WO2008010637A1 - Composition for attenuating neuropathic pain comprising a recombinant vector expressing gad65 - Google Patents
Composition for attenuating neuropathic pain comprising a recombinant vector expressing gad65 Download PDFInfo
- Publication number
- WO2008010637A1 WO2008010637A1 PCT/KR2007/001508 KR2007001508W WO2008010637A1 WO 2008010637 A1 WO2008010637 A1 WO 2008010637A1 KR 2007001508 W KR2007001508 W KR 2007001508W WO 2008010637 A1 WO2008010637 A1 WO 2008010637A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gad65
- neuropathic pain
- raav
- vector
- composition
- Prior art date
Links
- 208000004296 neuralgia Diseases 0.000 title claims abstract description 37
- 208000021722 neuropathic pain Diseases 0.000 title claims abstract description 37
- 239000013598 vector Substances 0.000 title claims abstract description 32
- 239000000203 mixture Substances 0.000 title claims abstract description 18
- 230000002093 peripheral effect Effects 0.000 claims abstract description 11
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 8
- 101000873786 Homo sapiens Glutamate decarboxylase 2 Proteins 0.000 claims description 30
- 102100035857 Glutamate decarboxylase 2 Human genes 0.000 claims description 26
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 241000702421 Dependoparvovirus Species 0.000 claims description 11
- 241000700605 Viruses Species 0.000 claims description 9
- 241000124008 Mammalia Species 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 3
- 210000003497 sciatic nerve Anatomy 0.000 abstract description 5
- 230000002459 sustained effect Effects 0.000 abstract description 4
- 230000001225 therapeutic effect Effects 0.000 abstract description 3
- 210000003594 spinal ganglia Anatomy 0.000 description 27
- 241000700159 Rattus Species 0.000 description 19
- 239000005090 green fluorescent protein Substances 0.000 description 17
- 208000004454 Hyperalgesia Diseases 0.000 description 13
- 230000000694 effects Effects 0.000 description 11
- 230000014509 gene expression Effects 0.000 description 11
- 239000002504 physiological saline solution Substances 0.000 description 11
- 239000007924 injection Substances 0.000 description 10
- 238000002347 injection Methods 0.000 description 10
- 208000002193 Pain Diseases 0.000 description 9
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 8
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 7
- 230000007423 decrease Effects 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 230000002458 infectious effect Effects 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 238000010171 animal model Methods 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 210000000653 nervous system Anatomy 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000004480 active ingredient Substances 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 210000003141 lower extremity Anatomy 0.000 description 4
- 230000000384 rearing effect Effects 0.000 description 4
- 210000000278 spinal cord Anatomy 0.000 description 4
- 230000000638 stimulation Effects 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 101100116172 Rattus norvegicus Gad2 gene Proteins 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000011532 immunohistochemical staining Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 210000002569 neuron Anatomy 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 238000007619 statistical method Methods 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- QWCKQJZIFLGMSD-GSVOUGTGSA-N D-alpha-aminobutyric acid Chemical compound CC[C@@H](N)C(O)=O QWCKQJZIFLGMSD-GSVOUGTGSA-N 0.000 description 2
- 101150014889 Gad1 gene Proteins 0.000 description 2
- 102100035902 Glutamate decarboxylase 1 Human genes 0.000 description 2
- 208000035154 Hyperesthesia Diseases 0.000 description 2
- 208000028389 Nerve injury Diseases 0.000 description 2
- 241000700584 Simplexvirus Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 206010053552 allodynia Diseases 0.000 description 2
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 2
- 238000000540 analysis of variance Methods 0.000 description 2
- 229910021538 borax Inorganic materials 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 210000001320 hippocampus Anatomy 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000003447 ipsilateral effect Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 238000001690 micro-dialysis Methods 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 230000008764 nerve damage Effects 0.000 description 2
- 210000004345 peroneal nerve Anatomy 0.000 description 2
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000004328 sodium tetraborate Substances 0.000 description 2
- 235000010339 sodium tetraborate Nutrition 0.000 description 2
- 208000020431 spinal cord injury Diseases 0.000 description 2
- 230000000946 synaptic effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 125000003821 2-(trimethylsilyl)ethoxymethyl group Chemical group [H]C([H])([H])[Si](C([H])([H])[H])(C([H])([H])[H])C([H])([H])C(OC([H])([H])[*])([H])[H] 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 241001269524 Dura Species 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 101100223310 Homo sapiens GAD1 gene Proteins 0.000 description 1
- 238000001282 Kruskal–Wallis one-way analysis of variance Methods 0.000 description 1
- 238000000585 Mann–Whitney U test Methods 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- WDVSHHCDHLJJJR-UHFFFAOYSA-N Proflavine Chemical compound C1=CC(N)=CC2=NC3=CC(N)=CC=C3C=C21 WDVSHHCDHLJJJR-UHFFFAOYSA-N 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 241000242739 Renilla Species 0.000 description 1
- 206010039670 Sciatic nerve injury Diseases 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004960 anterior grey column Anatomy 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 208000022371 chronic pain syndrome Diseases 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000003371 gabaergic effect Effects 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- 238000003365 immunocytochemistry Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000001722 neurochemical effect Effects 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 230000001473 noxious effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000006201 parenteral dosage form Substances 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 1
- 208000033808 peripheral neuropathy Diseases 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 210000002243 primary neuron Anatomy 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000011514 reflex Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000013222 sprague-dawley male rat Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- the present invention relates to a composition for attenuating neuropathic pain comprising a GAD65-expressing recombinant vector.
- Neuropathic pain refers to pain that stems from, or is caused by, primary lesions of various nervous systems or a dysfunction of the nervous systems and may be produced by multiple etiological factors (Bridges D., Br. J. Anaesth., 87: 12-26 (2001)).
- Peripheral neuropathic pain characterized by a diverse pathological processes, is accompanied by numerous episodes at different sites and at different times depending upon different disease states (Decosterd I. and Woolf CJ., Pain, 87: 149-158 (2000)).
- partial nerve injury appears to cause selective loss of GABAergic inhibitory synaptic current in the spinal cord.
- GABA glutamic acid dicarboxylase
- GAD65 is present in the membrane-bound form at the synapse and largely participates in the production of synaptic GABA for vesicular release.
- GAD67 is distributed throughout cells and is primarily responsible for the production of cytosolic GABA by secretion of GABA through non- vesicular mechanisms (Soghomonian JJ. and Martin D.L., Trends Pharmacol. ScL, 19: 500-505 (1998); Martin D.L. and AJ. T., Mechanisms controlling GABA synthesis and degradation in the brain.
- GABA in the nervous system the view at fifty years. Williams & Wilkins: Philadelphia, pp.25-41 (2000); Mi J. et al., J. Neurosci. Res., 57: 137-148 (1999); and Mackie M. et al., Neuroscience, 119: 461-472 (2003)).
- Moore et al reported that a concentration of GAD65 significantly decreases in the dorsal horn ipsilateral to the nerve injury site in a rat pain model, but expression of GAD67 does not exhibit such a decrease (Moore KA et al., J. Neurosci., 22: 6724-6731 (2002)).
- the present invention has been made in view of the above problems, and it is an object of the present invention to provide a pharmaceutical composition capable of effectively mitigating neuropathic pain.
- the above and other objects can be accomplished by the provision of a pharmaceutical composition for attenuating neuropathic pain of a mammal comprising a GAD65-expressing vector.
- the GAD65-expression vector used as an active ingredient of a pharmaceutical composition according to the present invention may be a virus vector (for example, DNA or RNA virus vector) containing a GAD65 gene.
- a virus vector may include Adenovirus vectors and Adeno- associated virus vectors.
- the Adeno- associated virus vector induces long-term gene expression and is therefore a vector system more suitable for the treatment of pain.
- the present invention constructed an rAAV-GAD65 vector for pain treatment having a gene map shown in FIG. 1 by the insertion of a GAD65 gene into an Adeno-associated virus vector under the control of a CMV promoter. The thus -constructed rAAV-GAD65 vector exerts sustained pain alleviation effects against neuropathic pain.
- the composition of the present invention may comprise one or more pharmaceutically acceptable carriers and vehicles, and optionally other therapeutically effective ingredients, in addition to the active ingredient GAD65-expression vector.
- the composition may be sterile and/or may contain adjuvants such as a preservative, a stabilizer, a wetting agent or an emulsification promoter, a salt for osmotic control, and/or a buffer.
- adjuvants such as a preservative, a stabilizer, a wetting agent or an emulsification promoter, a salt for osmotic control, and/or a buffer.
- the composition may be formulated into desired preparations by conventional methods known in the art.
- a typical parenteral dosage form is an injectable formulation that is preferably provided in the form of an isotonic aqueous solution or a suspension.
- the composition of the present invention may be administered to a subject, such as a mammal, e.g. a human, via any suitable route among various parenteral routes, according to gene therapy known to the public. In particular, most effective is admin- istration of the composition to dorsal root ganglions (DRGs) of the subject via direct injection.
- the composition may be given at a single dose or an equally divided multiple dose per day as 1 ng to 100 D/D BW/day, preferably 10 ng to 10 D/D BW/day of the active ingredient GAD-expression vector for a mammal including a human.
- a practical dose of the active ingredient will be determined taking into consideration various factors such as severity of pain, the selected administration route, age, sex, weight and conditions of a subject, and the like.
- composition of the present invention comprising a GAD65-expressing vector into a sciatic nerve leads to immediate therapeutic effects on peripheral neuropathic pain with a sustained and constant duration for several months. Therefore, the composition of the present invention is effective for attenuation of peripheral neuropathic pain.
- FIG. 1 is a gene map of an rAAV-GAD65 vector
- FIG. 2 is a fluorescence microscopic image showing expression profiles of green fluorescent protein (GFP) 3 or 8 weeks after direct injection of rAA V-GFP into L4 and L5 dorsal root ganglions (DRGs) of neuropathic pain model rats;
- GFP green fluorescent protein
- FIG. 3 is a micrograph (x 100) showing immunohistochemical staining results confirming expression of GAD65 in DRGs 8 weeks after injection of rAAV-GAD65 or physiological saline into L4 and L5 DRGs of neuropathic pain model rats;
- FIG. 4 is a graph showing effects of rAAV-GAD65 administration on mechanical allodynia in neuropathic pain model rats (non-treated control (•), rAAV-GAD65 administered (O), rAA V-GFP administered ( ⁇ ) and saline-administered control (A), and *: P ⁇ 0.05 and **: P ⁇ 0.01);
- FIG. 5 is a graph showing effects of rAAV-GAD65 administration on mechanical hyperalgesia in neuropathic pain model rats (non-treated control (•), rAAV-GAD65 administered (O), rAA V-GFP administered ( ⁇ ) and saline-administered control (A), and *: P ⁇ 0.05 and **: P ⁇ 0.01); and
- FIG. 6 is a bar graph showing a concentration of r -aminobutyric acid (GABA) measured by HPLC in dorsal horns of animals 8 weeks after injection of rAA V- GAD65 or physiological saline into DRGs of neuropathic pain model rats (**:P ⁇ 0.01). Best Mode for Carrying Out the Invention
- Example 1 Construction of recombinant Adeno-associated virus
- Adeno-associated virus used in the present invention was constructed and produced based on an AAV helper- free system (available from Stratagene, Kirkland, WA).
- rAAV2-JDK-GAD65 (hereinafter, referred to as "rAAV-GAD65") is a construct with the insertion of a gene coding for rat GAD65 into a pJDK plasmid that is an Adeno-associated virus containing a modified CMV promoter (JDK; SEQ. ID. NO.: 1) (Lee B. et al., Gene Ther., 12: 1215-1222 (2005)).
- cDNA of rat GAD65 was synthesized using RT-PCR (reverse transcription- polymerase chain reaction).
- RNA was extracted from the hippocampus using a Trizol solution (Invitrogen).
- SEQ. ID. NO.: 2 5'-GCCCTCGAG TTA- CAAATCTTGTCCCAGGCG-3'; shown with the Xbal recognition site underlined
- cDNA was synthesized. Thereafter, using the synthesized cDNA as a template and the above primer of SEQ. ID. NO.: 2 and a sequence of SEQ. ID.
- PCR was carried out to obtain a PCR product for rat GAD65.
- the PCR product was cleaved with Xbal and Xhol and was inserted into pBluescript SK(+) (Stratagene) cleaved with the same restriction enzymes to obtain pBluescript SK-GAD65.
- pBluescript SK-GAD65 was sequenced to re-confirm whether a nucleotide sequence was correctly incorporated.
- the pJDK plasmid and pBluescript SK-GAD65 were respectively cleaved with EcoRI(NEB) and a GAD65 DNA fragment was inserted into the cleaved pJDK plasmid using T4 DNA ligase (Takara Shuzo Co., Ltd., Tokyo, Japan).
- the resulting plasmid was used to transform E. coli XL-I Blue competent cells.
- Transformed cells were cultured on an LB plate containing 50 D/mL of kanamycin and selected to recover a plasmid pAAV- GAD65.
- a gene map of plasmid pAAV-GAD65 (rAAV-GAD65 virus was the same) is shown in FIG. 1, wherein TR represents a terminal repeat, P JDK represents a modified CMV promoter, e.g. JDK promoter, and Poly A represents a polyadenylation sequence.
- pAA V-GFP contains a humanized Renilla GFP(hrGFP) gene (Stratagene) under the control of a ubiquitous CMV promoter in the backbone of pAAV vector (Stratagene).
- pRepCap and pHelper vectors necessary for the construction of Adeno- associated virus were also purchased from Stratagene.
- the purified virus solution was dialyzed against a PBS buffer (PH 7.4), and the purified high-concentration rAAVs were stored in a PBS buffer containing 2% sorbitol at -8O 0 C (Kim S. J. et al., Oncol. Rep., 14: 1475-1479 (2005)).
- Example 2 Construction of animal model of neuropathic pain
- Rats were anesthetized with a sodium pentobarbital solution (50 D/D) and a segment of the left sciatic nerve was exposed at around the middle part of the femur. The surrounding tissues were carefully removed and the sciatic nerve was carefully fixed by a pincette. Three main parts of the sciatic nerve (tibial, shank and common fibular nerves) were clearly separated under a surgical microscope (Olympus, Japan). In order to construct an effective neuropathic pain model, the tibial and shank nerves were completely severed and firmly ligated and the fibular nerve was left intact. Following hemostatic treatment, the severed region was sutured together with muscle and skin.
- a sodium pentobarbital solution 50 D/D
- FIG. 2 is a fluorescence microscopic image (x 100) showing expression profiles of green fluorescent protein (GFP) 3 or 8 weeks after injection of rAAV-GFP into DRGs of the rats.
- GFP green fluorescent protein
- L4 and L5 DRGs emitted bright fluorescence due to accumulation of GFP after 3 weeks.
- GFP was continuously synthesized and a GFP signal was maintained positive at a time point of 8 week without significant changes.
- Rats were sacrificed 8 weeks later and T13-L1 spinal cord and L4-L5 DRGs were isolated, post- fixed, and equilibrated with a 30 % sucrose solution (Lee B. et al, supra ). Thereafter, cryosections were prepared, and each section was stained with polyclonal GAD65 antibodies (Chemicon, CA) and then was immunochemically stained with FITC-conjugated secondary antibodies (Santa Cruz Biotech).
- FIG. 3 is a micrograph (x 100) showing immunohistochemical staining results of
- Mechanical allodynia of each group was weekly measured from 1 week to 8 weeks by von Frey testing. Non- treated normal rats were employed as a control group.
- Rats were housed in acrylic cages (8x10x20 D) on top of a wire mesh grid which allowed access to the paws. After animals were allowed to acclimate to a new environment for 30 min, non-noxious mechanical stimuli were applied 10 times to side edges of the right and left hind limbs of rats using von Frey filaments (8 mN bending force), and total numbers of both rearing of the right and left hind limbs were measured.
- the rAAV-GAD65-administered group exhibited a sharp decrease of the allodynia response frequency to a range of 4.2+0.2 per 10 stimulations (a 49.4 % decrease relative to the physiological saline- administered control group). More importantly, rAAV-GAD65-administered group continuously exhibited significant alleviation of mechanical allodynia throughout the entire experimental time period. In contrast, the rAAV-GFP-administered group and the physiological saline-administered control group did not exhibit alleviation effects of mechanical allodynia. Further, no mechanical allodynia appeared in the non-treated control group.
- the rAAV-GAD65-administered group exhibited a sharp decrease (5.5+0.4 seconds (P ⁇ 0.05) in the rearing duration 1 week after injection of viruses; a 44% decrease relative to 12.5+1.0 seconds of the physiological saline- administered control group) with attenuation of symptoms arising from hyperalgesia.
- Rats anesthetized with urethane (1.25 D/D) were placed on a stereotaxic frame, a backside of the vertebra T 13 was exposed and fixed on a horizontal plane using a vertebral clamp. The dura was carefully open and a microdialysis probe (CM A/11, Sweden) was inserted into the vertebra. The probe was perfused with artificial cerebrospinal fluid (CSF) (145 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl , 1.O mM MgCl and 2.0 mM NaH PO , pH 7.4). A flow rate was carefully adjusted to 1.0 D/min using a CSF (CSF) (145 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl , 1.O mM MgCl and 2.0 mM NaH PO , pH 7.4). A flow rate was carefully adjusted to 1.0 D/min using a
- Peaks were respectively detected at an excitation wavelength of 340 nm and an emission wavelength of 460 nm, using RF-IOAxI (Shimazu Corp., Japan) at 3O 0 C and a flow rate of 0.7 mL/ min.
- rats of the rAAV-GAD65-administered group exhibited a significant increase in the GABA concentration of the spinal dorsal horn, i.e., 0.619+0.064 pmol/D (P ⁇ 0.01), whereas the physiological saline-administered control group showed no such an increase in the GABA concentration (0.284+0.065 pmol/D).
- composition of the present invention is effective for attenuation of peripheral neuropathic pain.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Environmental Sciences (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Provided is a pharmaceutical composition for attenuation of neuropathic pain comprising a GAD65-expressing recombinant vector. Direct introduction of the pharmaceutical composition of the present invention into a sciatic nerve leads to immediate therapeutic effects on peripheral neuropathic pain with a sustained and constant duration for several months. Therefore, the composition of the present invention is effective for the alleviation of peripheral neuropathic pain.
Description
Description
COMPOSITION FOR ATTENUATING NEUROPATHIC PAIN COMPRISING A RECOMBINANT VECTOR EXPRESSING
GAD65
Technical Field
[1] The present invention relates to a composition for attenuating neuropathic pain comprising a GAD65-expressing recombinant vector. Background Art
[2] Peripheral neuropathic pain is clinically common, yet little is known about development of effective therapeutic methods.
[3] Neuropathic pain refers to pain that stems from, or is caused by, primary lesions of various nervous systems or a dysfunction of the nervous systems and may be produced by multiple etiological factors (Bridges D., Br. J. Anaesth., 87: 12-26 (2001)). Peripheral neuropathic pain, characterized by a diverse pathological processes, is accompanied by numerous episodes at different sites and at different times depending upon different disease states (Decosterd I. and Woolf CJ., Pain, 87: 149-158 (2000)). Among intricate and complicated mechanisms underlying neuropathic pain, partial nerve injury appears to cause selective loss of GABAergic inhibitory synaptic current in the spinal cord. This feature contributes to phenotypes of neuropathic pain syndromes (Moore K.A. et al, J. Neurosci., 22: 6724-6731 (2002); Bennett GJ. et al., Neurochemical and anatomical changes in the dorsal horn of rats with an experimental peripheral neuropathy. In: Processing of sensory information in the superficial dorsal horn of the spinal cord, Plenum: New York, 1989, pp. 463-471; and Moore K. et al., Neurosci News, 4: 5-10 (2001)). r -aminobutyric acid (GABA) produced by glutamic acid dicarboxylase (GAD) is a principal inhibitory neurotransmitter present in the spinal dorsal horn, and also plays an important role in the ventral horn (Todd A. J. and Maxwell D. J., GABA in the mammalian spinal cord. In: GABA in the nervous systems: the view at fifty years (Martin D.L., Olsen R.W., eds), 2000).
[4] Genetic modification of neurons via the introduction of transgenes may be an effective therapy for the treatment of various neurological diseases, such as Alzheimer's disease, Parkinson's disease, and chronic pain syndromes that are intractable due to, or suffer from, little effective therapy. Previous reports suggest that introduction of vector-mediated foreign GAD and the resulting GABA lead to transient relief of neuropathic pain after spinal cord injury (SCI) (Liu J. et al., MoI. Ther. 10: 57-66 (2004)). To date, due to several advantages including long-term effective gene
transfer and minimum side effects (Rolling F. and Samulski RJ., MoL BiotechnoL, 3:9-15 (1995); Kaplitt M.G. et al., Nat. Genet., 8:148-154 (1994); Bankiewicz K.S. et al., Exp. Neurol., 144: 147-156 (1997); and Burger C. et al., Hum. Gene Ther., 16: 781-791 (2005)), a human Adeno-associated virus (hAAV)-based vector system has been most widely applied to the nervous system (Mandel RJ. et al., MoI. Ther., 13: 463-483)(2006)). Further, recombinant AA V2 (rAAV2) preferentially transforms the primary neurons of the central nervous system (Bankiewicz K.S. et al., supra; Mandel RJ. et al., J. Neuroscl, 18: 4271-4284 (1998); Wang L. et al, Gene Ther., 9: 381-389 (2002); Clark K.R. et al., J. Drug Target, 7: 269-283 (1999); and Burger C. et al., MoI Ther, 10: 302-317 (2004)).
[5] Two isoforms of mammalian GAD were identified and they are encoded by separate genes (Erlander M.G. et al., Neuron, 7: 91-100 (1991)). GAD65 is present in the membrane-bound form at the synapse and largely participates in the production of synaptic GABA for vesicular release. In contrast, GAD67 is distributed throughout cells and is primarily responsible for the production of cytosolic GABA by secretion of GABA through non- vesicular mechanisms (Soghomonian JJ. and Martin D.L., Trends Pharmacol. ScL, 19: 500-505 (1998); Martin D.L. and AJ. T., Mechanisms controlling GABA synthesis and degradation in the brain. In: GABA in the nervous system: the view at fifty years. Williams & Wilkins: Philadelphia, pp.25-41 (2000); Mi J. et al., J. Neurosci. Res., 57: 137-148 (1999); and Mackie M. et al., Neuroscience, 119: 461-472 (2003)). Moore et al reported that a concentration of GAD65 significantly decreases in the dorsal horn ipsilateral to the nerve injury site in a rat pain model, but expression of GAD67 does not exhibit such a decrease (Moore KA et al., J. Neurosci., 22: 6724-6731 (2002)).
[6] In recent years, Hao et al succeeded in construction of a recombinant Herpes simplex virus (HSV)-based vector encoding human GAD67 (QHGAD67), reporting that subcutaneous administration of QHGAD67 to foot pads of mice alleviates peripheral neuropathic pain (Hao S. et al., Ann. Neurol., 57: 914-918 (2005)). However, the pain relief effects reached to a maximum within several weeks by administration of QHGAD67, the action of which lasted only up to 2 to 5 weeks, and thereafter sharply disappeared.
[7] Therefore, there is a strong and continuous need in the art for the development of a therapeutic agent capable of exhibiting sustained pain-relief effects against peripheral neuropathic pain. Disclosure of Invention Technical Problem
[8] Therefore, the present invention has been made in view of the above problems, and
it is an object of the present invention to provide a pharmaceutical composition capable of effectively mitigating neuropathic pain. Technical Solution
[9] In accordance with an aspect of the present invention, the above and other objects can be accomplished by the provision of a pharmaceutical composition for attenuating neuropathic pain of a mammal comprising a GAD65-expressing vector.
[10] The GAD65-expression vector used as an active ingredient of a pharmaceutical composition according to the present invention may be a virus vector (for example, DNA or RNA virus vector) containing a GAD65 gene. Examples of such a virus vector may include Adenovirus vectors and Adeno- associated virus vectors. The Adeno- associated virus vector induces long-term gene expression and is therefore a vector system more suitable for the treatment of pain. The present invention constructed an rAAV-GAD65 vector for pain treatment having a gene map shown in FIG. 1 by the insertion of a GAD65 gene into an Adeno-associated virus vector under the control of a CMV promoter. The thus -constructed rAAV-GAD65 vector exerts sustained pain alleviation effects against neuropathic pain.
[11] Direct injection of the rAAV-GAD65 vector showing a high expression of GAD65 and GABA into dorsal root ganglions (DRGs) of the rat suffering from neuropathic pain due to sciatic nerve injury produces continuous GAD65 expression in DRGs lasting up to more than 8 weeks after injection of the vector, and the resultant GABA release, thereby resulting in significant alleviation of neuropathic pain. Such therapeutic effects immediately manifest and last for several months, during which the effects are substantially constant. Therefore, the composition of the present invention comprising a GAD65-expression vector is highly effective for alleviation of neuropathic pain, particularly peripheral neuropathic pain.
[12] The composition of the present invention may comprise one or more pharmaceutically acceptable carriers and vehicles, and optionally other therapeutically effective ingredients, in addition to the active ingredient GAD65-expression vector. The composition may be sterile and/or may contain adjuvants such as a preservative, a stabilizer, a wetting agent or an emulsification promoter, a salt for osmotic control, and/or a buffer. Further, the composition may be formulated into desired preparations by conventional methods known in the art. For example, a typical parenteral dosage form is an injectable formulation that is preferably provided in the form of an isotonic aqueous solution or a suspension.
[13] The composition of the present invention may be administered to a subject, such as a mammal, e.g. a human, via any suitable route among various parenteral routes, according to gene therapy known to the public. In particular, most effective is admin-
istration of the composition to dorsal root ganglions (DRGs) of the subject via direct injection. [14] The composition may be given at a single dose or an equally divided multiple dose per day as 1 ng to 100 D/D BW/day, preferably 10 ng to 10 D/D BW/day of the active ingredient GAD-expression vector for a mammal including a human. [15] However, it is to be understood that a practical dose of the active ingredient will be determined taking into consideration various factors such as severity of pain, the selected administration route, age, sex, weight and conditions of a subject, and the like.
Therefore, it will be appreciated that the scope of the present invention is not limited to the above- specified dosage range.
Advantageous Effects
[16] Direct introduction of a pharmaceutical composition of the present invention comprising a GAD65-expressing vector into a sciatic nerve leads to immediate therapeutic effects on peripheral neuropathic pain with a sustained and constant duration for several months. Therefore, the composition of the present invention is effective for attenuation of peripheral neuropathic pain. Brief Description of the Drawings
[17] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
[18] FIG. 1 is a gene map of an rAAV-GAD65 vector;
[19] FIG. 2 is a fluorescence microscopic image showing expression profiles of green fluorescent protein (GFP) 3 or 8 weeks after direct injection of rAA V-GFP into L4 and L5 dorsal root ganglions (DRGs) of neuropathic pain model rats;
[20] FIG. 3 is a micrograph (x 100) showing immunohistochemical staining results confirming expression of GAD65 in DRGs 8 weeks after injection of rAAV-GAD65 or physiological saline into L4 and L5 DRGs of neuropathic pain model rats;
[21] FIG. 4 is a graph showing effects of rAAV-GAD65 administration on mechanical allodynia in neuropathic pain model rats (non-treated control (•), rAAV-GAD65 administered (O), rAA V-GFP administered (Δ) and saline-administered control (A), and *: P<0.05 and **: P<0.01);
[22] FIG. 5 is a graph showing effects of rAAV-GAD65 administration on mechanical hyperalgesia in neuropathic pain model rats (non-treated control (•), rAAV-GAD65 administered (O), rAA V-GFP administered (Δ) and saline-administered control (A), and *: P<0.05 and **: P<0.01); and
[23] FIG. 6 is a bar graph showing a concentration of r -aminobutyric acid (GABA) measured by HPLC in dorsal horns of animals 8 weeks after injection of rAA V-
GAD65 or physiological saline into DRGs of neuropathic pain model rats (**:P<0.01). Best Mode for Carrying Out the Invention
[24] Reference Example: Statistical analysis
[25] All data were expressed as mean SEMs. Statistical analysis was carried out using analysis of variance (ANOVA) (electrophysiological recording data) or the Kruskal- Wallis One-way Analysis of Variance, and then the Mann- Whitney U-test was carried out for the comparison of behavior data between each group. Statistical significance was given for P<0.01 and P<0.05. All of statistical analyses were performed using SPSS (version 11.5, SPSS Inc., Chicago, IL).
[26]
[27] Example 1: Construction of recombinant Adeno-associated virus
[28] Adeno-associated virus used in the present invention was constructed and produced based on an AAV helper- free system (available from Stratagene, Kirkland, WA).
[29] rAAV2-JDK-GAD65 (hereinafter, referred to as "rAAV-GAD65") is a construct with the insertion of a gene coding for rat GAD65 into a pJDK plasmid that is an Adeno-associated virus containing a modified CMV promoter (JDK; SEQ. ID. NO.: 1) (Lee B. et al., Gene Ther., 12: 1215-1222 (2005)).
[30]
[31] Construction of rAAV-GAD65
[32] First, cDNA of rat GAD65 was synthesized using RT-PCR (reverse transcription- polymerase chain reaction).
[33] The hippocampus was removed from rats and total RNA was extracted from the hippocampus using a Trizol solution (Invitrogen). Using the extracted RNA as a template and a sequence of SEQ. ID. NO.: 2 (5'-GCCCTCGAG TTA- CAAATCTTGTCCCAGGCG-3'; shown with the Xbal recognition site underlined) complementary to 3 '-end of GAD mRNA as a primer, cDNA was synthesized. Thereafter, using the synthesized cDNA as a template and the above primer of SEQ. ID. NO.: 2 and a sequence of SEQ. ID. NO.: 3 (5'-GTCTCTAGA CCACCATG- GCATCTCCGGGCTCTG-3'; shown with the Xhol recognition site) complementary to 5 '-end of GAD mRNA as primers, PCR was carried out to obtain a PCR product for rat GAD65. The PCR product was cleaved with Xbal and Xhol and was inserted into pBluescript SK(+) (Stratagene) cleaved with the same restriction enzymes to obtain pBluescript SK-GAD65. pBluescript SK-GAD65 was sequenced to re-confirm whether a nucleotide sequence was correctly incorporated.
[34] In order to construct a pAAV-GAD65 vector containing endogenous GAD65 necessary in production of rAAV-GAD65 virus, the GAD65 gene of pBluescript SK- GAD65 was sub-cloned into a pJDK plasmid (courtesy of Duk-Kyung Kim, MD, PhD,
Sungkyunkwan University School of Medicine, Seoul, Korea; Byun J. et al., J. MoI. Cell Cardiol., 33: 295-305 (2001)) for the Adeno-associated virus. That is, the pJDK plasmid and pBluescript SK-GAD65 were respectively cleaved with EcoRI(NEB) and a GAD65 DNA fragment was inserted into the cleaved pJDK plasmid using T4 DNA ligase (Takara Shuzo Co., Ltd., Tokyo, Japan). The resulting plasmid was used to transform E. coli XL-I Blue competent cells. Transformed cells were cultured on an LB plate containing 50 D/mL of kanamycin and selected to recover a plasmid pAAV- GAD65. A gene map of plasmid pAAV-GAD65 (rAAV-GAD65 virus was the same) is shown in FIG. 1, wherein TR represents a terminal repeat, P JDK represents a modified CMV promoter, e.g. JDK promoter, and Poly A represents a polyadenylation sequence.
[35] pAA V-GFP contains a humanized Renilla GFP(hrGFP) gene (Stratagene) under the control of a ubiquitous CMV promoter in the backbone of pAAV vector (Stratagene).
[36] Meanwhile, pRepCap and pHelper vectors necessary for the construction of Adeno- associated virus were also purchased from Stratagene.
[37] In order to obtain a pure preparation of rAAVs, 293T cells (courtesy of Dr. J. Jung,
Harvard Medical School) cultured on a 10x10 D dish were transfected with pRepCap or pHelper in conjunction with pAAV-GAD65 or pAA V-GFP using a calcium phosphate- mediated transfection method (Nam Y.R. et al., Oncol. Rep., 12: 761-766 (2004)). This was followed by the single-step column purification (SSCP) (Auricchio A. et al. , Hum. Gene Ther., 12: 71-76 (2001)). The purified virus solution was dialyzed against a PBS buffer (PH 7.4), and the purified high-concentration rAAVs were stored in a PBS buffer containing 2% sorbitol at -8O0C (Kim S. J. et al., Oncol. Rep., 14: 1475-1479 (2005)).
[38] Total and infectious virus particles were respectively analyzed using an ELISA kit
(Progen Inc., Heidelberg, Germany) and immunocytochemistry for GAD65 (Chemicon, CA) (Nam Y. R. et al., supra). Mode for the Invention
[39] Example 2: Construction of animal model of neuropathic pain
[40] 8-10 week-old Sprague Dawley male rats weighing 180-200 g were placed in cages each housing five animals. Rats were raised in a breeding room maintained constantly at a temperature of 22+10C, humidity of 50+5% and a 12 h/12 h light/dark (L/ D) cycle. Animals were given free access to food and drinking water.
[41] Rats were anesthetized with a sodium pentobarbital solution (50 D/D) and a segment of the left sciatic nerve was exposed at around the middle part of the femur. The surrounding tissues were carefully removed and the sciatic nerve was carefully fixed by a pincette. Three main parts of the sciatic nerve (tibial, shank and common fibular
nerves) were clearly separated under a surgical microscope (Olympus, Japan). In order to construct an effective neuropathic pain model, the tibial and shank nerves were completely severed and firmly ligated and the fibular nerve was left intact. Following hemostatic treatment, the severed region was sutured together with muscle and skin.
[42]
[43] Experimental Example 1 : Transduction efficiency of rAAVs into DRGs
[44] In order to investigate a transduction efficiency of rAAVs into dorsal root ganglions
(DRGs), an animal model (n=6) was surgically established in the same manner as in Example 2 and 2 weeks later a part of the vertebra was removed from the anesthetized rat, thereby surgically exposing ipsilateral lumbar L4 and L5 DRGs. 3 D (1.3x10 infectious particles/mL) of an rAAV-GFP suspension in physiological saline was transferred to each DRG neurons via a glass micropipette connected to a Hamilton syringe for 20 min and the surgical sites were sutured again.
[45] FIG. 2 is a fluorescence microscopic image (x 100) showing expression profiles of green fluorescent protein (GFP) 3 or 8 weeks after injection of rAAV-GFP into DRGs of the rats. As can be seen, L4 and L5 DRGs emitted bright fluorescence due to accumulation of GFP after 3 weeks. Further, after introduction of rAAV-GFP into DRGs, GFP was continuously synthesized and a GFP signal was maintained positive at a time point of 8 week without significant changes.
[46]
[47] Example 3: Examination of GAD65 within DRGs in neuropathic pain model
[48] In order to examine GAD65 within DRGs in a neuropathic pain model, 3 D of an rAAV-GAD65 suspension in physiological saline (2.4x10 infectious particles/mL) (n=6) or 3 D (control) (n=5) of physiological saline was directly introduced in the same manner as in Experimental Example 1 into L4 or L5 DRGs of the animal model established as in Example 2.
[49] Rats were sacrificed 8 weeks later and T13-L1 spinal cord and L4-L5 DRGs were isolated, post- fixed, and equilibrated with a 30 % sucrose solution (Lee B. et al, supra ). Thereafter, cryosections were prepared, and each section was stained with polyclonal GAD65 antibodies (Chemicon, CA) and then was immunochemically stained with FITC-conjugated secondary antibodies (Santa Cruz Biotech).
[50] FIG. 3 is a micrograph (x 100) showing immunohistochemical staining results of
DRGs 8 weeks after administration of rAAV-GAD65. As can be seen, GAD65-specific immunohistochemical staining easily detected expression of GAD65 in DRGs, and the concentration of GAD65 was significantly higher in DRG with injection of rAAV-GAD65, as compared to the control group with an injection of physiological saline.
[51]
[52] Example 4: Attenuation of neuropathic pain-induced mechanical allodvnia and hy- peralgesia via expression of GAD65 in DRGs
[53] (1) Attenuation of mechanical allodynia
[54] According to the same manner as in Experimental Example 1, 3 D of an rAAV-
GAD65 suspension in physiological saline (2.4x10 infectious particles/mL) (n=10), 3 D of an rAA V-GFP suspension in physiological saline (1.3xlO7 infectious particles/mL) (n=4) or 3 D (control) (n=5) of physiological saline was directly introduced into L4 or L5 DRGs of the animal model established as in Example 2. Mechanical allodynia of each group was weekly measured from 1 week to 8 weeks by von Frey testing. Non- treated normal rats were employed as a control group.
[55] Rats were housed in acrylic cages (8x10x20 D) on top of a wire mesh grid which allowed access to the paws. After animals were allowed to acclimate to a new environment for 30 min, non-noxious mechanical stimuli were applied 10 times to side edges of the right and left hind limbs of rats using von Frey filaments (8 mN bending force), and total numbers of both rearing of the right and left hind limbs were measured.
[56] A frequency of hind limb withdrawal at 10-time mechanical stimulation was measured and recorded for four individual groups, an rAAV-GAD65-administered group (n=10), an rAAV-GFP-administered group (n=4), a physiological saline- administered control group (n=7), and a non-treated control group (FIG. 4). One week after administration, the rAAV-GFP-administered group and the physiological saline- administered control group exhibited allodynia response frequency of 8.8+0.2 and 8.5+0.3 per 10 stimulations, respectively. However, the rAAV-GAD65-administered group exhibited a sharp decrease of the allodynia response frequency to a range of 4.2+0.2 per 10 stimulations (a 49.4 % decrease relative to the physiological saline- administered control group). More importantly, rAAV-GAD65-administered group continuously exhibited significant alleviation of mechanical allodynia throughout the entire experimental time period. In contrast, the rAAV-GFP-administered group and the physiological saline-administered control group did not exhibit alleviation effects of mechanical allodynia. Further, no mechanical allodynia appeared in the non-treated control group.
[57]
[58] (2) Attenuation of mechanical hyperalgesia
[59] In order to confirm attenuation of mechanical hyperalgesia by expression of
GAD65 in DRGs, an rAAV-GAD65-administered group (n=6), an rAAV- GFP-administered group (n=5), a physiological saline-administered control group (n=7), and a non-treated control group were established as in Section 1, and a pinprick test was carried out using a bent needle (22-gauge) connected to a syringe. In this
connection, side edges of paw pads of right and left hind limbs were given pin stimulation having strength enough to evoke reflex withdrawal response also in animals other than the experimental group. Rearing duration was recorded using a stop watch.
[60] As a result, the rAAV-GAD65-administered group (n=6), the rAAV-
GFP-administered group (n=5), and the physiological saline-administered control group (n=7) exhibited significantly increased rearing duration of 9.8+0.4 seconds, 11+0.7 seconds and 10.5+lseconds, respectively, prior to administration (FIG. 5). Among these groups, only the rAAV-GAD65-administered group exhibited a sharp decrease (5.5+0.4 seconds (P<0.05) in the rearing duration 1 week after injection of viruses; a 44% decrease relative to 12.5+1.0 seconds of the physiological saline- administered control group) with attenuation of symptoms arising from hyperalgesia. Such attenuation effects by rAAV-GAD65 lasted through the entire experimental time period, and both of the rAAV-GFP-administered group and the physiological saline- administered control group did not show hyperalgesia-diminishing effects.
[61]
[62] Example 5: Determination of release of GABA from spinal dorsal horn
[63] In order to confirm whether the expression of GAD65 introduced into DRGs leads to a significant increase of GABA in the spinal dorsal horn, a concentration of GABA in the spinal dorsal horn was determined 8 weeks after administration of 3 D of an rAAV-GAD65 suspension in physiological saline (2.4x10 infectious particles/mL) (n=8) or 3 D (control) (n=7) of physiological saline to the animal model established as in Example 2, according to the same manner as in Experimental Example 1.
[64] Rats anesthetized with urethane (1.25 D/D) were placed on a stereotaxic frame, a backside of the vertebra T 13 was exposed and fixed on a horizontal plane using a vertebral clamp. The dura was carefully open and a microdialysis probe (CM A/11, Sweden) was inserted into the vertebra. The probe was perfused with artificial cerebrospinal fluid (CSF) (145 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl , 1.O mM MgCl and 2.0 mM NaH PO , pH 7.4). A flow rate was carefully adjusted to 1.0 D/min using a
2 4
CMA/102 pump (CMA/ microdialysis, Sweden). Thereafter, 30 D of each cerebrospinal fluid was collected, and mixed with 60 D of a working solution consisted of 3 mL of OPA mother liquid (2.7 D of O-phthaldialdehyde, 5 D of 2-mercaptoethanol and 9 mL of 0.1 M sodium tetraborate in 1 mL of MeOH) and 1 mL of sodium tetraborate, followed by HPLC analysis. The HPLC analysis was carried out using a reverse-phase column (AccQ-Tag, 3.9x150 D, Waters for amino acid analysis, Ireland) and a 0.02 M sodium acetate buffer (pH 4.6) containing 30% acetonitrile as a mobile phase. Peaks were respectively detected at an excitation wavelength of 340 nm and an emission wavelength of 460 nm, using RF-IOAxI (Shimazu Corp., Japan) at 3O0C and a flow rate of 0.7 mL/
min.
[65] As can be seen from FIG. 6, rats of the rAAV-GAD65-administered group exhibited a significant increase in the GABA concentration of the spinal dorsal horn, i.e., 0.619+0.064 pmol/D (P<0.01), whereas the physiological saline-administered control group showed no such an increase in the GABA concentration (0.284+0.065 pmol/D).
Industrial Applicability
[66] The composition of the present invention is effective for attenuation of peripheral neuropathic pain.
[67] Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
Claims
[1] A pharmaceutical composition for attenuating neuropathic pain of a mammal, comprising a GAD65-expressing recombinant vector.
[2] The composition according to claim 1, wherein the recombinant vector is a recombinant virus vector containing a GAD65 gene.
[3] The composition according to claim 2, wherein the recombinant virus vector is a recombinant Adeno-associated virus vector.
[4] The composition according to claim 1, wherein the recombinant vector is an rAAV-GAD65 vector having a gene map as shown in FIG. 1.
[5] The composition according to claim 1, wherein the neuropathic pain is peripheral neuropathic pain.
[6] The composition according to claim 1, wherein the mammal is a human.
[7] An injectable preparation comprising of the pharmaceutical composition of claim
1.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020060067264A KR20080007968A (en) | 2006-07-19 | 2006-07-19 | Composition for attenuating neuropathic pain comprising a recombinant vector expressing gad65 |
KR10-2006-0067264 | 2006-07-19 | ||
US11/691,478 US20080051357A1 (en) | 2006-07-19 | 2007-03-26 | Composition for attenuating neuropathic pain comprising a recombinant ventor expressing gad65 |
US11/691,478 | 2007-03-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2008010637A1 true WO2008010637A1 (en) | 2008-01-24 |
Family
ID=38956948
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/KR2007/001508 WO2008010637A1 (en) | 2006-07-19 | 2007-03-28 | Composition for attenuating neuropathic pain comprising a recombinant vector expressing gad65 |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2008010637A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110461367A (en) * | 2016-10-31 | 2019-11-15 | 可隆生命科学株式会社 | For alleviating or treating the composition of pain |
KR20200051679A (en) * | 2017-09-08 | 2020-05-13 | 더 리젠츠 오브 더 유니버시티 오브 캘리포니아 | Methods and compositions for the treatment of neuropathic pain |
EP3652309A4 (en) * | 2017-07-13 | 2021-04-28 | University of Miami | Method for managing pain |
CN113499450A (en) * | 2015-09-21 | 2021-10-15 | 可隆生命科学株式会社 | Composition for treating pain |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060099179A1 (en) * | 2000-05-23 | 2006-05-11 | Neurologix, Inc. | Glutamic acid decarboxylase (GAD) based delivery system |
US7060727B2 (en) * | 2002-12-11 | 2006-06-13 | Xenoport, Inc. | Prodrugs of fused GABA analogs, pharmaceutical compositions and uses thereof |
-
2007
- 2007-03-28 WO PCT/KR2007/001508 patent/WO2008010637A1/en active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060099179A1 (en) * | 2000-05-23 | 2006-05-11 | Neurologix, Inc. | Glutamic acid decarboxylase (GAD) based delivery system |
US7060727B2 (en) * | 2002-12-11 | 2006-06-13 | Xenoport, Inc. | Prodrugs of fused GABA analogs, pharmaceutical compositions and uses thereof |
Non-Patent Citations (1)
Title |
---|
GENCHENG HAN ET AL.: "Active tolerance induction and prevention of autoimmune diabetes by immunogene therapy using recombinant adenoassociated virus expressing glutamic acid decarboxylase 65 peptide GAD500-585", THE JOURNAL OF IMMUNOLOGY, 2005, pages 4516 - 4524 * |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113499450A (en) * | 2015-09-21 | 2021-10-15 | 可隆生命科学株式会社 | Composition for treating pain |
CN113499450B (en) * | 2015-09-21 | 2024-04-23 | 可隆生命科学株式会社 | Composition for treating pain |
CN110461367A (en) * | 2016-10-31 | 2019-11-15 | 可隆生命科学株式会社 | For alleviating or treating the composition of pain |
CN110461367B (en) * | 2016-10-31 | 2023-05-26 | 可隆生命科学株式会社 | Composition for alleviating or treating pain |
EP3652309A4 (en) * | 2017-07-13 | 2021-04-28 | University of Miami | Method for managing pain |
US11911450B2 (en) | 2017-07-13 | 2024-02-27 | University Of Miami | Method for managing pain |
KR20200051679A (en) * | 2017-09-08 | 2020-05-13 | 더 리젠츠 오브 더 유니버시티 오브 캘리포니아 | Methods and compositions for the treatment of neuropathic pain |
JP2020533313A (en) * | 2017-09-08 | 2020-11-19 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Methods and compositions for treating neuropathic pain |
EP3678709A4 (en) * | 2017-09-08 | 2021-06-09 | The Regents of the University of California | Method and composition for treating neuropathic pain |
JP7291125B2 (en) | 2017-09-08 | 2023-06-14 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Methods and compositions for treating neuropathic pain |
US11707535B2 (en) | 2017-09-08 | 2023-07-25 | The Regents Of The University Of California | Method and composition for treating neuropathic pain |
KR102640462B1 (en) | 2017-09-08 | 2024-02-23 | 더 리젠츠 오브 더 유니버시티 오브 캘리포니아 | Methods and compositions for treatment of neuropathic pain |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20080051357A1 (en) | Composition for attenuating neuropathic pain comprising a recombinant ventor expressing gad65 | |
US11725038B2 (en) | Targeted delivery of glycine receptors to excitable cells | |
RU2606012C2 (en) | Novel viral vector construct for neuron specific continuous dopa synthesis in vivo | |
JP6621747B2 (en) | Compositions and methods for controlling pain | |
KR101835490B1 (en) | Gene therapy for neurodegenerative disorders | |
ES2748282T3 (en) | Müller cell specific promoter | |
EA010055B1 (en) | Isolated nucleic acid encoding sp35 polypeptide, sp35 polypeptide and methods of use nucleic acid and polypeptide | |
JP2019520788A (en) | Anti-Ryk antibodies and methods of use thereof | |
ES2374061T3 (en) | COMPOSITIONS AND METHODS FOR REGULATED EXPRESSION OF PROTEIN IN THE INTESTINE. | |
WO1996005319A1 (en) | Genetically engineered cells that produce dopamine | |
US20040086494A1 (en) | Immune privileged cells for delivery of proteins and peptides | |
CN110709060A (en) | Gene therapy constructs and methods for treating hearing loss | |
WO2008010637A1 (en) | Composition for attenuating neuropathic pain comprising a recombinant vector expressing gad65 | |
CN109843913B (en) | Neuropeptide expression vectors and methods for treating epilepsy | |
JP2020527335A (en) | Gene therapy for eye diseases | |
KR20210143716A (en) | Hybrid promoters and their use in therapy, especially in the treatment of type II collagenopathy | |
JP2008506394A (en) | Retinal dystrophin transgenes and methods for their use | |
JP2002087983A (en) | Treating agent for amyotrophic lateral sclerosis | |
KR20220003566A (en) | A novel type of enzyme composition | |
WO2010037143A1 (en) | Vectors and methods of treating brain seizures | |
Mich et al. | AAV-mediated interneuron-specific gene replacement for Dravet syndrome | |
US20210220440A1 (en) | Method for treating trpv1-mediated diseases | |
US20040093623A1 (en) | Non-human animal disease models | |
KR100403893B1 (en) | Recombinant Virus Encoding Glutamate Dicarboxylase (GAD) Activity | |
RU2748383C1 (en) | METHOD FOR THERAPY FOR TAY-SACHS DISEASE AND SANDHOFF DISEASE USING GENETICALLY MODIFIED HUMAN MESENCHYMAL STEM CELLS WITH OVEREXPRESSION OF β-HEXOSAMINIDASE A |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 07745673 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
NENP | Non-entry into the national phase |
Ref country code: RU |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 07745673 Country of ref document: EP Kind code of ref document: A1 |