Pharmaceutical composition for treating pain

Abstract

The recovery, from mechanical and thermal pain hypersensitivity, of a diabetic pain model mouse according to the present invention, can be interpreted by dividing a correlation mechanism, between a GABAergic neuronal system and related pain genes through transcription factor (Ascl1 / Lhx6) activity, into peripheral nervous system and central nervous system, and thus can be variously used in the fields of preventing, alleviating, and treating diabetic neuropathic pain.

Description

Pharmaceutical composition for pain treatment

[0001] The present invention relates to a composition for treating pain.

[0002] Neuropathy, particularly diabetic neuropathy, is a common symptom in patients with diabetes. Risk factors for diabetic neuropathy include poor blood sugar control, hypertension, smoking, age, obesity, and cardiovascular disease. Exacerbation of diabetic neuropathy symptoms can significantly reduce a patient's quality of life. It is estimated that more than 20% of all diabetic patients experience diabetic pain. Patients suffering from diabetic pain exhibit a variety of symptoms, including allodynia, hyperalgesia, paresthesia (dysesthesia), and numbness. Diabetic neuropathy pain is caused by increased sensitivity of pain neurotransmission in the peripheral and central nervous systems.

[0003] Antidepressants such as amitriptyline are known to treat diabetic neuropathic pain. However, amitriptyline has side effects such as dry mouth, urinary tract obstruction, constipation, and increased intraocular pressure. Anticonvulsants such as gabapentin inhibit voltage-gated calcium channels and reduce allodynia, but they also cause side effects such as drowsiness, dizziness, and lethargy. Narcotic analgesics such as morphine have been proven to provide pain relief, but they also suffer from the risk of increased dosage due to tolerance and increased drug dependence.

[0004] As such, a satisfactory treatment that can treat or slow the progression of neuropathic pain has not yet been developed.

[0005] Accordingly, the inventors of the present invention conducted research to develop a new neuropathic pain treatment agent, and as a result, confirmed that pain (e.g., diabetic nerve pain) can be treated, alleviated, or prevented through the activity of transcription factors that regulate the function of the GABA neuronal system, thereby completing the present invention.

[0006] Accordingly, an object of the present invention is to provide a pain treatment agent comprising a transcription factor that regulates the function of the GABA neuron system.

[0007] Furthermore, the present invention identifies pain modulating factors in dorsal ganglion neurons of the peripheral nervous system using RNA sequencing, and provides biomarkers for confirming their interrelationships. Transcription factors Ascl1 and lhx6 are considered biomarkers, and measuring their expression levels can be used to diagnose pain conditions or evaluate treatment responses. Furthermore, changes in the expression level of GABA receptors (GABAB1-2) also reflect neuroinhibitory action and pain sensitization, and thus can be utilized as indicators of pain modulation.

[0008] In addition, the present invention provides a biomarker that can compare the degree of pain relief in the peripheral and central nervous systems and identify differences in pain relief by confirming that specific transcription factors that regulate the function of the GABA neuron system are related to pain. Accordingly, the expression level of pain-related channels (TRPV1, Nav1.7) as biomarkers can be used to evaluate pain status and treatment effects, and the neuroinflammation status can be monitored and pain control effects can be confirmed through changes in the expression of inflammatory (TNF-alpha, IL-1 beta, IL-6) and anti-inflammatory (IL-4, 10, 13) cytokines. In addition, the activation status of the MAPK signaling pathway (ERK, p38, JNK) can be measured and utilized as a biomarker to evaluate the activation of pain and inflammatory pathways.

[0009] Additionally, the present invention provides a method for treating, alleviating, or preventing neuropathic pain using transcription factors associated with the activity of the GABA neuron system expressed in the peripheral nervous system or central nervous system.

[0010] To achieve the above purpose, the present invention relates to a composition for treating pain comprising a transcription factor that regulates the expression of the GABAergic neuron system.

[0011] Specifically, the present invention relates to a composition for treating pain, comprising: an Ascl1 protein of SEQ ID NO: 1 or an Lhx6 protein of SEQ ID NO: 2; a polynucleotide encoding the Ascl1 protein or the Lhx6 protein; or an expression vector comprising the polynucleotide.

[0012] In addition, the present invention relates to a composition for treating pain, comprising: an Ascl1 protein of SEQ ID NO: 1 and an Lhx6 protein of SEQ ID NO: 2; a polynucleotide encoding the Ascl1 protein and a polynucleotide encoding the Lhx6 protein; or an expression vector comprising a polynucleotide encoding the Ascl1 protein and an expression vector comprising a polynucleotide encoding the Lhx6 protein.

[0013] In the present invention, the expression vector may be a viral vector or a non-viral vector, wherein the viral vector may be an adeno-associated virus (AAV) vector, an adenovirus vector, an alphavirus vector, a herpes simplex virus vector, a vaccinia vector, a Sendai virus vector, a flavivirus vector, a radobovirus vector, a retrovirus vector, or a lentivirus vector.

[0014] The present invention provides a composition that can treat, alleviate or prevent pain, such as neuropathic pain, inflammatory pain, cancer pain, post-surgical pain, phantom limb pain, burn pain, gout pain, osteoarthritis pain, trigeminal neuralgia pain, acute herpes and post-herpetic pain, causalgia pain, idiopathic pain, fibromyalgia, chemotherapy-induced pain, diabetic neuropathic pain, migraine, arthralgia or rheumatoid arthritis pain.

[0015] More specifically, the present invention relates to a composition for treating, alleviating, or preventing pain (particularly diabetic pain) comprising the Ascl1 and lhx6 genes, which are transcription factors that regulate the expression of the GABA neuron system. The composition according to the present invention can treat, alleviate, or prevent pain by modulating the function of the GABA neuron system and the activity of pain-related factors in the peripheral and / or central nervous systems.

[0016] In the present invention, the difference in gene expression level (expression profile) using RNA sequencing was analyzed into a structured model according to cellular mechanism, molecular function, and intracellular and extracellular location using gene ontology, and through the analysis, the results of gene function analysis (functional annotation) were obtained, thereby confirming the intracellular metabolic pathway of pain genes in particular using the KEGG method.

[0017]

[0018] In the present invention, a decrease in the expression of transcription factors such as Ascl1, Lhx6, Dlx1,2,6 and Nkx1.2, 2.2 involved in the function of the GABA neuron system was confirmed in a diabetic neurodegenerative pain-induced condition.

[0019] In the present invention, two classified functions included in the GABA neuron system function in a diabetic nerve pain-inducing condition were classified, namely, the GABA neuron function that secretes the inhibitory neurotransmitter GABA and the GABA receptor function that binds to and is activated by GABA, and the decrease in expression was compared and confirmed.

[0020] In the present invention, it was confirmed that in a diabetic peripheral neuropathy pain-inducing condition, the expression of pro-inflammatory cytokines (e.g., TNF-alpha, IL-1beta, IL-6) genes increased and the expression of anti-inflammatory cytokines (e.g., IL-4, IL-10, IL-13) genes decreased.

[0021] In the present invention, an increased expression pattern of pain-related receptors (channels) TRPV1, TRPA1, Piezo1, Nav1.7, and Nav1.8 was confirmed in a diabetic neurodegenerative pain-induced condition.

[0022]

[0023] In the present invention, transcription factor (Ascl1 / lhx6) activity restores the expression of genes associated with reduced GABA neuron system function and alleviates pain hypersensitivity in pain model mice.

[0024] It can be inferred that changes in the activity of GABA neuron system function and pain-related genes and receptor (channel) are interrelated through the transcription factor (Ascl1 / lhx6) activity experiment according to the present invention.

[0025] In the present invention, transcription factor (Ascl1 / lhx6) activity suppresses changes in GABA neuron system function and pain-related gene and receptor (channel) activity, thereby alleviating pain hypersensitivity in pain model mice.

[0026] In the present invention, the mechanism of alleviating diabetic pain hypersensitivity by transcription factor (Ascl1 / lhx6) activity was identified by dividing it into peripheral and central nervous systems.

[0027] The pharmaceutical composition according to the present invention has the effects of preventing, alleviating and treating diabetic neuropathy pain.

[0028] The recovery of mechanical and thermal pain hypersensitivity of diabetic pain model mice according to the present invention can be interpreted by classifying the mechanism of the interaction between the GABA neuron system and related pain genes through the activation of transcription factors (Ascl1 / lhx6) into peripheral and central nervous systems, and thus can be utilized in various fields of prevention, improvement, and treatment of diabetic neuropathic pain.

[0029] Figures 1 to 28 are the results of experiments using dorsal root ganglia (DRG) in the peripheral nervous system.

[0030] Figure 1 is a diagram showing the confirmation of increased pain sensitivity in diabetic mice due to streptozotocin (abbreviated as STZ).

[0031] Figures 2 to 5 are diagrams showing the results of analyzing changes in gene expression in dorsal root ganglion neurons of diabetic mice using RNA seq.

[0032] Figures 6 to 9 are diagrams showing the results of analysis confirming a decrease in the expression of genes and transcription factors specifically related to GABAergic neurons in dorsal root ganglion neurons of diabetic mice.

[0033] Figures 10 to 12 are diagrams showing the results of overexpression of the selected transcription factor Ascl1 / lhx6 reducing the increase in pain sensitivity.

[0034] Figures 13 and 14 are diagrams showing the results of overexpression of the transcription factor Ascl1 / lhx6 specifically increasing the expression of genes related to GABAergic neurons.

[0035] Figures 15 to 18 are diagrams showing the results of overexpression of the transcription factor Ascl1 / lhx6, which increases proinflammatory cytokines and reduces pain-related TRPV1 and Nav1.7.

[0036] Figures 19 to 25 are diagrams showing the results of confirming the regulation of expression of pain and inflammation-related genes when the transcription factor Ascl1 / lhx6 is overexpressed through RNA seq.

[0037] Figures 26 to 28 are diagrams showing the results of analyzing the degree of excitability of dorsal root ganglion neurons through the patch clamp method to determine increases and decreases in pain sensitivity.

[0038] Figures 29 to 39 are the results of experiments using the spinal cord in the central nervous system.

[0039] Figures 29 to 31 are diagrams showing the results of confirming and analyzing a decrease in the expression of transcription factors and specific genes specifically related to GABAergic neurons in the spinal cord of diabetic mice.

[0040] Figures 32 to 35 are diagrams showing that overexpression of the transcription factor Ascl1 / lhx6 results in increased and decreased pro / anti-inflammatory cytokines and increased and decreased glial cell activity in the spinal cord.

[0041] Figures 36 to 39 are diagrams showing that overexpression of the transcription factor Ascl1 / lhx6 results in a decrease in increased MAPK pathway activity in the spinal cord.

[0042] Figure 40 is a drawing illustrating an outline of an experiment to produce a mouse diabetic neuropathy pain model and to evaluate the inhibition of hyperalgesia according to administration of the composition of the present invention.

[0043] Figure 41 is a list of primer sequences used in performing PCR.

[0044]

[0045] The present invention relates to a composition for treating pain, comprising: an Ascl1 protein of SEQ ID NO: 1 or an Lhx6 protein of SEQ ID NO: 2; a polynucleotide encoding the Ascl1 protein or the Lhx6 protein (for example, a polynucleotide represented by SEQ ID NO: 3 or 4); or an expression vector comprising the polynucleotide.

[0046] In addition, the present invention relates to a composition for treating pain, comprising: an Ascl1 protein of SEQ ID NO: 1 and an Lhx6 protein of SEQ ID NO: 2; a polynucleotide encoding the Ascl1 protein and a polynucleotide encoding the Lhx6 protein (for example, a polynucleotide represented by SEQ ID NO: 3 and a polynucleotide represented by SEQ ID NO: 4); or an expression vector comprising the polynucleotides.

[0047]

[0048] In the present invention, pain may be nociceptive pain, psychogenic pain, inflammatory pain, or pathological pain, and the pathological pain may be neuropathic pain, cancer pain, chemotherapy-induced pain, postoperative pain, trigeminal neuralgia pain, idiopathic pain, diabetic neuropathic pain, or migraine.

[0049] Pain according to the present invention includes nociceptive pain, psychogenic pain, inflammatory pain associated with tissue damage and immune cell infiltration, and pathological pain (functional pain such as fibromyalgia, irritable bowel syndrome, and tension headaches) caused by damage to the nervous system or its abnormal function. Pain may also include anatomically distinct pain such as neck pain, middle back pain, lower back pain, or tailbone pain. Pain may also include neuropathic pain, migraine, and the like. Neuropathic pain is a chronic neurological disorder caused by damage to the nervous system due to various causes such as trauma, inflammation, ischemic damage, or metabolic waste products, and may result from damage or disease affecting the somatosensory system. It is a type of non-malignant chronic pain that is generally caused by abnormalities in the nerves, spinal cord, and brain and is estimated to affect more than 1% of the population.

[0050] In the pharmaceutical composition of the present invention, the effective amount of the compound may vary depending on the type of the patient's affected area, application site, number of treatments, treatment time, formulation, patient's condition, type of adjuvant, etc. The amount used is not particularly limited, but may be 0.01 μg / kg / day to 10 mg / kg / day. The daily amount may be administered once a day, or divided into 2 to 3 times a day at appropriate intervals, or intermittently at intervals of several days.

[0051] In the pharmaceutical composition of the present invention, the compound may be contained in an amount of 0.1 to 100 wt% based on the total weight of the composition. The pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. In addition, solid or liquid formulation additives may be used in the manufacture of the pharmaceutical composition. The formulation additives may be either organic or inorganic. Examples of the excipients include lactose, sucrose, sucrose, glucose, cornstarch, starch, talc, sorbitol, crystalline cellulose, dextrin, kaolin, calcium carbonate, and silicon dioxide. Examples of binders include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, dextrin, and pectin. Examples of lubricants include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any colorant that is generally approved for addition to pharmaceuticals can be used. These tablets and granules can be appropriately coated with sugar, gelatin, or other coatings as needed. In addition, preservatives, antioxidants, etc. can be added as needed.

[0052] The pharmaceutical composition of the present invention can be prepared in any formulation commonly manufactured in the art, and the form of the preparation is not particularly limited.

[0053] The pharmaceutical composition of the present invention can be administered orally or parenterally, and preferably parenterally, by intravenous injection, subcutaneous injection, intracerebroventricular injection, intracerebrospinal fluid injection, intrathecal injection, transforaminal injection, intramuscular injection, and intraperitoneal injection.

[0054] The present invention provides a method for alleviating and / or treating pain, comprising administering a therapeutically effective amount of the pharmaceutical composition to a patient in need of pain alleviation and / or treatment. The method may further comprise, prior to the administering step, a step of identifying the patient as a patient in need of pain alleviation and / or treatment. The term "therapeutically effective amount" may be based on the amount of active gradient that can achieve the desired effect, pain alleviation and / or treatment.

[0055]

[0056] Hereinafter, to facilitate understanding of the present invention, the present invention will be described in detail based on examples. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit the spirit or scope of the present invention in any way. These examples are provided to better explain the present invention to those of average skill in the art.

[0057] Experimental method

[0058] Experimental Example 1. Creation of an animal model of diabetic neuropathy pain.

[0059] In this experimental example, a diabetic pain animal model is created to test the degree of increased mechanical and thermal hyperalgesia sensitivity.

[0060] 1-1. Preparation for animal testing

[0061] To establish a diabetic neuropathy pain model, adult male C57BL / 6N mice were purchased from Orient bio (Seongnam, Korea) and housed three per cage on a 12-h light / dark cycle for at least 1 week prior to starting the experimental procedures.

[0062] 1-2. Animal model production

[0063] A diabetic neuropathy pain animal model was created by intraperitoneally injecting streptozotocin (STZ) (200 mg / kg; Sigma Aldrich) dissolved in citrate buffer (pH=4.5) (Furman BL 2015, Curr Protoc Pharmacol) into adult male C57BL / 6N mice in Experimental Example 1-1. The control group was injected with an equal volume of vehicle. Blood glucose concentrations were measured 3 and 7 days after STZ injection using an Accu-Chek Compact Plus blood glucose meter (Roche Diagnostics, Meylan, France). All mice were measured to have hyperglycemia of >300 mg / dL (16.7 mmol / l), confirming the induction of diabetes. Among the animals injected with STZ, those that did not show hyperglycemia 3 days after injection were excluded from the study.

[0064] 1-3. Animal experiments on mechanical and thermal pain hypersensitivity

[0065] To assess hindpaw pain sensitivity, paw withdrawal threshold (PWT) to mechanical stimulation was measured in mice using von Frey filaments (NC12775-99, North Coast Medical, Morgan Hill, CA, USA), and thermal hyperalgesia was measured. Paw withdrawal latency (PWL) was recorded using a Hargreaves radiant heat device (IITC Life Sciences, Woodland Hills, CA, USA). The 50% paw withdrawal threshold (PWT) was calculated using the up-down method (Rahman et al., 2022, Int J Mol Sci).

[0066] Animals were placed in a Plexiglass chamber with a 4x3 mm wire mesh grid floor and allowed to acclimate for 30 min. Calibrated von Frey filaments of different sizes (g) were applied perpendicularly to the plantar surface of the right hind paw with sufficient force to flex the filament for 6 s or until the paw withdrew. A rapid withdrawal or paw flinching was interpreted as a positive response, indicating an animal model with pain hypersensitivity. If no response was obtained, the next higher force filament was applied. If a response was obtained, the next lower force filament was applied.

[0067] Thermal hyperalgesia was measured by recording PWL using a Hargreaves radiant heat device (Rahman et al., 2022). The cutoff value was set at 20 seconds to prevent tissue damage. Mechanical allodynia and thermal hyperalgesia behavioral tests were performed 60 minutes before STZ or vehicle injection (baseline day 0) and on days 3, 7, 14, 21, and 28 after STZ (or vehicle) injection. The experimental results are shown in Figure 1.

[0068] Experimental Example 2. Lentivirus Production

[0069] 2-1 Genetic information

[0070] Two genes, Ascl1 (Achaete-scute family bHLH transcription factor 1 (NM_008553) (Origene, CAT#: MR227072) and Lhx6 (LIM homeobox protein 6 (NM_008500) (Origene, CAT#:MR226749), were purchased from Origene and used for experiments.

[0071] 2-2. Lentivirus production

[0072] To produce lentiviral vectors, three lentiviral constructs were designed: Lenti-CMV-mAscl1-P2A-EGFP, Lenti-CMV-mLhx6-P2A-dTomato, and Lenti-CMV-GFP. Lentiviral vectors with these constructs were packaged and produced at the KIST (Korea Institute of Science and Technology) virus facility. The titers of GFP, Ascl1, and Lhx6 lentiviruses were 1.16x10^11 vg / ml, 5.29x10^10 vg / ml, and 5.37x10^10 vg / ml, respectively.

[0073] 2-3. Intrathecal injection

[0074] In Experimental Steps 1-3, after confirming a stable diabetic state and a diabetic-induced neuropathic pain behavior test on the 7th day after STZ injection, a single intrathecal lentivirus was administered via acute needle puncture into the L5-L6 intervertebral space (Sung-Min Hwang et al., 2023, Biomed Pharmacother). The lentiviral preparation was concentrated to a titer of 7 × 10^9 / ml by tangential cross-flow ultrafiltration and administered. The method and timing of PWT measurement were the same as those described in Experimental Example 1-3 above. When the combined Lenti-Ascl1 or Lenti-lhx6 lentiviral vector was intrathecally administered, the vector was administered at a dose of 7 × 10^7 TU (total 1.4 × 10^7 / 20μl / mouse). The evaluation of neurobehavioral changes was conducted in a time-dependent manner as before.

[0075] Experimental Example 3. Dorsal Root Ganglion (DRG) Cell Isolation and Culture

[0076] DRGs were isolated from mice (6–9 weeks old) and incubated with collagenase A (0.2 mg / mL, Roche, Basel, Switzerland) / dispase-II (2.4 units / mL, Roche) at 37°C for 90 min. After mechanical dissociation by gentle pipetting, DRG cells were plated on poly-D-lysine-coated coverslips and grown in neurobasal culture medium containing 10% fetal bovine serum (Gibco, Waltham, MA, USA) and 2% B27 supplement (Invitrogen, Carlsbad, CA, USA). Neurons were cultured in a 5% CO2 incubator at 37°C for 12 h before patch clamp experiments.

[0077] Experimental Example 4. Quantitative RT-PCR (quantitative real-time PCR)

[0078] Gene expression analysis using quantitative real-time polymerase chain reaction (Q-PCR) was performed as follows.

[0079] Total RNA was extracted from DRG using Trizol (Thermo Fisher; catalog # 15596026). cDNA was synthesized using a reverse transcription kit (MMLV (Invitrogen; 28025013)) according to the manufacturer's instructions. qPCR was performed using the SYBR Green PCR kit (Bio-rad; 170-8882AP). All designed primer sequences are listed in Figure 41.

[0080] PCR was performed for 40 cycles at 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, 95°C for 10 s, and 65°C for 5 s. The results were normalized to the mRNA level of GAPDH (glyceraldehyde 3-phosphate dehydrogenase), which was used as an internal control.

[0081] Experimental Example 5. Patch Clamp

[0082] Patch-clamp analysis was performed at 25°C using an MPC-200 manipulator (Sutter Instrument, Novato, CA, USA) and a Multiclamp 700B amplifier (Molecular Devices, San Jose, CA, USA). Patch pipettes were prepared using borosilicate capillaries (Chase Scientific Glass Inc., Rockwood, TN, USA). The pipette resistance was 2–5 megohms, and series resistance was compensated (>80%). Data were low-pass filtered at 2 kHz and sampled at 10 kHz. Voltage-clamp recordings were performed at a holding potential of −70 mV. The pipette solution for voltage-clamp experiments contained 122.5 mM K-gluconate, 12.5 mM KCl, 0.2 mM EGTA, 8 mM NaCl, 2 mM MgATP, 0.3 mM Na3GTP, and 10 mM HEPES, and the pH was adjusted to 7.3 with KOH. The external solution contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, and 2 mM EGTA. In current-clamp mode, action potentials were evoked by current injection. Resting membrane potential was measured without current injection. The rheobase (the amount of current required to elicit an action potential) was determined by applying a step protocol (0 to 200 pA in 10 pA increments with a pulse duration of 500 ms). Capsaicin was applied via the same focal perfusion system. The recording chamber (300 μl) was continuously perfused (3–4 ml / min) with the focal perfusion system. The perfusion exchange time was 1–2 seconds after merging the manifold in the recording chamber.

[0083] Experimental Example 6. Western Blot

[0084] Spinal cord samples were homogenized in ice-cold tissue protein extraction reagent (NP-40, Elpis Biotech; EBA-1049, Korea) supplemented with 1% protease inhibitor mix. Protein concentration was measured using a bicinchoninic acid assay kit (Thermo fisher; 23227), and samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride membranes (Millipore; ipvh00010). The blots were blocked with 5% nonfat milk in Tris-buffered saline (TBST) containing Tween and incubated overnight with phospho-MAPK antibodies and antibodies in the MAPK antibody sampler kit (1:1000, Cell Signaling; 9910 and 9926). Each membrane was washed with TBST and incubated with the secondary antibody, anti-rabbit Ig-HRP (1:10000, Cell Signaling; 9910), for 1 h. After washing with TBST, the immunocomplexes were detected by chemoluminescence analysis using a Pierce western blotting kit (Beyotime, China). Quantitative densitometry analysis was performed using a UVP BioSpectrum multispectral imaging system (GE Healthcare Life Sciences, Image Quant LAS 4000).

[0085] Experimental Example 7. RNA Sequencing

[0086] Starting with 1 μg of RNA from each sample, libraries were constructed using the Illumina platform (KappaBiosystem) according to the manufacturer's instructions (individual barcodes included). Library quality was verified using Qubit 2.0, Agilent 2100, and Q-PCR. After passing quality filters, libraries were pooled onto a HiSeq 4000 at Macrogen (http: / / www.macrogen.com) in Seoul, South Korea, based on effective concentration and sequenced. This process yielded read lengths of 100 nt. Data quality was verified by Macrogen and generated in FASTq format.

[0087]

[0088] Experimental results

[0089] Peripheral Nervous System: Experimental Results Using the Dorsal Root Ganglia (DRG)

[0090] Experimental Results 1. Animal Experiment on Mechanical and Thermal Pain Hypersensitivity in STZ-Induced Diabetic Mice

[0091] 1-1. STZ was injected into the peritoneal cavity of mice, and pain responses were measured using the von Frey and Hargreaves tests on days 3, 7, 14, 21, and 28 after injection.

[0092] 1-2. The STZ group showed significant reductions in mechanical thresholds and thermal latency 3 days after administration, and the reductions persisted for up to 28 days after administration (see Figure 1). Therefore, it was confirmed that a diabetic neuropathy mouse model had been established in STZ-induced mice.

[0093]

[0094] Experimental Results 2. Changes in the expression of various pain-related functional genes, including the GABA neuronal system, through RNA seq analysis in the DRG of diabetic mice.

[0095] 2-1. To find the cause of pain induction in the STZ treatment group, RNA-seq was performed to explore the transcriptome profile of gene expression changes in the control and STZ treatment group DRG.

[0096] 2-2. To obtain an overview of the gene expression profiles of the STZ-treated group compared to the control group, hierarchical clustering and volcano plot analyses were used. Hierarchical clustering analysis of the identified differentially expressed genes (DEGs) revealed that the three control and STZ groups were clustered into separate groups, and the number of DEGs was confirmed from the volcano plot (see Figures 2 and 3).

[0097] 2-3. A total of 591 DEG genes were identified, of which 364 genes were upregulated and 227 genes were downregulated (see Fig. 4).

[0098] 2-4. Based on RNA-seq results, KEGG enrichment pathway analysis revealed that MAPK cascade, cytokines, and inflammatory responses were upregulated in GO (gene ontology) analysis, while GABAergic neuronal function was downregulated. Ultimately, we confirmed that the conditions of the control and experimental groups could influence pain induction (see Figure 5).

[0099]

[0100] Experimental Results 3. Confirmation of changes in the GABA neuron system and transcription factor gene expression levels in the dorsal root ganglion (DRG) of diabetic mice. (See Figures 6 to 8.)

[0101] 3-1. GABAergic neuron regulatory genes, GAD65.67 and vGAT, were significantly downregulated in the STZ-treated group compared to the control group. In contrast, GABA receptor subunit-related genes were either unchanged or slightly increased. Therefore, these results suggest that GABAergic neuron synaptic function was downregulated in the STZ-treated group. (See Figures 6 to 8.)

[0102]

[0103] 3-2. Furthermore, we examined whether various GABA neuronal system-regulating transcription factors were expressed differently in the DRG of STZ-induced diabetic mice. As a result, two transcription factors, Ascl1 and Lhx6, showed a significant decrease in expression levels on day 28 in the STZ-treated group, while other transcription factors, Dlx1,2,6 and NKx2.1,2.2, showed no significant changes in expression (see Figure 9).

[0104]

[0105] Experimental results 4. The effects of intrathecal injection of lentivirus expressing Ascl1 and lhx6 (hereinafter, lentivirus expressing Ascl1 and lhx6 are abbreviated as A / L) on mechanical allodynia and thermal hyperalgesia in diabetic mice were observed.

[0106] 4-1. Ascl1 and Lhx6 were administered to STZ-induced diabetic mice.

[0107] 4-2. Cultured DRG neurons were infected in vitro with lentiviruses expressing Ascl1 or lhx6, either alone or in combination at an appropriate concentration (7*10^6 TU), and the expression levels of various individual genes were compared. Treatment with Ascl1 or lhx6 alone resulted in a slight increase in gene expression. However, the group treated with Ascl1 and lhx6 together induced higher gene expression levels of GAD65.67 and vGAT compared to DRG neurons treated with lentiviruses expressing Ascl1 or lhx6 alone (see Figure 10).

[0108] 4-3. Based on these results, we tested whether the pain experienced in the STZ treatment group could be alleviated by intrathecal injection of lentiviruses expressing Ascl1 and lhx6 together at an appropriate concentration (7*10^7 TU). As a result, we confirmed that the A / L treatment group significantly reduced the increase in mechanical threshold and thermal latency experienced in the STZ treatment group. (See Figure 11)

[0109]

[0110] Experimental results 5. Confirmation of changes in the expression level of GABA-related genes in the DRG of diabetic mice when lentivirus (A / L) expressing Ascl1 / lhx6 was injected into the spinal cord.

[0111] 5-1. To verify specific genes that attenuate pain induction after transcription factor overexpression, gene expression levels were compared within three groups (control group, STZ treatment group, and A / L treatment group).

[0112] 5-2. The A / L treatment group significantly increased the reduced gene expression levels of GAD65.67, vGAT, and GABAB1-2, genes related to GABAergic neurons, which were observed in the STZ treatment group, but did not affect GABAARs, genes related to GABA receptor subunits. These results suggest that the transcription factor primarily enhances GABAergic neuron function among DRG neurons. (See Figures 13 and 14.)

[0113]

[0114] Experimental results 6. Effect of intrathecal injection of lentivirus expressing Ascl1 / lhx6 on the expression levels of proinflammatory cytokines, anti-inflammatory cytokines, and pain-inducing channels (or receptors) in the DRG of diabetic mice.

[0115] Because the 6-1 RNA seq results showed that inflammatory cytokine function was upregulated in the STZ treatment group, we further confirmed the expression levels of pro-inflammatory and anti-inflammatory cytokines.

[0116] 6-2. Gene expression levels of proinflammatory cytokines, such as TNF-alpha, IL-beta, and IL-6, significantly increased in the STZ treatment group, and this increased gene expression level was suppressed in the A / L treatment group. However, the expression of anti-inflammatory cytokines IL-4, IL-10, and IL-13 did not show significant changes in the STZ treatment group. (See Figures 15 and 16.)

[0117] 6-3. As a result of examining the expression levels of pain-inducing channels or receptors, TRPV1, TRPA1, Piezo1, Nav1.7, and Nav1.8, it was confirmed that while other receptors did not show significant changes, TRPV1 and Nav1.7 increased in the STZ group, and the level of increase was significantly reduced in the A / L treatment group (see Figures 17 and 18).

[0118]

[0119] Experimental Results 7. Hierarchical Cluster Analysis and Protein-Protein Interaction (PPI) Network Analysis Using Gene Expression Difference Profiles

[0120] To further discover differentially expressed genes by transcription factor activation in the 7-1 control, STZ treatment, and A / L treatment groups, hierarchical cluster and protein-protein interaction (PPI) network analyses were performed.

[0121] 7-2. We identified 66 genes that were upregulated and 84 genes that were downregulated in the STZ treatment group, and confirmed that these regulations were reversed in the A / L treatment group (see Figures 19 to 22).

[0122] 7-3 These genes were analyzed through hierarchical clustering and protein-protein interaction (PPI) network analysis to determine their expression levels and interaction networks. Among them, a total of 15 pain- and inflammation-related genes were selected, and seven downregulated genes (JunD, Gng3, zfp771, Id3, FosB, C-fos, and Erg1) and eight upregulated genes (Kif7, Foxk1, Gatad2b, Arid1b, Cic, Hivep1, Nr3c2, and Foxn3) were identified. (See Figures 23 to 25.)

[0123] 7-4. To assess the reliability of the RNA seq results, 15 genes were re-evaluated using qRT-PCR, and five genes (JunD / Gng3 / Fosb / C-fos / Foxk1) were selected. These genes are believed to play a key role in regulating diabetic pain sensitivity in the peripheral nervous system, based on GABAergic neuron function and Ascl1 / lhx6 activation (see Figures 24 and 25).

[0124]

[0125] Experimental Results 8. Confirmation of the Effect of Ascl1 / lhx6 on Ganglion Neuron Hyperexcitability after Exposure to Hyperglycemia (HG) Conditions (See Figures 26 to 28)

[0126] 8-1. To investigate why Ascl1 / lhx6 activation reduces pain hypersensitivity in STZ-induced mice, dorsal root ganglion nerve excitability was assessed using patch clamp after culturing in high glucose (HG) medium for 24 h.

[0127] 8-2. The HG condition induced an increase in the number of action potentials, a decrease in the resting membrane potential (RMP), and an increase in the action potential rheobase. Application of Ascl1 / lhx6 activation reversed each change to normal levels. (See Figure 26.)

[0128] 8-3. The effect of Ascl1 / lhx6 activation on TRPV1 activation was also evaluated. Capsaicin-induced inward currents were increased in HG medium, and Ascl1 / lhx6 activation attenuated this phenomenon. These results suggest that Ascl1 / lhx6 activation influences DRG properties under HG conditions. (See Figure 27.)

[0129] 8-4. The effect of Ascl1 / lhx6 activation on sodium current activation was also evaluated. We confirmed that sodium current inward current was increased in HG medium, and that Ascl1 / lhx6 activation reduced this phenomenon. These results suggest that Ascl1 / lhx6 activation influences DRG properties under HG conditions. (See Figure 28.)

[0130]

[0131] Central Nervous System: Experimental Results on the Spinal Cord (see Figures 29 to 39)

[0132] Experimental Results 9. Evaluation of the expression level of genes related to the GABA neuron system in the spinal cord of diabetic mice when a lentivirus expressing Ascl1 / lhx6 was injected intrathecally.

[0133] 9-1. In spinal cord tissue, the GABAergic neuron-regulating transcription factors Ascl1 and Lhx6 were significantly downregulated in the STZ-treated group compared to the control group. Furthermore, downregulation of GABA receptor subunits GABAB1 and 2 was confirmed. (See Figures 29 and 30.)

[0134] 9-2. In the A / L treatment group, it was confirmed that the levels of GABAergic neuronal regulatory transcription factors (Ascl1 and Lhx6) and receptor (GABAB1-2) gene changes induced in the STZ treatment group were reversed to normal levels. (See Figure 31)

[0135]

[0136] Experimental Results 10. Effects of intrathecal injection of lentivirus expressing Ascl1 / lhx6 on the levels of proinflammatory cytokines, anti-inflammatory cytokines, and glial activity in the spinal cord of diabetic mice (see Figures 32 to 35)

[0137] 10-1. In spinal cord tissue, gene expression levels of proinflammatory cytokines such as TNF-alpha, IL-beta, and IL-6 significantly increased in the STZ group, while expression levels of anti-inflammatory cytokines IL-4, IL-10, and IL-13 showed a significant decrease in the STZ group. (See Figures 32 and 33)

[0138] 10-2. We examined changes in the expression of genes related to microglia and astrocytes (Iba1 and GFAP), and confirmed that they were overexpressed in the STZ-treated group 28 days after STZ treatment. (See Figures 32 and 33.)

[0139] 10-3. The A / L treatment group reduced the increased expression of proinflammatory cytokines and increased the decreased expression of anti-inflammatory cytokines compared to the STZ treatment group. (See Figures 34 and 35)

[0140] 10-4. The A / L treatment group was confirmed to have reduced the overexpressed microglia and astrocytes in the STZ treatment group (see Figures 34 and 35).

[0141]

[0142] Experimental Results 11. Evaluation of the effect of intrathecal injection of lentivirus expressing Ascl1 / lhx6 on the expression level of MAPK signaling pathway in the spinal cord of diabetic mice (see Figures 36 to 39)

[0143] 11-1. As a result of evaluating the activation of inflammation-related MAPK signaling in spinal cord tissue, it was confirmed that the phosphorylation levels of MAPK-related ERK, p38, and JNK were increased in the STZ group.

[0144] 11-2. It was confirmed that the A / L treatment group significantly suppressed the activation level of ERK, p38, and JNK phosphorylation levels associated with increased MAPK signaling in the STZ treatment group.

[0145] The pharmaceutical composition of the present invention can be used for the treatment of pain.

[0146]

[0147] [Sequence List Free Text]

[0148] Sequence number 1: Ascl1 protein code: NP_004307

[0149] 1 messakmesg gagqqpqpqp qqpflppaac ffataaaaaaa aaaaaaaqsa qqqqqqqqqq

[0150] 61 qqapqlrpaa dgqpsggghk sapkqvkrqr ssspelmrck rrlnfsgfgy slpqqqpaav

[0151] 121 arrnerernr vklvnlgfat lrehvpngaa nkkmskvetl rsaveyiral qqlldehdav

[0152] 181 saafqagvls ptispnysnd lnsmagspvs syssdegsyd plspeeqell dftnwf

[0153] 서열번호 2: Lhx6 protein code: NP_001229262

[0154] 1 mrrglcrrsa enpdagpvma qpgsgckatt rclegtappa maqsdaeala galdkdegqa

[0155] 61 spctpstpsv csppsaassv psagknicss cgleildryl lkvnnliwhv rclecsvcrt

[0156] 121 slrqqnscyi knkeifckmd yfsrfgtkca rcgrqiyasd wvrrargnay hlacfacfsc

[0157] 181 krqlstgeef glveekvlcr ihydtmienl kraaengngl tlegavpseq dsqpkpakra

[0158] 241 rtsftaeqlq vmqaqfaqdn npdaqtlqkl admtglsrrv iqvwfqncra rhkkhtpqhp

[0159] 301 vppsgappsr lpsalsddih ytpfsspera rmvtlhgyie shpfsvltlp alphlpvgap

[0160] 361 qlplsr

[0161] 서열번호 3 : Ascl1 mRNA code: NM_008553

[0162] 1 agcactctct cacttctggc cagggaacgt ggaaggcgca ccgacaggga tccggccagg

[0163] 61 gagggcgagt gaaagaagga aatcagaaag gaagggagtt aacaaaataa taaaaacagc

[0164] 121 ctgagccacg gctggagaga ccgagacccg gcgcaagaga gcgcagcctt agtaggagag

[0165] 181 gaacgcgaga cgcggcagag cgcgttcagc actgactttt gctgctgctt ctgctttttt

[0166] 241 ttttcttaga aacaagaagg cgccagcggc agcctcacac gcgagcgcca cgcgaggctc

[0167] 301 ccgaagccaa cccgcgaagg gaggagggga gggaggagga ggcggcgtgc agggaggaga

[0168] 361 aaaagcattt tcactttttt tgctcccact ctaagaagtc tcccggggat tttgtatata

[0169] 421 ttttttaact tccgtcaggg ctcccgcttc atatttcctt ttctttccct ctctgttcct

[0170] 481 gcacccaagt tctctctgtg tccccctcgc gggccccgca cctcgcgtcc cggatcgctc

[0171] 541 tgattccgcg actccttggc cgccgctgcg catggaaagc tctgccaaga tggagagcgg

[0172] 601 cggcgccggc cagcagcccc agccgcagcc ccagcagccc ttcctgccgc ccgcagcctg

[0173] 661 tttctttgcc acggccgcag ccgcggcggc cgcagccgcc gcagcggcag cgcagagcgc

[0174] 721 gcagcagcag cagcagcagc agcagcagca gcagcaggcg ccgcagctga gaccggcggc

[0175] 781 cgacggccag ccctcagggg gcggtcacaa gtcagcgccc aagcaagtca agcgacagcg

[0176] 841 ctcgtcttcg cccgaactga tgcgctgcaa acgccggctc aacttcagcg gctttggcta

[0177] 901 cagcctgccg cagcagcagc cggccgccgt ggcgcgccgc aacgagcgcg agcgcaaccg

[0178] 961 cgtcaagttg gtcaacctgg gctttgccac ccttcgggag cacgtcccca acggcgcggc

[0179] 1021 caacaagaag atgagtaagg tggagacact gcgctcggcg gtcgagtaca tccgcgcgct

[0180] 1081 gcagcagctg ctggacgagc atgacgcggt gagcgccgcc ttccaggcag gcgtcctgtc

[0181] 1141 gcccaccatc tcccccaact actccaacga cttgaactcc atggccggct cgccggtctc

[0182] 1201 atcctactcg tcggacgagg gctcttacga cccgctcagc cccgaggagc aggagcttct

[0183] 1261 cgacttcacc aactggttct gaggggctcg gcctggtcag gccctggtgc gaatggactt

[0184] 1321 tggaagcagg gtgatcgcac aacctgcatc tttagtgctt tcttgtcagt ggcgttggga

[0185] 1381 gggggaaa agaaaaga aaaaaga agagaagaa gaaagagaa gaaaaaaaaaa

[0186] 1441 agaaaacag tcaaccaacc ccatcgccaa ctaagcgagg catgcctgag agacatggct

[0187] 1501

[0188] 1561 gaagcaac tgggacctga gtcaatgcgc aaaatgcagc ttgtgtgcaa aagcagtggg

[0189] 1621

[0190] 1681 agctgaaagt tcttgctcgg gtcccttcac ctcctcgccc tttcttaaag tgcagttctt

[0191] 1741 agccctctag aaacgagttg gtgtctttcg tctcagtagc ccccacccca ataagctgta

[0192] 1801 gacattggtt tacagtgaaa ctatgctatt ctcagccctt tgaaactctg cttctcctcc

[0193] 1861 agggcccgat tcccaaaccc catggcttcc ctcacactgt cttttctacc attttcatta

[0194] 1921 tagaatgctt ccaatctttt gtgaattttt tattataaaa aatctatttg tatctatcct

[0195] 1981 aaccagttcg gggatatatt aagatatttt tgtacataag agagaaagag agagaaaaat

[0196] 2041 tttagaagt tttgtacaaa tggtttaaaa tgtgtatatc ttgatacttt aacatgtaat

[0197] 2101 gctattacct ctgcatattt tagatgtgta gttcacctta caactgcaat tttccctatg

[0198] 2161 tggttttgta aagaactctc ctcataggtg agatcaagag gccaccagtt gtacttcagc

[0199] 2221 accaatgtgt cttactttat agaaatgttg ttaatgtatt aatgatgtta ttaaatactg

[0200] 2281 ttcaagaaga acaaagttta tgcagctact gtccaaactc aaagtggcag ccagttggtt

[0201] 2341 ttgataggtt gccttttgga gatttctatt actgcctttt tttttcttac tgttttatta

[0202] 2401 caaacttaca aaaatatgta taaccctgtt ttatacaaac tagtttcgta ataaaacttt

[0203] 2461 ttcctttttt taaaatgaaa a

[0204] 서열번호 4 : Lhx6 mRNA code: NM_008500

[0205] 1 gatttagtaa agacacaggc gaatcaagag gaggcgaggc cggtattgtc cgtctgaata

[0206] 61 ggcgctgata gcgccgatgc gccgggggtt gtgccggcgc agcgctgaga atcccgacgc

[0207] 121 ggggccggtg atggcccagc cagggtccgg ctgcaaagcg accacccgct gtcttgaagg

[0208] 181 gaccgcgccg cccgccatgg ctcagtctga cgccgaggcc ctggcaggag ctctggacaa

[0209] 241 ggacgagggt caggcctccc catgtacgcc cagcacgcca tctgtctgct caccgccctc

[0210] 301 tgccgcctcc tccgtgccgt ctgcaggcaa gaacatctgc tccagctgcg gcctcgagat

[0211] 361 cctggaccga tatctgctca aggtcaacaa cctcatctgg cacgtgcggt gcctcgagtg

[0212] 421 ctccgtgtgt cgcacgtcgc tgaggcagca gaacagctgc tacatcaaga acaaggagat

[0213] 481 cttctgcaag atggactact tcagccgatt cgggaccaag tgtgcccggt gcggccgaca

[0214] 541 gatctacgcc agcgactggg tgcggagagc tcgcggcaac gcctaccacc tggcctgctt

[0215] 601 cgcctgcttc tcgtgcaagc gccagctgtc cactggtgag gagttcggcc tggtcgagga

[0216] 661 gaaggtgctc tgccgcatcc actacgacac catgattgag aacctcaaga gggccgccga

[0217] 721 gaacgggaac ggcctcacgt tggagggggc agtgccctcg gaacaggaca gtcaacccaa

[0218] 781 gccggccaag cgcgcgcgga cgtccttcac cgcggaacag ctgcaggtta tgcaggcgca

[0219] 841 gttcgcgcag gacaacaacc ccgacgctca gacgctgcag aagctggcgg acatgacggg

[0220] 901 cctcagccgg agagtcatcc aggtgtggtt tcaaaactgc cgggcgcgtc ataaaaagca

[0221] 961 cacgccgcaa cacccagtgc cgccctcggg ggcgcccccg tcccgccttc cctccgccct

[0222] 1021 gtccgacgac atccactaca ccccgttcag cagccccgag cgggcgcgca tggtcaccct

[0223] 1081 gcacggctac attgagagtc atcctttttc agtactaacg ctgccggcac ttccgcatct

[0224] 1141 gcccgtgggc gccccacagc tgcccctcag ccgctgagat ccagtgtcca agctgcggcc

[0225] 1201 aggagtccac ccacctccgc atccaccccc gtccgccatc ctgcccacca ccaggtcggt

[0226] 1261 tcccgaggcc tggcctttcc ctctcctgct gagaaccaga acccaccagg agcaccacag

[0227] 1321 agtcctcctc ttggaaggca gaactccctg aaatctggaa tcagggtgga aacagcctgt

[0228] 1381 ttttcccatt taaacaggag tcctcttcaa cttcagctga ttacaataac aaaaggcgga

[0229] 1441 attgaattgt gcgatgccaa cggccttctc atttacaggt ttttttcccc cacattggcc

[0230] 1501 tttatttact acttccttgg aaccatctct gaattctgaa tagctgacaa cccccaatgt

[0231] 1561 tatccactct gttgcttttg tctggaaaac tctacagtgt ttgtgggatg tccccaaagg

[0232] 1621 aaagctatgt tctaatttta tcatttccat ctgtctggtt atgtcaagtt aattcagaaa

[0233] 1681 gagaagagac agtgaccaac cctgagaggc ctaatagggc agagatggag gcctgcccag

[0234] 1741 actaggaggc agcggggata gacagggaat ggggagaaga aagaccccca ttggtttgga

[0235] 1801 aatcaaggag agggcggtga catattggac cagaagaggc actagccatt ttaaggagag

[0236] 1861 gaaagagaaa actctggggt cagggagaga ccctaccccc acctaattat ccagcatata

[0237] 1921 tgtaagaaac atagcagcga tggtattcga tctgtgccat gactcttctg aatgtttgga

[0238] 1981 caggttagag ttggggaccc ctgttggcca cttgttgacc tctcatagtg gtgcttgggc

[0239] 2041 caggtcttct caatggaagg ggaatccctt ataggggaga gggaacagag cccagtgaaa

[0240] 2101 tggcagtcag aatgttaacc ctggatccat ctctaagtag agagagggtg cccattgcct

[0241] 2161 aggtgagtgt gccaagctca ggattccaac tggtgcctct gagcttccca atcaatactt

[0242] 2221 cctggagcca gccccaccca cccctgagaa cagaggtcag acacagctgc gtaacatcca

[0243] 2281 tcctgctaca actcttccac cccaaacaaa agggctcagg ctacacacga ccatgattta

[0244] 2341 tgttttcagg ggatgcccat ttgtcccaag cttatcctgt aattctagaa ttacctggtg

[0245] 2401 tcctgatgca ttttccacta gaggttgcta atcagcatgt tttagcccaa gtccgccttc

[0246] 2461 ctgctgtggt taacctgtta tgttgctttt ggaaggagac tctaagacag ggaaagcaag

[0247] 2521 ttcatggtac atacgcagcc attgtctctg tttttaccca tggcagacat tgctaatcaa

[0248] 2581 tggcagctct atttcactga gtctggataa ggtttcagag ttcaaatgct tgacgttggc

[0249] 2641 acttaacatg aaagcctata ggtcattctt gctctgggat ctacaggcag ggtaggcaca

[0250] 2701 ggtgcagcct aagaagggaa cctgcttcct ctcccttcca aagacagtga cagctgactg

[0251] 2761 agggcaaaga gcaggcacca ctcagaacgt ggtgagtaca gctcagctca gcactcagtc

[0252] 2821 agtggtaact tgtgcccagc cctgtgctag gcgctgacat taacaggagc aaccagggcc

[0253] 2881 caattcctgg ccttggagct caatctttc ctttgatttt tgctcctgat catcaaggcc

[0254] 2941 ccagtggcaa ccatgtggta agtggccaac caagccctac ccagggtcac ccaacacact

[0255] 3001 ctgccttgag cctctcctca gggtctattc cttgcgtgga ttatgtggcc gtagcatgtt

[0256] 3061 acagttcaaa catgtctcca ctaccctgtt aagagcagcc tgggaacgta caggccatca

[0257] 3121 agactattta tttaaataca aaaaaagggg aaaacacaca cacggaaaa aaattgtaag

[0258] 3181 cacttttttt gtaaaaccaa tgtctgtttt gttacatacc tttcatgtcg tgctttgtaa

[0259] 3241 atgtcttatt tgtgtaataa agttaatgca agtagtg ctggcactga aatcca

Claims

1. A composition for treating pain, comprising the Ascl1 protein of sequence number 1 or the Lhx6 protein of sequence number 2; a polynucleotide encoding the Ascl1 protein or the Lhx6 protein; or an expression vector comprising the polynucleotide.

2. A composition for treating pain, comprising: an Ascl1 protein of sequence number 1 and an Lhx6 protein of sequence number 2; a polynucleotide encoding the Ascl1 protein and a polynucleotide encoding the Lhx6 protein; or an expression vector comprising a polynucleotide encoding the Ascl1 protein and an expression vector comprising a polynucleotide encoding the Lhx6 protein.

3. A pharmaceutical composition according to claim 1, wherein the expression vector is a viral vector or a non-viral vector.

4. A pharmaceutical composition according to claim 3, wherein the viral vector is an adeno-associated virus (AAV) vector, an adenovirus vector, an alphavirus vector, a herpes simplex virus vector, a vaccinia vector, a Sendai virus vector, a flavivirus vector, a radobovirus vector, a retrovirus vector, or a lentivirus vector.

5. A pharmaceutical composition according to claim 4, wherein the pain is neuropathic pain, inflammatory pain, cancer pain, post-surgical pain, phantom limb pain, burn pain, gout pain, osteoarthritis pain, trigeminal neuralgia pain, acute herpes and post-herpetic pain, caustic pain, idiopathic pain, fibromyalgia, anticancer drug-induced pain, diabetic neuropathic pain, migraine, arthralgia, or rheumatoid arthritis pain.

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