected cell lysate (Figure 13, left panel), indicating that the purified anti-PNPGl antibody can recognize PNPGl protein.

[00354] Confocal microscopy. Confocal microscopy was used to confirm the membrane localization of V5-tagged PNPGl. CHO cells were transfected with pcDNA-PNPGl. A mouse anti-V5 antibody and FITC-conjugated anti-mouse IgG secondary antibody (Sigma, St. Louis, MO) were used to detect recombinant rat PNPGl. Briefly, CHO cells were plated on a cover slip in a 6 well plate and transiently transfected with pcDNA-PNPGl using Lipofectamine Plus™ Reagent according to the manufacturer's protocol (Cat. No. 10964-013, Invitrogen, Carlsbad, CA). Two days after transfection, the cells were rinsed with phosphate buffered saline (PBS), fixed with 3.7% formaldehyde at room temperature (RT) for 20 min, rinsed again with PBS, incubated in 0.2% Triton in PBS for 10 min at RT, incubated with anti-V5 antibody (1:50 in 0.1 % Triton-PBS, 25 μl/each slip) at RT for 1 hr, washed three times with PBS for 3 min each, incubated with FITC- conjugated anti-mouse IgG (1: 200 dilution) as secondary antibody for 45 min to 1 hr, and washed three times with PBS for 3 min each. The cover slips were mounted on glass slides and imaged using a Nikon Eclipse E800, Cl-confocal microscope system (Nikon USA, Melville, NY) with 60X amplification lenses. FITC-labeled protein can be observed on the edge of cells (Figure 6), indicating that transiently expressed PNPGl is a membrane associated protein. Expression of V5-tagged PNPGl protein was also observed in the endoplasmic reticulum and other intracellular compartments as well, as is often observed with high levels of recombinantly-expressed protein associated with transient transfections.

[00355] In situ hybridization. The present inventors confirmed that PNPGl is expressed in naϊve DRG neurons by in situ hybridization (Figure 7). ³⁵S-UTP labeled antisense RNA probes were generated using T7 RNA polymerase from a PCR template. The PCR template was generated using rat PNPGl-specific primers, MB0187 and MB0188 (Table 2; SEQ ID NOS: 27 and 28, respectively), containing T7 and T3 RNA polymerase promoter sequences from a rat cDNA library. The in situ hybridization protocol was done according to Frantz et. al. (J. Neuroscience 1994; 14: 5725) except that the proteinase K step was omitted. DRG from Sprague Dawley rats were dissected and frozen in TBS Tissue Freezing Medium™ (Triangle Biomedical Sciences, Durham, NC). Twenty micron frozen sections were fixed with 4% paraformaldehyde onto Fisher Scientific Superfrost glass slides. Tissue sections were washed with PBS and treated with 0.25% acetic anhydride in 0.1 M triethanolamine and dehydrated through an ethanol series (50% , 70%, 2 X 95%). Sections were incubated with 6 X 10⁶ cpm/ml of ³⁵S-labeled RNA probe in hybridization buffer (62.5 % formamide, 12.5% dextran sulfate, 0.0025% polyvinylpyrolidone, 0.0025% ficoll, 0.0025% bovine serum albumin, 375 mM NaCl, 12.5 mM Tris pH = 8, 1.3 mM EDTA, 10 mM dithiothreitol (DTT), 150 μg/ml E. coli tRNA) at 60°C for 16 hours. Sections were then treated with RNaseA, 50 μg/ml in 10 mM Tris/0.5 M NaCl. These were then washed through a series of SSC (described above) washes containing 1 mM DTT (2X SSC, IX SSC, 0.5X SSC, 0.1X SSC). A final wash in 0.1X SSC, 1 mM DTT was for 30 min at 65°C. Sections were then dehydrated through an ethanol series (50% , 70%, 95%, 3X 100%), air-dried, and dipped in Kodak NTB2 emulsion and exposed for 2 weeks. Slides were developed using Kodak D19 developer and Rapid Fix. Slides were counterstained with Hematoxylin and Eosin (see Figure 7). 6.6. siRNAs Targeting Rat PNPGl Reduce the Level of Recombinant PNPGl Protein Expression in HEK 293 Cells.

[00356] Knock down of PNPGl expression by siRNA in vitro. HEK-293 cells were transfected with pcDNA-PNPGl and pSilencer 2.0-U6 engineered to express either an siRNA targeting PNPGl or one targeting the Mu opioid receptor gene (used as a negative control) (see Figure 8 for diagram of constructs). pSilencer 2.0- U6 vector (Cat. No. 7209, Ambion Inc. , Austin, TX) is a mammalian expression vector for siRNA-induced gene silencing. Two oligonucleotides, MB0475 and MB0476 (Table 3, SEQ ID NOS: 29 and 30, respectively), were synthesized, annealed and ligated into the Bam HI and Hind III sites of the pSilencer 2.0-U6 vector (Figure 12 A). Table 3. List of siRNA sequences (with nucleotide sequences shown from 5' to 3'):

[00357] The resulting construct (pSi-PNPGl) expresses an siRNA starting with a G and including 19 nt sense and antisense sequence (corresponding to nt 135 to 154 from the AUG start of rat PNPGl ORF), separated by a 7 nt loop sequence (Figure 12A). As depicted in Figure 12B this siRNA is able to target nt 133 starting with AA, to nt 154 of PNPGl mRNA. In order to demonstrate effective siRNA- mediated reduction of PNPGl expression, HEK 293 cells were co-transfected with varying ratios of pcDNA-PNPGl (expressing epitope-tagged PNPGl) and pSi- PNPGl or pSi-Mu, expressing the test or negative control siRNA, respectively (Figure 8). The amount of resulting PNPGl protein expression was measured by Western blot at 72 hours after transfection. Compared to the negative control, siRNA expressed from pSi-PNPGl effectively knocked down PNPGl protein expression. *> 7. Deposit of Biological Materials with the ATCC

[00358] Plasmid pPNPGl comprising a nucleic acid molecule having a nucleotide sequence encoding the rat PNPGl protein was deposited with the American Type Culture Collection (ATCC) at 10801 University Boulevard, Manassas, VA 20110-2209, USA on October 24, 2003, and has been assigned ATCC Accession No.PTA-5617. The rat PNPGl sequence was obtained by fully sequencing Invitrogen Rat UI EST clone UI-R-BJ2-bor-e-06-0-UI (Invitrogen Cat.#99002). This EST clone came out of government-sponsored research: the I.M.A.G.E. Consortium at Washington University (see http://image.llnl.gov/). It was this same clone that was deposited with the ATCC (the backbone vector is pT7T3-Pac, Invitrogen).

[00359] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[00360] All references cited herein, including all patents, published patent applications, and published scientific articles, are incorporated by reference in their entireties for all purposes.

Claims

WHAT IS CLAIMED IS:

1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a mammalian PNPGl protein, with the proviso that the nucleotide sequence is not the sequence consisting of SEQ ID NO: 3 or SEQ ID NO:5.

2. The isolated nucleic acid molecule of claim 1, comprising a nucleotide sequence encoding a rat PNPGl protein.

3. The isolated nucleic acid molecule of claim 2, wherein the rat PNPGl protein comprises the amino acid sequence of SEQ ID NO:2.

4. The isolated nucleic acid molecule of claim 3, wherein the rat PNPGl protein comprising the amino acid sequence of SEQ ID NO:2 is encoded by the nucleotide sequence of SEQ ID NO:l or a degenerate variant thereof.

5. An isolated single-stranded nucleic acid molecule having a nucleotide sequence that is the complement of a nucleotide sequence of one strand of any of the nucleic acid molecules of claims 1 to 4.

6. The isolated nucleic acid molecule of claim 1, which is a DNA molecule.

7. The isolated nucleic acid molecule of claim 1, which is an mRNA molecule.

8. An isolated nucleic acid molecule comprising a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of the PNPGl- encoding nucleic acid molecule of claim 1, with the proviso that the nucleotide sequence is not the sequence consisting of SEQ ID NO: 3 or SEQ ID NO:5.

9. An oligonucleotide molecule that hybridizes under standard hybridization conditions to a nucleic acid molecule of claim 3.

10. A recombinant expression vector comprising the nucleic acid molecule of claim 1 fused to a nucleic acid molecule encoding an epitope tag.

11. A recombinant expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encodmg the amino acid sequence of SEQ ID NO: 2 fused to a nucleotide sequence encoding an epitope tag:

12. A recombinant vector comprising a nucleic acid molecule encoding a PNPGl protein, wherein said nucleic acid molecule encoding a PNPGl protein has been amplified using primers MB0473 (SEQ ID NO: 25) and MB0474 (SEQ ID NO: 26) from the pPNPGl vector which has been deposited with the American Type Culture Collection (ATCC Accession No. PTA-5617).

13. A recombinant vector which has the same PNPGl-encoding sequence as the vector of claim 12, wherein said PNPGl-encoding sequence is fused to a nucleotide sequence encoding an epitope tag.

14. A recombinant. vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:4.

15. A recombinant vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:6.

16. The recombinant vector of any one of claims 10-15, which is a plasmid, cosmid, phage, or a viral vector.

17. The recombinant vector of any one of claims 10-15, further comprising a selectable marker.

18. A host cell comprising the recombinant vector of any one of claims

10-15.

19. The host cell of claim 18, which is a eukaryotic cell.

20. The host cell of claim 19, which is a mammalian cell.

21. The host cell of claim 20, which is a rat, mouse or human cell.

22. The host cell of claim 18, which is a prokaryotic cell.

23. The host cell of claim 18, wherein expression of the nucleic acid molecule is constitutive.

24. The host cell of claim 18, wherein expression of the nucleic acid molecule is inducible.

25. An isolated polypeptide comprising the amino acid sequence of a mammalian PNPGl protein.

26. The isolated polypeptide of claim 25, wherein the mammalian PNPGl protein is a rat, mouse or human PNPGl protein.

27. The isolated polypeptide of claim 26, wherein the rat PNPGl protein comprises the amino acid sequence of SEQ ID NO: 2; the mouse PNPGl protein comprises the amino acid sequence of SEQ ID NO:4; and the human PNPGl protein comprises the amino acid sequence of SEQ ID NO:6.

28. A host cell which has been genetically modified to express or overexpress the polypeptide of claim 25.

29. An isolated cellular membrane fraction prepared from the host cell of claim 28, which cellular membrane fraction comprises a PNPGl protein.

30. An antibody or antibody fragment that specifically binds to a polypeptide of claim 25.

31. A mammalian cell that has been genetically modified so that its normal expression of a PNPGl-encoding gene has been reduced or eliminated.

32. A mammalian cell that has been genetically modified so that its normal expression of a PNPGl-encoding gene has been increased.

33. A genetically modified mammal that comprises cells of claim 31 or 32.

34. A method for producing a genetically modified mammal, comprising genetically modifying one or more cells of said mammal such that the expression of the PNPGl gene in said mammal has been modified compared to the expression of the PNPGl gene in a wild-type mammal.

35. The method of claim 34, wherein said genetically modified cells are embryonic stem (ES) cells and wherein the intact genetically modified mammal is prepared utilizing said ES cells.

36. The method of claim 34, wherein said mammal is a mouse.

37. The method of claim 34, wherein the expression of the PNPGl gene has been reduced or eliminated in at least some cells of said genetically modified mammal.

38. The method of claim 34, wherein the expression of the PNPGl gene has been increased in at least some cells of said genetically modified mammal.

39. An antisense oligonucleotide molecule that can specifically inhibit expression of a mammalian PNPGl gene.

40. A ribozyme molecule that can specifically inhibit expression of a mammalian PNPGl gene.

41. An interfering RNA molecule that can specifically inhibit expression of a mammalian PNPGl gene.

42. A method for detecting a pain response in a test cell subjected to a treatment or stimulus or suspected of having been subjected to a treatment or stimulus, said method comprising:

(a) determining the expression level in the test cell of a nucleic acid molecule encoding a PNPGl protein; and

(b) comparing the expression level of the PNPGl-encoding nucleic acid molecule in the test cell to the expression level of the same nucleic acid molecule in a control cell not subjected to a treatment or stimulus; wherein a detectable change in the expression level of the PNPGl-encoding nucleic acid molecule in the test cell compared to the expression level of the PNPGl- encoding nucleic acid molecule in the control cell indicates that the test cell is exhibiting a pain response.

43. The method of claim 42, wherein the test and control cells naturally express a PNPGl-encoding nucleic acid molecule.

44. The method of claim 42, wherein the test and control cells have been genetically modified to express a PNPGl-encoding nucleic acid molecule.

45. The method of claim 42, wherein the test and control cells are both from the central nervous system or the peripheral nervous system.

46. The method of claim 42, wherein the test and control cells are both from the dorsal root ganglion (DRG).

47. The method of claim 42, wherein the test and control cells are neuronal cells.

48. The method of claim 42, wherein PNPGl-encoding nucleic acid encodes a rat, mouse or human PNPGl protein.

49. The method of claim 48, wherein the rat PNPGl protein comprises the amino acid sequence of SEQ ID NO:2; the mouse PNPGl protein comprises the amino acid sequence of SEQ ID NO:4; and the human PNPGl protein comprises the amino acid sequence of SEQ ID NO:6.

50. The method of claim 42, wherein the expression level of the nucleic acid molecule in each of the test and control cells is determined by quantifying the amount of PNPGl-encoding mRNA present in the two cells.

51. The method of claim 42, wherein the expression level of the nucleic acid molecule in each of the test and control cells is determined by quantifying the amount of PNPGl protein produced in each of the two cells.

52. A method for identifying a candidate compound useful for modulating the expression of a PNPGl-encoding nucleic acid, said method comprising:

(a) contacting a first cell with a test compound under conditions sufficient to allow the cell to respond to said contact with the test compound;

(b) determining in the cell prepared in step (a) the expression level of a PNPGl-encoding nucleic acid molecule; and

(c) comparing the expression level of the PNPGl-encoding nucleic acid molecule determined in step (b) to the expression level of the PNPGl-encoding nucleic acid molecule in a second (control) cell that has not been contacted with the test compound;

wherein a detectable change in the expression level of the PNPGl-encoding nucleic acid molecule in the first cell in response to contact with the test compound compared to the expression level of the PNPGl-encoding nucleic acid molecule in the second cell that has not been contacted with the test compound, indicates that the test compound modulates the expression of the PNPGl-encoding nucleic acid and is a candidate compound.

53. The method of claim 52, wherein the test compound decreases the expression of the PNPGl-encoding nucleic acid molecule.

54. The method of claim 52, wherein the test compound increases the expression of the PNPGl-encoding nucleic acid molecule.

55. The method of claim 52, wherein the test compound is a small inorganic molecule, a small organic molecule, a polypeptide, a nucleic acid molecule, or a chimera or derivative thereof.

56. The method of claim 52, wherein the cells used naturally express a

PNPGl-encoding nucleic acid molecule.

57. The method of claim 52, wherein the cells used express a PNPGl- encoding nucleic acid molecule in response to a specific stimulus.

58. The method of claim 52, wherein the cells used have been genetically modified to express a PNPGl-encoding nucleic acid molecule.

59. The method of claim 52, wherein the cells used are from the central nervous system or the peripheral nervous system.

60. The method of claim 52, wherein the cells used are from the dorsal root ganglion (DRG).

61. The method of claim 52, wherein the cells used are neuronal cells.

62. The method of claim 52, wherein the PNPGl-encoding nucleic acid encodes a mammalian PNPGl protein.

63. The method of claim 62, wherein the PNPGl-encoding nucleic acid encodes a rat, mouse or human PNPGl protein.

64. The method of claim 63, wherein the rat PNPGl protein comprises the amino acid sequence of SEQ ID NO:2; the mouse PNPGl protein comprises the amino acid sequence of SEQ ID NO:4; and the human PNPGl protein comprises the amino acid sequence of SEQ ID NO:6.

65. The method of claim 52, wherein the cells used are from an animal model of pain.

66. The method of claim 52, wherein the cells used are from a human or companion animal subject.

67. The method of claim 52, wherein the expression level of the PNPGl- encoding nucleic acid molecule is determined by quantifying the amount of PNPGl- encoding mRNA present in the cells.

68. The method of claim 52, wherein the expression level of the PNPGl- encoding nucleic acid molecule is determined by quantifying the amount of PNPGl protein present in the cells.

69. A method for identifying a candidate compound useful for modulating the expression of a PNPGl protein, said method comprising:

(a) contacting a first cell with a test compound under conditions sufficient to allow the cell to respond to said contact with the test compound;

(b) determining in the cell prepared in step (a) the expression level of a PNPGl protein; and

(c) comparing the expression level of the PNPGl protein determined in step (b) to the expression level of the PNPGl protein in a second (control) cell that has not been contacted with the test compound;

wherein a detectable change in the expression level of the PNPGl protem in the first cell in response to contact with the test compound compared to the expression level of the PNPGl protein in the second cell that has not been contacted with the test compound, indicates that the test compound modulates the expression of the PNPGl protein and is a candidate compound.

70. The method of claim 69, wherein the test compound decreases the expression of the PNPGl protein.

71. The method of claim 69, wherein the test compound increases the expression of the PNPGl protein.

72. The method of claim 69, wherein the test compound is a small inorganic molecule, a small organic molecule, a polypeptide, a nucleic acid molecule, or a chimera or derivative thereof.

73. The method of claim 69, wherein the cells used naturally express a PNPGl protein.

74. The method of claim 69, wherein the cells used express a PNPGl protein in response to a specific stimulus.

75. The method of claim 69, wherein the cells used have been genetically modified to express a PNPGl protein.

76. The method of claim 69, wherein the cells used are from the central nervous system or the peripheral nervous system.

77. The method of claim 69, wherein the cells used are from the dorsal root ganglion (DRG).

78. The method of claim 69, wherein the cells used are neuronal cells.

79. The method of claim 69, wherein the PNPGl protein is a mammalian PNPGl protein.

80. The method of claim 79, wherein the PNPGl protein is a rat, mouse or human PNPGl protein.

81. The method of claim 80, wherein the rat PNPGl protein comprises the amino acid sequence of SEQ ID NO:2; the mouse PNPGl protein comprises the amino acid sequence of SEQ ID NO: 4; and the human PNPGl protein comprises the amino acid sequence of SEQ ID NO:6.

82. The method of claim 69, wherein the cells used are from an animal model of pain.

83. The method of claim 69, wherein the cells used are from a human or companion animal subject.

84. The method of claim 69, wherein the expression level of the PNPGl protein is determined using antibodies of claim 29.

85. A method for monitoring the efficacy of an analgesic treatment in a cell comprising:

(a) administering to said cell an analgesic compound under conditions sufficient to allow the cell to respond to said compound;

(b) determining in the cell prepared in step (a) the expression level of a PNPGl-encoding nucleic acid molecule; and

(c) comparing the expression level of the PNPGl-encoding nucleic acid molecule determined in step (b) to the expression level of the PNPGl-encoding nucleic acid molecule in a second (control) cell that has not been contacted with the analgesic compound;

wherein a detectable change in the expression level of the PNPGl-encoding nucleic acid molecule in the first cell in response to contact with the analgesic compound compared to the expression level of the PNPGl-encoding nucleic acid molecule in the second (control) cell that has not been contacted with the analgesic compound is indicative of the activity of the analgesic compound.

86. The method of claim 85, wherein the cells used are from the central nervous system or the peripheral nervous system.

87. The method of claim 85, wherein the cells used are from the dorsal root ganglion (DRG).

88. The method of claim 85, wherein the cells used are neuronal cells.

89. The method of claim 85, wherein the cells used are from an animal model of pain.

90. The method of claim 85, wherein the cells used are from a human or companion animal subject.

91. The method of claim 85, wherein the expression level of the PNPGl- encoding nucleic acid molecule is determined by quantifying the amount of PNPGl- encoding mRNA present in the cells.

92. The method of claim 85, wherein the expression level of the PNPG1- encoding nucleic acid molecule is determined by quantifying the amount of PNPGl protein present in the cells.

93. A method for identifying a candidate compound capable of binding to a PNPGl protein, said method comprising:

(a) contacting a PNPGl protein with a test compound under conditions that permit binding of the test compound to the PNPGl protein; and

(b) detecting binding of the test compound to the PNPGl protein.

94. The method of claim 93, wherein the test compound is a small inorganic molecule, small organic molecule, a polypeptide, a nucleic acid molecule, or a chimera or derivative thereof.

95. The method of claim 93, wherein the PNPGl protein is: (a) present in an intact cell that expresses the PNPGl protein; (ii) present in a membrane fraction comprising the PNPGl protein; or (iii) is an isolated PNPGl protein that is unassociated with a cell or membrane fraction.

96. The method of claim 93, wherein the PNPGl protein is produced in a cell that naturally expresses a PNPGl-encoding nucleic acid molecule.

97. The method of claim 93, wherein the PNPGl protein is produced in a cell that expresses a PNPGl-encoding nucleic acid molecule in response to a specific stimulus.

98. The method of claim 93, wherein the PNPGl protein is produced in a cell that has been genetically modified to express a PNPGl-encoding nucleic acid molecule.

99. The method of claim 93, wherein the PNPGl protein is produced in a cell from the central nervous system or the peripheral nervous system.

100. The method of claim 93, wherein the PNPGl protein is produced in a cell from the dorsal root ganglion (DRG).

101. The method of claim 93, wherein the PNPGl protein is produced in a neuronal cell.

102. The method of claim 93, wherein the PNPGl protein is a mammalian PNPGl protein.

103. The method of claim 102, wherein the mammalian PNPGl protein is a rat, mouse or human PNPGl protein.

104. The method of claim 103, wherein the rat PNPGl protein comprises the amino acid sequence of SEQ ID NO:2; the mouse PNPGl protein comprises the amino acid sequence of SEQ ID NO:4; and the human PNPGl protein comprises the amino acid sequence of SEQ ID NO:6.

105. The method of claim 95, wherein the intact cell used is from an animal model of pain.

106. The method of claim 95, wherein the intact cell used is from a human or companion animal subject.

107. A method for treating a condition that can be treated by modulating expression of a PNPGl-encoding nucleic acid molecule or a PNPGl protein, comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound that modulates expression of a PNPGl-encoding nucleic acid molecule or a PNPGl protein.

108. The method of claim 107, wherein the compound decreases expression of a PNPGl-encoding nucleic acid molecule or a PNPGl protein.

109. The method of claim 107, wherein the compound increases expression of a PNPGl-encoding nucleic acid molecule or a PNPGl protein.

110. The method of claim 107, wherein the treated condition is a pain or a related disorder.

111. The method of claim 107, wherein the treated condition is selected from the group consisting of neuropathic pain, nociceptive pain, chronic pain, inflammatory pain, pain associated with cancer, and pain associated with rheumatic disease.

112. The method of claim 107, wherein the treated condition is selected from the group consisting of addiction, seizure, stroke, ischemia, a neurodegenerative disorder, anxiety, depression, headache, asthma, rheumatic disease, osteoarthritis, retinopathy, inflammatory eye disorders, pruritis, ulcer, gastric lesions, uncontrollable urination, an inflammatory or unstable bladder disorders, inflammatory bowel disease, irritable bowel syndrome (IBS), irritable bowel disease (IBD), gastroesophageal reflux disease (GERD), functional dyspepsia, functional chest pain of presumed oesophageal origin, functional dysphagia, non- cardiac chest pain, symptomatic gastroesophageal disease, gastritis, aerophagia, functional constipation, functional diarrhea, burbulence, chronic functional abdominal pain, recurrent abdominal pain (RAP), functional abdominal bloating, functional biliary pain, functional incontinence, functional ano-rectal pain, chronic pelvic pain, pelvic floor dyssenergia, unspecified functional ano-rectal disorder, cholecystalgia, interstitial cystitis, dysmenorrhea, and dyspareunia.