KR101252891B1 - Recombinant Vector Containing siRNA Targeing GCH1 and Pharmaceutical Composition for Attenuating Neuropathic Pain Comprising The Same - Google Patents

Abstract

The present invention relates to a recombinant vector comprising an siRNA targeting GCH1 (GTP cyclohydrolase 1) and a pharmaceutical composition for neuropathic pain relief comprising the recombinant vector.

Recombinant vectors comprising siRNA targeting GCH1 of the present invention can deliver the siRNA to Dorsal Root Ganglia (DRG), and down-regulates the expression of GCH1 to suppress neuropathic pain due to nerve damage At the same time, it can also suppress inflammatory responses in the spine due to nerve damage.

GCH1, siRNA, adeno-associated virus, neuropathic pain

Description

Recombinant Vector Containing siRNA Targeing GCH1 and Pharmaceutical Composition for Attenuating Neuropathic Pain Comprising The Same}

The present invention relates to a therapeutic agent for neuropathic pain, and more particularly, to a recombinant vector comprising an siRNA targeting GCH1 and a pharmaceutical composition for neuropathic pain relief comprising the recombinant vector.

Neuropathic pain is caused by diseases of the peripheral nervous system (PNS) or central nervous system (CNS), especially caused by brain damage (Baron R. Mechanisms of disease: neuropathic pain: a clinical perspective.Nat Clin Pract Neurol 2006; 2: 95-106). In particular peripheral neuropathic pain is defined as pain caused by abnormal activity of the peripheral afferent (Bridges D, Thompson SWN, Rice ASC. Mechanisms of neuropathic pain. British Journal of Anaesthesia 2001; 87: 12-26). Abnormalities in the PNS induce a variety of changes in both the peripheral and CNS and cause pain. This results in amplified sensory transmission (central sensitization) leading to changes in spinal nerve activity and excitability (Woolf CJ, Salter MW. Neuronal Plasticity: Increasing the Gain in Pain. Science 2000; 288: 1765-1768). Neuropathic pain is caused by various neuro-physiological processes (Zimmermann M. Pathobiology of neuropathic pain. European Journal of Pharmacology 2001; 429: 23-37). This pain is mediated by receptors on A-delta and C-nerve fibers present in skin, bone, connective tissue and muscle. This receptor senses different information such as harmful compounds, heat and mechanical stimuli and then delivers it to the brain. Prolonged and consistent pain caused by this sensing can be a serious difficulty. These abnormal sensations are usually caused by allodynia and hyperalgesia (Baron R. Mechanisms of disease: neuropathic pain: a clinical perspective.Nat Clin Pract Neurol 2006; 2: 95-106; Woolf CJ, Salter . MW Neuronal Plasticity: Increasing the Gain in Pain Science 2000; 288:. 1765-1768). Allodynia generally exhibits pain response to irritating stimuli (eg, light contact). Hyperalgesia is increased sensitivity to stimulus that causes normal pain.

Currently, drug therapy of neuropathic pain is largely based on two types of drugs: non-steroidal anti-inflammatory drugs (NSAIDs) and opioids (Kenneth C. Pharmacotherapy for neuropathic pain. Pain Practice 2006; 6: 27-33). Although these drugs have been established for a long time, they have the drawback that they often show partial and temporary pain relief. In addition, repeated use of the drug can cause side effects such as depression and tissue toxicity. Therefore, different strategies for pain relief are needed (Kenneth C. Pharmacotherapy for neuropathic pain.Pain Practice 2006; 6: 27-33; Pohl M, Braz J. Gene therapy of pain: emerging strategies and future directions.European Journal of Pharmacology 2001; 429: 39-48).

As another strategy for alleviating chronic pain, gene-based therapies have a number of benefits, such as a single treatment being effective and less invasive (Pohl M, Braz J. Gene therapy of pain: emerging strategies and future directions. European Journal of Pharmacology 2001; 429: 39-48). In addition, the effect lasts for a long time and has relatively few side effects. This requires the selection of appropriate therapeutic genes and the development of more efficient gene expression systems, including gene delivery vehicles.

The present inventors administered a recombinant adeno-associated viral vector containing a siRNA targeting GCH1 to the sciatic nerve, and as a result, siRNA was delivered to the posterior root and suppressed neuropathic pain due to nerve damage by down-regulating the expression of GCH1. In addition, it was confirmed that it can also inhibit the inflammatory response in the spine due to nerve damage and came to complete the present invention.

Accordingly, the present invention is to provide a recombinant vector comprising the siRNA.

In addition, the present invention is to provide a pharmaceutical composition for neuropathic pain relief comprising the recombinant vector.

GCH1 (GTP cyclohydrolase I) is one of the factors associated with neuropathic pain. GCH1 is a 260 kDa homologous subunit depolymer that catalyzes the conversion of GTP to dihydroneopterin triphophate or neopterin. GCH1 is known as an early rate limiting enzyme in BH4 biosynthesis. BH4 is an essential cofactor of nitrate synthase in phenylalanine hydroxylase involved in tyrosine synthesis, tyrosine hydroxylase involved in catecholamine production, tryptophan hydroxylase involved in serotonin synthesis and nitric acid synthesis. Studies using neuropathic pain animal models have shown that BH4 synthase is activated in damaged sensory nerves. Direct injection of BH4 or similar molecules can increase the animal's response to pain-causing stimuli. Therefore, in order to alleviate neuropathic pain, the production of BH4 should be effectively suppressed.

On the other hand, RNA interference (RNAi) is a natural mechanism for selectively inhibiting the expression of target genes. Mediators of sequence specific mRNA degradation are small interfering RNAs of 19-23 nucleotides produced by cleavage of ribonuclease III from longer ds RNA. The cytoplasmic RNA-induced silencing complex (RISC) binds to siRNA and directs the degradation of mRNA containing a sequence complementary to one of the siRNAs. The application of RNA interference in mammals has the effect of therapeutic gene silencing. Despite the advantages of siRNAs, siRNAs have limitations in clinical applications in that they must be prepared in vitro and knockdown genes are typically delivered by transient transfection for 6-10 days. The shRNA (small-hairpin RNA) expression system of the present invention can solve the aforementioned disadvantages.

In one aspect, the present invention relates to a recombinant vector comprising an siRNA targeting GCH1.

In the present invention, siRNAs targeting GCH1 (GTP cyclohydrolase I) include (1) siRNAs targeting 275 to 293 nucleotides of GCH1; (2) siRNAs targeting 293 to 311 nucleotides of GCH1; (3) siRNAs targeting 318 to 336 nucleotides of GCH1; (4) siRNAs targeting 427 to 445 nucleotides of GCH1; (5) siRNAs targeting 487 to 505 nucleotides of GCH1; (6) siRNAs targeting 515 to 533 nucleotides of GCH1; (7) siRNAs targeting 516 to 534 nucleotides of GCH1; (8) siRNAs targeting 656 to 674 nucleotides of GCH1; And (9) siRNAs targeting 658 to 676 nucleotides of GCH1.

In the present invention, siRNA targeting GCH1 has a sequence complementary to a part of the GCH1 gene, and may degrade mRNA or inhibit translation of the GCH1 gene. When the complementarity is 80-90%, translation of mRNA can be inhibited, and when it is 100%, mRNA can be degraded.

Thus, siRNAs targeting GCH1 in the present invention are directed to 275 to 293 nucleotides of GCH1, to 293 to 311 nucleotides of GCH1, to 318 to 336 nucleotides of GCH1, to 427 to 445 nucleotides of GCH1, and 487 to 505 nucleotides of GCH1. 80%, preferably 90%, particularly preferred for 515-533 nucleotides of GCH1, 516-534 nucleotides of CH1, 656-674 nucleotides of GCH1 or complementary sequences for 658-676 nucleotides of GCH1. Preferably, it includes a base sequence having 100% homology.

In one embodiment, the siRNA of (1) consists of the nucleotide sequence shown in SEQ ID NO: 1 and its complementary nucleotide sequence, and the siRNA of (2) consists of the nucleotide sequence shown in SEQ ID NO: 2 and its complementary nucleotide sequence The siRNA of (3) consists of the nucleotide sequence shown in SEQ ID NO: 3 and its complementary nucleotide sequence, and the siRNA of (4) consists of the nucleotide sequence shown in SEQ ID NO: 4 and its complementary nucleotide sequence, The siRNA of (5) consists of the nucleotide sequence shown in SEQ ID NO: 5 and its complementary nucleotide sequence, and the siRNA of (6) consists of the nucleotide sequence shown in SEQ ID NO: 6 and its complementary nucleotide sequence, (7 ) SiRNA consists of the nucleotide sequence shown in SEQ ID NO: 7 and its complementary nucleotide sequence, and siRNA of (8) consists of the nucleotide sequence shown in SEQ ID NO: 8 and its complementary nucleotide sequence, (9) siRNA is a nucleotide sequence shown in SEQ ID NO: 9 and its It may be made of a beam base sequence. Each base sequence and its complementary base sequence may be palindrom connected by a loop region of 5 to 15 bp to form a hairpin structure.

The siRNA of the present invention can be prepared according to the preparation method of RNA molecules known in the art. As a method for preparing RNA molecules, chemical synthesis methods and enzymatic methods can be used. For example, the chemical synthesis of RNA molecules can use the methods described in the literature (Verma and Eckstein, Annu. Rev. Biochem. 67, 99-134, 1999), and the enzymatic synthesis of RNA molecules is T7, T3. And phage RNA polymerases such as SP6 RNA polymerase are disclosed in the literature (Milligan and Uhlenbeck, Methods Enzymol . 180: 51-62, 1989).

The recombinant vector of the present invention can be constructed by a recombinant DNA method known in the art.

Viruses (or viral vectors) useful for delivering siRNAs for GCH1 in the present invention include baculoviridiae, parvoviridiae, picornoviridiae, herpesviridiae. , Poxviridiae, adenoviridiae, and the like, but are not limited thereto.

In the present invention, it is most preferable to use an Adeno Associated Virus (AAV). Adeno-associated viruses rarely cause immune responses and cytotoxicity. In particular, adeno-associated virus serotype 2 (serotype 2) is capable of efficient gene transfer to the neurons of the CNS, and also can effectively express the transgene in the nervous system for a long time.

In addition, the non-viral vector useful for delivering siRNA for GCH1 in the present invention means all the vectors commonly used for gene therapy, except for the viral vector described above, such as various plasmids and liposomes that can be expressed in eukaryotic cells. Etc.

On the other hand, in the present invention, siRNA targeting GCH1 is preferably operably linked to at least a promoter in order to be properly transcribed in the delivered cell. The promoter may be any promoter capable of functioning in eukaryotic cells, but a human H1 polymerase-III promoter is particularly preferable. For efficient transcription of siRNA targeting GCH1, additional regulatory sequences including leader sequence, polyadenylation sequence, promoter, enhancer, upstream activation sequence, signal peptide sequence and transcription terminator may be added as needed. It may also include.

As used herein, the term “operably linked” means that the binding between nucleic acid sequences is functionally related. The case where any nucleic acid sequence is operably linked is when any nucleic acid sequence is positioned to be functionally related to another nucleic acid sequence. In the present invention, when any transcriptional regulatory sequence affects the transcription of siRNA targeting GCH1, the transcriptional regulatory sequence is said to be operably linked to the siRNA.

In another aspect, the present invention relates to a neuropathic pain pharmaceutical composition comprising a recombinant vector of the present invention and a pharmaceutically acceptable carrier.

The recombinant vector according to the present invention can deliver the siRNA to Dorsal Root Ganglia (DRG) even when administered to the sciatic nerve, and at the same time suppresses neuropathic pain caused by nerve injury and at the spine due to nerve injury. It can suppress the inflammatory response. In other words, by suppressing the expression of GCH1 induced by nerve damage to suppress neuropathic pain, down-regulation of GCH1 also reduces the inflammatory response in the spine.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in the preparation of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, silicic acid Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils, and the like. It is not. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

The pharmaceutical compositions of the present invention may be administered via routes commonly used in gene therapy, with parenteral administration being preferred, using, for example, intravenous, intraperitoneal, intramuscular, subcutaneous or topical administration. May be administered. In particular, administration to the sciatic nerve is most preferred.

In the present invention, the pharmaceutical composition may allow the recombinant vector to be administered to mammals including humans in an amount of 1 ng to 100 μg, preferably 10 ng to 10 μg per kg of body weight per day. However, suitable dosages of the pharmaceutical compositions of the present invention may depend on factors such as the formulation method, mode of administration, age, weight, sex, degree of disease symptom, food, time of administration, route of administration, rate of excretion and response to the patient. It is to be determined by the method that the dosage is not intended to limit the scope of the invention in any way.

The pharmaceutical compositions of the present invention are prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating into a multi-dose container. The formulations here may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of extracts, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

The pharmaceutical compositions of the present invention can be lyophilized to increase stability at room temperature, reduce the need for expensive cold storage, and prolong self-life. The lyophilization process may consist of successive stages of freezing, primary drying and secondary drying. The secondary drying process after freezing the composition is to increase the pressure and heat it for sublimation of water vapor. The secondary drying step is to evaporate the residual moisture absorbed from the dry matter.

Lyophilized formulations may include excipients and lyoprotectants. Excipients include, but are not limited to, buffers of 0.9% NaCl and 10 mM sodium phosphate (pH 7.0) or 10 mM sodium citrate (pH 7.0). Lyophilizers serve to protect biological molecules during the freezing and drying process and to impart mechanical support to the final product, such as PBS (pH 7.0), PBS / 4%, 12% or 15% trehalose. Etc. can be mentioned.

Gene therapy according to the present invention for the treatment of neuropathic pain has the following effects as compared to drug therapy: (1) long-term expression of the gene allows neuropathic pain relief to be achieved with a single treatment; (2) The therapeutic gene can be delivered to the target site. According to the present invention, the recombinant vector administered to the sciatic nerve could effectively deliver the therapeutic gene to the posterior root; (3) NSAIDs and opioids are visible, ie free from side effects such as depression and cytotoxicity.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Substances and methods

Example 1: Cell Culture and Transfection

293T cells were provided by Dr J. Jung (Harvard Medical School, USA). 293T cells were DMEM (Dulbecco's) supplemented with 10% FBS (fetal bovine serum), L-glutamine (2 mM), penicillin (100 IU / ml) and streptomycin (50 μg / ml) at 37 ° C. in a 5% CO ₂ incubator. stored in modified Eagle's medium) (Gibco BRL, Carlsbad, Calif.) medium.

Cells were seeded in 6-spheres 24 hours before transformation. The next day cells were transformed with plasmid DNA (0.5 μg) containing siRNA (100 mM) and GCH1 gene in OPTI-MEM medium (Invitrogen, Carlsbad, Calif.) Containing serum-free lipofectamine reagent (Invitrogen, Carlsad, Calif.) I was. After 3 hours growth medium containing 10% serum was added without removing the transfection mixture. The next day cells were recovered and lysed for 15 minutes using lysis buffer (Intron, Seoul, Korea). Total protein was analyzed from the supernatants after centrifugation and Western blot analysis was performed with antibodies as described previously.

Example 2: Design and Sequence of siRNA

First, a conserved cDNA sequence (CCDS) software program was used to identify exons of the rGCH1 gene. The software estimates the exon of the gene, taking into account the information obtained from humans and mice. The software identified a GCH1 exon consisting of six exons, similar in humans and mice (FIG. 1).

It was used as the siRNA designed software developed by the school that MWG Biotech AG software (www.mwg-biotech.acom) or CAPSID (C onvenient A pplication P rogram for si RNA esign D) for the design of effective siRNA. After determining the siRNA sequence, 21 nucleotide double-stranded siRNA molecules with dTdT 3'-overhangs containing nonspecific control siRNAs were synthesized by Bioneer (Seoul, Korea) with a "ready-to-use" option. siRNA double stranded oligonucleotides were resuspended in nuclease-free water as directed by the manufacturer. Nonspecific siRNAs labeled with Cy3-fluorescent dye were used as controls to investigate transfection efficiency into cells. The sequences of all siRNAs used in this example are shown in Table 1 and FIG. 1.

SiRNA sequences of the invention siRNA Targeted sequence (5'-3 ') start GC (%) SEQ ID NO: rGCH1-ex1 CCAUGCAGUUCUUCACCAA 275 47.4 One rGCH1-ex 1-2 AGGGAUACCAGGAGACCAU 293 49.2 2 rGCH1-ex 2 UGUCCUGAACGAUGCUAUA 318 42.1 3 rGCH1-ex 3 GUCCAUAUUGGUUAUCUUC 427 36.8 4 rGCH1-ex 4 GUGGAAAUCUACAGUAGAA 487 36.8 5 rGCH1-ex 5-1 UUCAAGAACGCCUUACCAA 515 42.1 6 rGCH1-ex 5-2 UCAAGAACGCCUUACCAAA 516 42.1 7 rGCH1-ex 6-1 UGCAGAAAAUGAACAGCAA 656 36.8 8 rGCH1-ex 6-2 CAGAAAAUGAACAGCAAGA 658 36.8 9 Nonspecific AUUCUAUCACUAGCGUGAC - 42.1 10

* The siRNA names are named by quoting the exons each siRNA targets.

Example 3: Construction of Recombinant Adeno-associated Virus (rAAV) Expressing Small Hairpin GCH1 (sh1GCH1) Induced by pH1

3-1: Construction of pSP72-pH1

Human H1 polymerase-III promoter (pH1) as a promoter for shRNA expression was amplified by PCR based methods. That is, the sequence of pH1 is primer 5'-CCA TGG AAT TCG AAC GCT GA-3 '(SEQ ID NO: 11) and 5'-GGG AAA GAG TGG TCT CAT AC-3 from human genomic DNA isolated from HeLa cells. (SEQ ID NO: 12) was amplified by PCR. The PCR product thus amplified was used as a template for the sense primer ( Eco RI linker) 5'-ATC GAA TTC ATA TTT GCA TGT CGC TAT GTG-3 '(SEQ ID NO: 13) and the antisense primer ( Bam HI linker) 5'-ATC GGA Secondary PCR was performed using TCC GAG TGG TCT CAT ACA GAAC-3 '(SEQ ID NO: 14). PCR products were digested with Eco RI / Bam HI and isolated from agarose gels. Then, pSP72-pH1 was prepared by cloning to the EcoR I / BamH I position of the cloning vector pSP72 (Promega, Madison, Wis.).

3-2: Construction of pSP72-pH1-shGCH1

PSP72-pH1-shGCH1 was prepared by constructing an siRNA 'rGCH1-ex 4' targeting exon 4 to include Bam HI and Hind III 5'- or 3'- overhangs and then ligating to the pSP72-pH1 vector. The sense primer sequence with Bam HI linker for this is 5'-GAT CCC GTG GAA ATC TAC AGT AGA A TT CAA GAG A TT CTA CTG TAG ATT TCC AC T TTT TTG GAA A-3 '(SEQ ID NO: 15) The antisense primer sequence with Hind III linker is 5′-AGC TTT TCC AAA AAA GTG GAA ATC TAC AGT AGA A TC TCT TGA A TT CTA CTG TAG ATT TCC AC G-3 ′ (SEQ ID NO: 16). The nucleotides specific for GCH1 in the sequence in the sequence are underlined and the loop structure is shown in bold.

3-3: Construction of pSP72-XP-rAAV-MCS

PHpa-Trs-SK, the original backbone of self-complementary AAV, was provided by McCarty (Ohio State University). Modifications were made to new vectors with multi-cloning sites. First, a pSP72-XP-rAAV vector was constructed. It cleaved the essential region of AAV, including internal repeat (ITR), CMV promoter, reporter gene GFP and SV40 poly A signal from pHpa-Trs-SK using X ho I and Pvu II, and then ligated with pSP72 vector. It was constructed by. Next, a linker with multiple cloning positions was produced. This linker is 5'- CGG GAG ATC TTC CTG CAG GAT ATC TGG ATC CAC GAA GCT TCC CAC CGG TTC TAG AGC G-3 '(SEQ ID NO: 17), which is a sense oligonucleotide sequence having a Kpn I restriction enzyme site; 5'-TCG ACG CTC TAG AAC CGG TGG GAA GCT TCG TGG ATC CAG ATA TCC TGC AGG AAG ATC TCC CGG TAC-3 '(SEQ ID NO: 18). The linker was constructed from the previously prepared pSP72-XP-rAAV with KpnI and SalI, and then the linker was ligated to prepare pSP72-XP-rAAV-MCS.

3-4: Construction of rAAV-shGCH1

removal of the CMV promoter to the Kpn I and Xba I digestion from pSP72-XP-rAAV-MCS, and then made dundan (blunt end) by DNA polymerase (New England BioLabs, Beverly, MA). pH1-shGCH1 was allowed to lie adjacent to the BGH poly A position (present in pSP72-XP-rAAV-MCS). The pSP72-XP-rAAV-MCS vector then cleaved the poly A tail with Sal I. Inserts for shGCH1 expression, pH1-shGCH1, were obtained by digesting with Eco RV and Xho I from the pSP72-pH1-shGCH1 vector. The vector and insert were then blunted with DNA polymerase (New England BioLabs, Beverly, Mass.). The thus prepared vector and insert were blunt ligated. It is designed not to express GFP but to express only shGCH1. This was named pSP72-XP-rAAV-pH1-shGCH1 for the plasmid and rAAV-shGCH1 for the virus.

3-5: Construction of rAAV-GFP-shGCH1

To detect the cells into which the vector was introduced, a dual gene expression rAAV vector of GFP and shGCH1 containing a CMV promoter for expression of the GFP expression cassette was finally constructed independent of pH1 for expression of shGCH1. The cloning strategy is as follows: The pSP72-XP-rAAV-MCS vector (also called the pSP72-XP-rAAV-GFP vector) was cleaved with Eco RI and the insert pH1-shGCH1 was eco RV from the pSP72-pH1-shGCH1 vector. and digested with Xho I. The vectors and inserts were then blunted with DNA polymerase (New England BioLabs, Beverly, Mass.). This vector can express shGCH1 and GFP. This vector was named pSP72-XP-rAAV-GFP-pH1-shGCH1 for the plasmid and rAAV-GFP-shGCH1 for the virus (FIG. 3A). The pSP72-XP-rAAV-GFP vector was also named rAAV-GFP in the case of viruses.

Example 4: Preparation and Titer Determination of Recombinant Self-Complementary Adeno Adjunct Virus Vector Serotype 2

pHpa-trs-SK for preparing rAAV2 was provided by Dr J. Samulski (University of North Carolina) as a self-complementary AAV plasmid vector. The pRepCap plasmid containing the capsid gene of serotype 2 was determined by Dr. C. High (University of Pennsylvania). rAAV is a triple transfection process and calcium phosphate method using pHpa-trs-SK with pXX6 (Stratagene, La Jolla, Calif.) encoding the helper genes of GFP or GFP-shGCH1, pRepCaps and adenoviruses under a bidirectional promoter. Produced by

For further production of rAAV 293T cells were transfected in 20 × 10 cm dishes. Intracellular rAAV was isolated and purified by the CsCl gradient step. Pure groups of rAAV were prepared from cell lysates including supernatant by two successive CsCl gradient steps. After dialysis in 10 mM Tris buffer (pH 7.9) containing 2 mM MgCl ₂ and 2% sorbitol, rAAV was possibly stored at -80 ° C. The total number of virus particles was confirmed by ELISA kit (Progen Inc., Heidelberg, Germany) and real time qPCR analysis.

Example 5: Total RNA Extraction and RT-PCR

Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA) from L4-L5 posterior root injured nerve. The extracted RNA was dissolved in deionized water. Total RNA samples (250 ng / μl) were manufacturer's protocol using superscript III enzyme (Invitrogen, Carlsbad, Calif.) And 20 μl of 100 pmole Oligo d (T) 20 (Invitrogen, Carlsbad, Calif.) As the final volume. RT (reverse transcription) reaction was performed. The RT reaction ran at 50 ° C. for 50 minutes and was inactivated at 70 ° C. for 15 minutes. One tenth of the reaction product was then subjected to 20 μl PCR amplification reaction (Invitrogen, Carlsbad, CA). The reaction profile was performed once for 3 minutes at 94 ° C for 35 seconds at 94 ° C for 30 seconds, at 56 ° C for 30 seconds, and at 72 ° C for 45 seconds. The sequence of the GCH1-specific primer of the rat has a sense of 5'-GGA TAC CAG GAG ACC ATC TCA-3 '(SEQ ID NO: 19) and an antisense of 5'-GTC CTC CTT GAA GTC GAT GC-3' (SEQ ID NO: : 20). The GFP specific primer sequence has a sense of 5'-CCT TCA TTG ACC TCA ACT AC-3 '(SEQ ID NO: 21) and an antisense of 5'-GGA AGG CCA TGC CAG TGA GC-3' (SEQ ID NO: 22) to be. PCR products were isolated by electrophoresis on 1% agarose gel and observed by ethidium bromide staining.

Example 6: Western Blot Analysis

The cells were recovered and lysed with 100 μl of lysis buffer (Intron, Seoul, Korea). The lysate was boiled with 5X sample buffer at 100 ° C for 5 minutes, and the denatured protein was separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by semi-drier (Bio-Rad). Transfer to PVDF membrane at 20V for 30 minutes. After treatment with block solution (0.4% Tween 20 and 5% dry milk in PBS) for 1 hour at room temperature, the membrane was treated with primary antibodies: GCH1 (1: 1000, Abnova Abnova, Taipei, Twawan), β-actin (1: 5000 , Sigma, St. Louis, MI) and GFP (1: 1000, Santa Cruz) overnight. After washing with PBST or TBST, a secondary antibody (Jackson laboratory) conjugated with horse radish peroxidase was applied and the blot was expressed with an improved chemiluminescent reagent (Pierce Biotechnology, Rockford, IL).

For tissues, ipsilateral L4, 5 DRG, and spinal dip (SCDH, T13-L2) were prepared. Each sample was homogenized with lysis buffer (20 mM Tris, pH 7.4, 50 mM NaCl, 1% Triton, and protease inhibitors) and debris removed by centrifugation. The supernatant can be stored and stored at -80 ° C. Samples were measured for protein concentration using a BCA (Bio-ad, Richmond, CA) assay. Protein samples (30 μg each) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed as described above.

Example 7: Animal Care, Virus Administration, and Behavioral Testing

7-1: Care of Animals

Adult male Sprague Dawley rats weighing 180-200 g were housed 3 per cage and allowed to eat freely in food and water. Animal use and care was approved by the University Board, and rats were administered on a 12-hour day / night cycle in a room with optimal temperature and humidity control.

7-2: Virus Administration

Rats were anesthetized with Zoletil (50 mg / kg) and Rompun (25 mg / kg) to inject rAAV into the sciatic nerve. The left sciatic nerve segment was then exposed. Under the surgical microscope (Olympus, Japan) the surrounding tissue was carefully removed and the sciatic nerve exposed. 3 μl of rAAV was slowly delivered to the sciatic nerve for 5 minutes through a glass micropipette connected with a Hamilton syringe. After 5 minutes the pipette was removed.

7-3: Building the SNI Model

Rats were anesthetized with Zoletil (50 mg / kg) and Rompun (25 mg / kg) to produce a sparsed nerve injury (SNI) model as shown in FIG. 4A. The left sciatic nerve was then exposed. Of the three stranded sciatic nerves, the total fibula and tibial nerves were ligated tightly and completely transversely, while the abdominal nerves remained intact. The wound was irrigated with saline and sutured with two layers of surgical skin sutures.

7-3: Behavioral Check

Rat allodynia was confirmed by applying a series of von Frey filaments in the ascending and descending order (up-and-down method) to the sole surface behind the forefoot (range 0.01-1.66 g). Rapid movement of the back of the leg is considered positive. Movement threshold values of 4.0 g or less were classified as allodynia.

Example 8: Tissue Immunoassay

After completing the appropriate procedure, rats were anesthetized and infused with 125 ml of normal saline and 250 ml of 4% paraformaldehyde chilled with ice. After injection DRG (L4, L5) and spine (T13-L2) were removed and cryprotected at 30 ° C sucrose solution in phosphate buffer for 5 days at 4 ° C and embedded in OCT embedding medium. DRG and spinal sections were cut to 10 μm thickness and stored frozen until use.

For fluorescence detection, tissue sections were left standing at 4 ° C. for 24 hours with primary antibodies: GCH1 (1: 500, Abnova, Taipei, Twawan), Iba-1 (1: 500, Wako, Japan). Alexa 488 conjugated secondary antibody (1: 1000, Invitrogen) and Alexa 555 conjugated secondary antibody (1: 1000, Invitrogen) were applied to each section for 1 hour at room temperature. Each section was washed with TBS and sampled with Vectashield with DAPI (Vector laboratories, Burlingame, Calif.).

result

1. Production of siRNA that attenuates GCH1 specific for rat GCH1

SiRNA and plasmid pcDNA3.1 / rGCH1 were cotransformed into 293T cells to investigate the silencing efficacy of siRNA candidates obtained through Example 2. These cells were harvested at 24 hours after cotransformation. GCH1 expression in cell lysates was assessed by Western blotting analysis using GCH1 specific antibodies. As shown in FIG. 2, all siRNAs tested in this example showed gene silencing effects as compared to nonspecific nonspecific siRNA or virtually treated controls. siRNAs targeting exon 1-1, exon 2, exon 3, exon 4, exon 5-1 of rGCH1 were more effective than others. Thus, this data confirmed that expression of rGCH1 can be effectively downregulated by siRNA targeting 1-5 exons of rGCH1.

2. Construction of rAAV expressing shGCH1 with GFP and Down-regulation Effect of GCH1

rAAV-GFP-shGCH1 was constructed from Example 3 to examine shGCH1 by rAAV. rAAV-GFP-shGCH1 can produce both shGCH1 and GFP using a bidirectional promoter as shown in FIG. 3A. Here, rAAV, rAAV-GFP was used as a control. RAAV-GFP-shGCH1 was effectively produced as indicated by the total particle production yield in FIG. 3B: rAAV-shGCH1 (3.3 × 10 ¹¹ TP / ml) and rAAV-GFP-shGCH1 (3.0 × 10 ¹¹ TP / ml). In addition, rAAV-GFP-shGCH1 treatment in HeLa cells in vitro induced GFP expression while down-regulating GCH1 expression (FIGS. 3C and 3D). Therefore, rAAV-GFP-shGCH1 inhibited the expression of rGCH1 gene but did not inhibit the expression of reporter gene. Therefore, the data means that gene delivery is properly performed by the rAAV used in the present invention.

3. Establishment of animal models of neuropathic pain and successful gene transfer to DRG following administration of rAAV to the sciatic nerve

The SNI model produced in Example 7 was confirmed to show mechanical allodynia within 3 days after surgery (FIG. 4B).

Meanwhile, an animal experiment as shown in FIG. 5 was attempted to investigate the therapeutic efficacy of rAAV-GFP-shGCH1. The effect of gene delivery was measured by examining the production of reporter gene GFP. RT-PCR was performed on GFP from DRG samples and 11 of 15 samples were GFP positive, indicating a 70% gene transfer success rate (FIG. 6A and data not shown). There was no significant difference in success rate with time after virus injection. GFP protein was detected from the rAAV-GFP and rAAV-GFP-shGCH1 treatment groups (FIG. 6B). Efficient gene delivery to DRG was possible by injecting rAAV into the sciatic nerve spaced apart from DRG.

4. Relief of neuropathic pain symptoms by rAAV-GFP-shGCH1 with bidirectional promoter

The beneficial effect from the downregulation of rGCH1 by rAAV-GFP-shGCH1 in DRG was confirmed by performing the von Frey test (up-and-down method, Example 7). As shown in FIGS. 7A and 7B, a significant decrease in pain symptoms was observed in the group injected with rAAV-GFP-shGCH1 (9.9 × 10 ⁷ TP at 3 μL). This data shows that spaced injections of rAAV-GFP-shGCH1 can significantly inhibit mechanical allodynia (day 3, P <0.01; day 7, P <0.05; day 10, P <0.01; day 14, P <0.01 vs. SNI model, respectively). This effect lasted for the entire experimental period in the group injected with rAAV-GFP-shGCH1. In contrast, there was no pain reduction in the rAAV-GFP injection group. The mechanical allodynia pattern of each group is shown in Figure 7a. The results showed that the SNI model and the rAAV-GFP injection group were mostly sensitive and showed a forefoot threshold of less than 4 g. The shGCH1 infusion group showed a paw movement threshold value of more than 4 g, confirming that it inhibited the development of neuropathic pain induced by GCH1.

5. Reduced expression of GCH1 in DRG after rAAV-GFP-shGCH1 treatment

RT-PCR analysis showed that the rAAV-GFP-shGCH1 treatment group was similar to the normal group in the amount of GCH1 (FIG. 8A). In contrast, significant induction of GCH1 mRNA was observed in the SNI model and the rAAV-GFP treated group. Consistent with the RT-PCR results, the GCH1 protein amount was similar to the normal group in the rAAV-GFP-shGCH1 treatment group. As shown in FIG. 8B, the amount of GCH1 protein was increased 2 days after injury and the increased amount was maintained up to 14 days. Tissue immunoassay for GCH1 showed that the amount of GCH1 was not increased by surgery in the rAAV-GFP-shGCH1 treatment group (FIG. 8C). Considering behavioral tests and GCH1 expression patterns, this data reaffirms that GCH1 induction is closely associated with the development of neuropathic pain.

6.Inhibition of microglia activation by rAAV-GFP-shGCH1 in spine

It is known that inflammation caused by nerve damage causes neuropathic pain, so that tissues using Iba-1 antibodies can detect macrophages and microglia to investigate whether GCH1 downregulation is associated with activation of inflammation in the spine. Immunoassay was performed. Consistent with the results from DRG, the SNI model and the rAAV-GFP injection group showed increased activation of microglia, especially in the ipsilateral dorsal spine of the spinal cord (FIG. 9). The number of Iba-1 positive cells was significantly increased, and morphological changes of microglia were also observed. The data show that selective knockdown of GCH1 can effectively inhibit the activity of microglia in the spine.

Figure 1 shows the exon (A) and sequence (B) of GCH1 of rats and siRNA position (underlined) for each exon.

Figure 2 shows the results of Western blot analysis of 293T cells cotransformed with siRNA for GCH1 of rats and GCH1 of the present invention.

Figure 3a shows a schematic diagram of rAAV-GFP-shGCH1 according to the present invention.

3B shows the total titer of rAAV produced from 293T cells transformed with rAAV-GFP or rAAV-GFP-shGCH1.

3C shows the results of fluorescence microscopy confirming that GFP is expressed in HeLa cells treated with rAAV-GFP or rAAV-GFP-shGCH1.

3D shows that expression of GCH1 is down regulated in HeLa cells treated with rAAV-GFP or rAAV-GFP-shGCH1.

4A is a simplified illustration of the process of establishing various models of neuropathic pain, including the SNI model used in the present invention.

FIG. 4B shows the results of Von Fray behavior analysis for mechanical allodynia in normal or SNI models.

5 shows an overview of experimental strategies for verifying the therapeutic effect of rAAV-GFP-shGCH1 in vivo.

Figure 6a shows the results of RT-PCR analysis of the expression level of GFP in DRG isolated from normal, SNI model, rAAV-GFP or SNI model administered rAAV-GFP-shGCH1 to the sciatic nerve, respectively.

Figure 6b is the result of confirming the expression level of GFP in DRG separated from the SNI model, rAAV-GFP or SA model in which rAAV-GFP-shGCH1 was administered to the sciatic nerve, respectively, by confocal microscopy.

Figure 7a shows the results of analyzing the mechanical allodynia pattern for each SNI model administered normal, SNI model, rAAV-GFP or rAAV-GFP-shGCH1.

FIG. 7B shows the mean of the forefoot threshold for each of the SNI models administered normal, SNI model, rAAV-GFP or rAAV-GFP-shGCH1.

FIG. 8A shows the results of RT-PCR analysis of GCH1 and GFP mRNA for DRG isolated from normal, SNI model, SNI model administered with rAAV-GFP or rAAV-GFP-shGCH1, respectively.

8B shows the results of Western blot analysis of GCH1 for DRG isolated from normal, SNI model, SNI model administered with rAAV-GFP or rAAV-GFP-shGCH1, respectively.

FIG. 8C shows the results of GCH1 protein specific immunohistochemistry for DRG isolated from normal, SNI model, SNI model administered with rAAV-GFP or rAAV-GFP-shGCH1.

Figure 9 shows the results of microglia activity for spine isolated from normal, SNI model, SNI model administered with rAAV-GFP or rAAV-GFP-shGCH1, respectively.

<110> University of Ulsan Foundation For Industry Cooperation <120> Recombinant Vector Containing siRNA Targeting GCH1 and          Pharmaceutical Composition for Attenuating Neuropathic Pain          Comprising The Same <160> 22 <170> Kopatentin 1.71 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 1 ccaugcaguu cuucaccaa 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 2 agggauacca ggagaccau 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 3 uguccugaac gaugcuaua 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 4 guccauauug guuaucuuc 19 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 5 guggaaaucu acaguagaa 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 6 uucaagaacg ccuuaccaa 19 <210> 7 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 7 ucaagaacgc cuuaccaaa 19 <210> 8 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 8 ugcagaaaau gaacagcaa 19 <210> 9 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 9 cagaaaauga acagcaaga 19 <210> 10 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 10 auucuaucac uagcgugac 19 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ccatggaatt cgaacgctga 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gggaaagagt ggtctcatac 20 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 atcgaattca tatttgcatg tcgctatgtg 30 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 atcggatccg agtggtctca tacagaac 28 <210> 15 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gatcccgtgg aaatctacag tagaattcaa gagattctac tgtagatttc cacttttttg 60 gaaa 64 <210> 16 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 agcttttcca aaaaagtgga aatctacagt agaatctctt gaattctact gtagatttcc 60 acg 63 <210> 17 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> MCS <400> 17 cgggagatct tcctgcagga tatctggatc cacgaagctt cccaccggtt ctagagcg 58 <210> 18 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> MCS <400> 18 tcgacgctct agaaccggtg ggaagcttcg tggatccaga tatcctgcag gaagatctcc 60 cggtac 66 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 ggataccagg agaccatctc a 21 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gtcctccttg aagtcgatgc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 ccttcattga cctcaactac 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 ggaaggccat gccagtgagc 20  

Claims (13)

Targeting GCH1 (GTP cyclohydrolase 1) SiRNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 and its complementary nucleotide sequence; SiRNA consisting of the nucleotide sequence shown in SEQ ID NO: 3 and its complementary nucleotide sequence; SiRNA consisting of the nucleotide sequence shown in SEQ ID NO: 4 and its complementary nucleotide sequence; SiRNA consisting of the nucleotide sequence shown in SEQ ID NO: 5 and its complementary nucleotide sequence; And Recombinant adeno-associated viral vectors each comprising an siRNA consisting of the nucleotide sequence shown in SEQ ID NO: 7 and its complementary nucleotide sequence, Wherein each recombinant adeno accessory viral vector comprises a human H1 polymerase-III promoter, siRNA is operably linked to the human H1 polymerase-III promoter, comprises a CMV promoter, and a reporter gene is linked to the CMV promoter The operatively connected pharmaceutical composition for neuropathic pain relief, characterized in that the human H1 polymerase-III promoter and the CMV promoter is a bidirectional promoter. delete delete delete The method of claim 1, The adeno-associated virus is serotype 2, rAAV-GFP-shGCH1 pharmaceutical composition for neuropathic pain relief, characterized in that. delete delete delete The method of claim 1, The neuropathic pain is a neuropathic pain relief pharmaceutical composition, characterized in that the peripheral neuropathic pain. delete delete delete delete

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