Traitement en cours

Veuillez attendre...

PATENTSCOPE sera indisponible durant quelques heures pour des raisons de maintenance le mardi 26.10.2021 à 12:00 PM CEST
Paramétrages

Paramétrages

Aller à Demande

1. WO2007108621 - UTILISATION D'ARNSI POUR INHIBER L'EXPRESSION DU NF-ϰB/RELA

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]

[DESCRIPTION]
[Invention Title]
SIRNA FOR INHIBITING NF-KB/RELA EXPRESSION

[Technical Field]
The present invention relates to siRNA for inhibiting NF-κB/RelA, more particularly, to siRNA for inhibiting NF-κB/RelA and a pharmaceutical composition for treating rheumatoid arthritis.

[Background Art]
Rheumatoid arthritisC'RA"), whose pathological symptoms include thickening of synovial membrane and infiltration of inflammatory cells, is a chronic, systemic inflammatory disease attacking the joints. In the early stage of the disease, T cells are activated by immune response that is geared up to promote inflammation in the joints, however, as the disease is progressed, the influence of T cells is reduced and the joints are rapidly ruined by the activation of cartilage destructive enzymes and bone destruction factors which are caused by abnormal proliferation and activation of synovial fibroblast.
In the early stage of RA, new blood vessels are generated(angiogenesis) from the joint area, where inflammatory cells are infiltrated into the joints and releases cytokines to activate synovial cells. The activated synovial cells proliferate and form thickened cellular tissues in the exterior of synovial cells to generate a new tissue, i.e., pannus. Pannus secretes various cytokines and proteases and promotes differentiation of macrophage and osteoclast by which the cartillage are gradually destroyed. As the destruction of cartillage, the distance between bones become short ultimately to lead to destruction of bones by the collision of pannus and bones(see: Bresnihan et al . , Ann. Rheum Dis., 59:506-511, 2000; Firestein, Arthritis Rheum., 39:1781-1790, 1996; Firestein et al . , Arthritis Rheum., 46:298-308, 2002).

On the other hand, NF-κB(nuclear factor-kappa B) is a protein which promotes the expression of several proteins such as TNF- α (tumor necrosis factor- α), interleukinsCIL-I, IL-6, IL-8, etc.), GM-CSF(granulocyte-macrophage colony stimulating factor), cFLIP(cellular FLICE-inhibitory protein), iNOS(inducible nitric oxide synthase), ICAM-Kintercellular adhesion molecule 1), E-selectin, MHC class I, II, RANTES(Regulated upon Activation, Normal T-cell Expressed and Secreted), and BIRC3(Baculoviral IAP Repeat-Containing 3) that are associated with immune and inflammatory responses, some of which are activated by the said proteins associated with immune and inflammatory responses(see: Voraberger et al . , J. Immunol., 147:2777-2786, 1991).
NF- KB is classified into five NF- KB subgroups, i.e., p50/pl05(NF- K Bl), p52/pl00(NF-κB2), c-Rel, ReIB and p65(RelA). NF- KB has two classic forms, homo-dimer or hetero-dimer of various forms, and the NF- KB dimers are transferred to nucleus and promote expression of the said proteins. The NF- KB is bound with IKB protein(inhibitor KB) in the cytoplasm under a normal, non-stressful condition. However, NF- KB is separated from NF- KB/I KB complex and transferred to nucleus and promotes expression of the said proteins associated with immune and inflammatory responses, when signal transduction in response to an extracelluar signal leads IKB to be phosphorylated and inactivated by IKK(IKB kinaseXseeJ Rothwarf et al . , Nature, 395:297-300, 1998; Yamaoka et al . , Cell, 93:1231-1240, 1998).
Since the said proteins which are associated with immune and inflammatory responses play a crucial role in the pyreticosis and progress of RA, NF- KB plays a central role to the RA, and the regulation of NF- KB activity is closely related with the therapeutic strategies for acute and chronic inflammatory diseases. Actually, it has been known that p65(RelA)(hereinafter referred to as ^F-?/RelA?, one of NF- KB family members, plays a crucial role in the pathogenesis of RA, and activated NF- K B/RelA is observed in the FLSCfibroblast-like synoviocyte) of RA patients, and several anti-rheumatic drugs currently used on clinic are effectuated by the inactivation of NF-κB/RelA.
For example, glucocorticoids increase the production of I? to inhibit the activity of NF-κB(see_: Auphan et al . , Science, 270:286-290, 1995), and aspirin, sodium salicylate, gold compounds, sulfasalazine and methotrexate inhibit the activity of IKK to prevent the degradation of I? which, in turn, inhibits the activity of NF- κB(see'- Yin et al . , Nature, 396:77-80, 1998; Jeon et al . , J. Immunol., 164:5981-5989, 2000; Wahl et al . , J. Clin. Invest., 101:1163-1174, 1998; Majumdar et al . , J. Immunol., 167:2911-2920, 2001; Tak et al , Arthritis Rheum., 44:1897-1907, 2001); and, IMD-0560, an inhibitor of IKK, reduces the production of cytokines such as IL-6, IL-8 and inhibits the activity of NF-κB/RelA in FLS cells to alleviate the symptoms of RA(see_: Okazaki et al . , J. Rheumatol., 32:1440-1447, 2005).
These results suggest that anti-rheumatic drugs can be effectuated to treat RA in the region of RA by inhibiting the activity of NF-κB/RelA. Actually, most of anti-rheumatic drugs currently used are effectuated by inhibiting the activity of NF-κB/RelA in the protein level or preventing the degradation of I? to inhibit the activity of NF-κB/RelA. The prior antirheumatic drugs are, however, proven to be less satisfactory in the senses that: in the case of those inhibiting the activity of NF-κB/RelA in the protein level, they inhibit the activities of proteins other than NF- K B/RelA; and, in the case of those preventing the degradation of I?, they can prevent only one degradation mechanism of I? and can not prevent the other degradation mechanisms, finally to delay the relapse of RA by delaying the degradation.
Under the circumstances, a variety of researches have been made to solve the problems of prior anti-rheumatic drugs and to develop a new drug target which can inactivate NF-κB/RelA in the gene level in cells to cause RA like FLS, however, however, most of new anti-rheumatic drugs developed so far, have not been practically applied in the art, since they failed in the specific inhibition of NF-κB/RelA in the gene level of FLS cell and worked even on the other normal tissues.

<3> Accordingly, there are strong reasons for exploring and developing an anti-rheumatic agent for treating rheumatoid arthritis by inhibiting NF- K B/RelA expression in the gene level.
<13>
[Disclosure]
[Technical Problem]
<i4> The present inventors have made an effort to develop an ant i-rheumatic drug for rheumatoid arthritis which can inhibit NF-κB/RelA expression in the gene level, and discovered that rheumatoid arthritis can be treated by the introduction of a chemically synthesized siRNA which binds with mRNA transcribed from a gene coding for NF-κB/RelA to inhibit the said mRNA into synovial fibroblast, finally to inhibit NF-κB/RelA expression in the cell.

<15>
[Technical Solution]
A primary object of present invention is, therefore, to provide siRNA which binds with mRNA transcribed from a gene coding for NF-κB/RelA to inhibit NF-κB/RelA expression.

<i6> The other object of the invention is to provide a pharmaceutical composition for treating rheumatoid arthritis which comprises an active ingredient of the said siRNA.
<19>
[Description of Drawings]
<20>
<43>
[Best Mode]
<44> Small interfering RNAC'siRNA") , a double-stranded RNA with a nucleotide sequence of 20-25bp, is known to cause RNA interference(RNAi) to inhibit protein expression. As a mechanism of inhibiting protein expression, it has been known that siRNA which is formed by the digestion of double- stranded RNA(dsRNA) in cells with dicer, a RNAse, or chemically synthesized siRNA binds with a protein in the cell to form a RNA-induced silencing complexC'RISC"), which is subsequently bound with mRNA having a sequence complementary to the said siRNA to digest or inactivate the bound mRNA. It has been reported that siRNA, a double-stranded RNA(dsRNA) can be bound with mRNA, a single-stranded RNA(ssRNA), since siRNA in the RISC is present in a form of ssRNA(see_: Hamilton & Baulcumbe, Science, 286:950-952, 1999; Novina & Sharp, Nature, 430:161-164, 2004).
<45> On the other hand, it has been expected that siRNA for the inhibition of gene expression can be applied for the study on the biological function of a certain gene and the development of a method for treating a gene-associated diseases in infectious viruses or cancer cells(see_: McCaffrey et al., Nature Biotechnology, 21:639-644, 2003; Giladi et al . , Molecular Therapy, 8:769-776, 2003; Konishi et al., Hepatology, 38:842-850, 2003).
<46>
<47> In this connection, the present inventors have made extensive studies on the inhibition of NF-κB/RelA, a causative protein of rheumatoid arthritis, and tried to inhibit the said protein by introducing siRNA designed for binding with mRNA of NF-κB/RelA in a target cell, while focusing on the inhibition of NF-κB/RelA expression by means of siRNA.
<48> First, the present inventors designed siRNA having sequences of SEQ. ID. Nos. 1 to 30, based on the sequence of NF-κB/RelA mRNA(NM_021975)(SEQ. ID. No. 53) available from GenBank, and chemically synthesized the siRNA.
<49>
<50> 5 ' -cgaauggcucgucuguagu-3 ' (SEQ. ID. No . 1) ,
<5i> 5 ' -ggagcacagauaccaccaa-3 ' (SEQ . ID . No . 2) ,
<52> 5 ' -gcauccagaccaacaacaa-3 ' (SEQ. ID. No . 3) ,
<53> 5 ' -ccuuccaaguuccuauaga-3 ' (SEQ . ID . No . 4) ,
<54> 5 ' -ggacuacgaccugaaugcu-3 ' (SEQ. ID. No . 5) ,
<55> 5 ' -ucugcuuccaggugacagu-3 ' (SEQ. ID . No . 6) ,
<56> 5 ' -guccuuccucaucccaucu-3 ' (SEQ. ID. No . 7) , <57> 5 ' -cucaucccaucuuugacaa-3 ' (SEQ. ID. No. 8),
<58> 5'-caucccaucuuugacaauc-3' (SEQ. ID. No. 9),
<59> 5 ' -ucuuc cuacugugugacaa-3 ' (SEQ. ID. No. 10),
<60> 5 ' -cuccuuuucgcaagcugau-3 ' (SEQ. ID. No. 11),
<6i> 5'-cugaugugcaccgacaagu-3' (SEQ. ID. No. 12),
<62> 5'-cucagugagcccauggaau-3' (SEQ. ID. No. 13),
<63> 5 ' -ccauggaauuccaguaccu-3 ' (SEQ. ID. No. 14),
<64> 5 ' -gucaccggauugaggagaa-3 ' (SEQ. ID. No. 15),
<65> 5 ' -ggauugaggagaaacguaa-3 ' ( SEQ . ID. No . 16 ),
<66> 5 ' -gauugaggagaaacguaaa-3 ' (SEQ . ID . No . 17) ,
<67> 5 ' -aggacauaugagaccuuca-3 ' (SEQ. ID. No. 18),
<68> 5'-ggacauaugagaccuucaa-3' (SEQ. ID. No. 19),
<69> 5 ' -ccuucaagagcaucaugaa-3 ' (SEQ. ID. No. 20),
<70> 5 ' -ccugagcaccaucaacuau-3 ' (SEQ. ID. No. 21),
<7i> 5'-guuucccaccaugguguuu-3' (SEQ. ID. No. 22),
<72> 5 ' -ucccaccaugguguuuccu-3 ' (SEQ. ID. No. 23),
<73> 5'-caccaugguguuuccuucu-3' (SEQ. ID. No. 24),
<74> 5 ' -gcagcugcaguuugaugau-3 ' ( SEQ . ID . No . 25 ),
<75> 5 ' -ggaguacccugaggcuaua-3 ' (SEQ. ID. No. 26),
<76> 5 ' -gaguacccugaggcuauaa-3 ' (SEQ. ID. No. 27),
<77> 5 ' -guacccugaggcuauaacu-3 ' (SEQ. ID. No. 28),
<78> 5 ' -ugaguc agauc agcuc cua-3 ' ( SEQ . ID. No . 29 ),
<79> 5 ' -gagucagaucagcuccuaa-3 ' (SEQ. ID. No. 30).
<80>
<8i> Transformation of tumor cells with each of the synthesized siRNAs resulted in the decrease in NF-κB/RelA mRNA level, among them, the level of

NF-κB/RelA mRNA was relatively low in each of the cells transformed with siRNAs of SEQ. ID. Nos. 2, 3, 8, 9, 14, 16, 17, 26, 27, 28 and 30, and, the lowest level was examined in the cell transformed with siRNA of SEQ. ID. No. 17.
<82> Moreover, it was found that in case of fibroblast-like synoviocyte(FLS) transformed with siRNA of SEQ. ID. No. 17, the induction level of expression of proteins such as IL-lβ , IL-6, IL-8, BIRC3 and RANTES whose expression is induced by NF-κB/RelA, was decreased by the treatment with TNF- α .
<83> In addition, microscopic examination has revealed that FLS cells transformed with siRNA of SEQ. ID. No. 17 were dead by the treatment of TNF- α implying that siRNA of SEQ. ID. No. 17 can be used for the treatment of RA caused by NF-κB/RelA, since the said transformants were regarded as the same as abnormally thickened synovial cells.
<84>
<85> In one aspect of the present invention, a pharmaceutical composition for treating rheumatoid arthritis comprises an active ingredient of siRNA having one of nucleotide sequences represented as SEQ. ID. Nos. 1 to 30 and pharmaceutically acceptable carrier thereof.
<86> In the other aspect of the invention, 2'-0-methyl or phosphorothioate may be introduced at each of bases of the nucleotide sequences of the siRNAs, since the variants prepared by introducing 2'-0-methyl or phosphorothioate at each of bases of the siRNAs maintained their stabilities in blood serum for a long time and suppressed immune response caused by siRNA in vivo, while inhibiting the expression of NF-κB/RelA in a similar level as non-modified siRNAs. In addition, siRNAs may further comprise 3' overhang having one or two unpaired nucleotides at 3'-terminal of the nucleotide sequence.
<87>
<88> Based on the previous reports that the reduced activity of NF- KB may lead to cell death(see_: Bai et al., Arthritis Rheum., 50:3844-3855, 1988; Beg & Baltimore, Science, 274:782-784, 1999), it was assumed that rheumatoid arthritis could be treated more effectively, by the use of siRNAs inhibiting NF-κB/RelA expression and NF- KBl expression as well to lead to the cell death. The said assumption was experimentally evidenced that the expression of inflammatory cytokine was inhibited and the death of abnormally proliferated FLS cells was induced in the FLS cells transformed with siRNA inhibiting NF-κB/RelA expression(SEQ. ID. No. 17) as well as siRNA inhibiting NF- κBl expression(SEQ. ID. No. 51), implying that rheumatoid arthritis can be treated more effectively.
<89>
<90> In yet one aspect of the present invention, the pharmaceutical composition for treating rheumatoid arthritis comprises an active ingredient of siRNA having one of sequences represented as SEQ. ID. Nos. 1 to 30 which inhibit NF~κB/RelA expression and a sequence represented as SEQ. ID. No. 51 which inhibits NF- κBl expression, and pharmaceutically acceptable carrier thereof.
<91>
<92> The pharmaceutical composition which comprises an active ingredient of siRNA which binds with mRNA transcribed from a gene coding for NF-κB/RelA and pharmaceutically acceptable carrier thereof may be administered to the loci of inflammation in an injectable form or directly applied to the affected part in a inunctum or patch preparation. The injectable form may be formulated with an injectable composition after forming particles of nano- size with liposomes or polymers, and the inunctum or patch may be formulated with a gel composition or an adhesive composition for percutaneous administration, in accordance with the formulation method conventional in the art. The injectable composition includes, not limited thereto, preferably isotonic solutions or suspensions, which are sterilized and/or contain excipients(e.g. , preservatives, stabilizers, wetting agents, emulsifiers, salts for osmotic pressure or buffers), the gel composition contains a gelling agent such as carboxymethyl cellulose, methyl cellulose, acrylic acid polymer, carbopol and pharmaceutically acceptable carrier and/or liposome preparation, and the adhesive composition for percutaneous administration comprises an active ingredient layer containing adhesive layer, absorption layer for absorbing sebaceum and drug-containing layer containing pharmaceutically acceptable carrier and/or liposome preparation.

<94> Effective Dose of siRNA
<95>
<96> Though the effective dose of siRNA is variable depending on the patient's body weight, age, sex, healthy condition, diet, duration of medication or way of administration, siRNA as an active ingredient of a pharmaceutical composition may be parenteral Iy administered to a patient in an amount of 0.5 to 2.5mg/kg/day in a single dose or three times, which is individualized by experience of the skilled in the art.
<97>
<98> Acute Toxicity of siRNA
<99>
<ioo> Individual death rates within 24 hours were assessed for beagle dogs of 6 to 7weeks old after parenteral administration of siRNAs of present invention, where 8 female dogs having a body weight of 6 to 8kg and 8 male dogs having a body weight of 7 to 9kg were employed, respectively. As a result, no mortal dog was detected up to the level of 50mg/kg, indicating that siRNA as an active ingredient of a pharmaceutical composition was sufficiently safe within the range of effective dose.
<101>
<iO2> The pharmaceutical composition for treating rheumatoid arthritis of present invention directly inhibits NF-κB/RelA expression in the gene level as well as induces the death of synovial cells which proliferate abnormally, which makes possible its practical application for the treatment of rheumatoid arthritis in a more efficient manner.
<103>
[Mode for Invention]
<iO4> The present invention is further illustrated in the following Examples, which should not be taken to limit the scope of the invention.
<105>
<iO6> Example 1 : Preparat ion of siRNA bound wi th NF- κ B/RelA mRNA <iO8> A target nucleotide sequence to be bound with siRNA was designed using a mRNA sequence of NF-κB/RelA(NM_021975, SEQ. ID. No. 53) which is available from GenBank, and siRNA to be bound with the said sequence was prepared.
<iO9> First, a target nucleotide sequence to be bound with siRNA on NF- K B/RelA mRNA sequence which is available from GenBank was designed using Turbo si-Designer siRNA design programCBioneer Corp., Korea). Then, 30 kinds of siRNAs to be bound with NF-κB/RelA mRNA thus designed were chemically synthesized by the successive linkage of phosphodiester bond which is a backbone of DNA structure using β-cyanoethyl phosphoramidite(see: Shinha et al., Nucleic Acids Research, 12:4539-4557, 1984).
<πo> Specifically, RNA sequences(sense RNAs) of SEQ. ID. Nos. 1 to 30 and their complementary RNA sequences(antisense RNAs) were designed, respectively, and a series of processes of deblocking, coupling, oxidation and capping were carried out repetitively, by using RNA synthesizer(Perseptive Biosystems 8909, PE Biosystems, USA) on a solid support bound with a nucleotide, to give a reaction mixture containing the sense RNAs and antisense RNAs, which was then applied to HPLC LC918(Jaρan Analytical Industry, Japan) equipped with Diasogel C18 column(Daiso, Japan), to separate the desired RNAs. Then, chemical identification was conducted to confirm whether they were identical to the desired sequences by the aid of MALDI-TOF Mass Spectrometry(Shimadzu, Japan). And then, the same amounts of the sense RNAs and antisense RNAs were mixed with a binding buffer(30mM HEPES-KOH(pH 7.4), 10OmM potassium acetate, and 2mM magnesium acetate), and reacted at 90°C for 2min and at 37°C for lhr, to prepare double stranded SiRNAs(SEQ. ID. Nos. 1 to 30) in which each of sense RNAs and antisense RNAs was bound, respectively.
<1 1 1>
<i i2> Example 2 : Screening of siRNA inhibi t ing the expression of NF~ κ B/RelA in a tumor cel l l ine(HeLa)
<113>
<ii4> A tumor cell line, HeLa, human uterine cancer cell, was transformed with each of siRNAs prepared in Example 1 and the expression patterns of the transformed cells were examined to screen siRNA with a low expression level of NF-κB/RelA mRNA.
<115>
<ii6> Example 2-1: Cultivation of human uterine cancer eel 1(HeLa)
<117>
<ii8> Human uterine cancer cell, HeLa cell(CCL-2) obtained from American Type Culture Col lectionCATCC) was cultured in RPMI medium(GIBCO/Invitrogen, USA, 10%(v/v) fetal bovine serum, penicillin 100unit/ml, and streptomycin 100 βg/mt) at 37°C under an environment of 5%(v/v) CO2 gas.
<119>
<12O> Example 2-2: Transformation of HeLa cell with siRNA
<121>
5
<122> 2.5 x 10 HeLa cell(CCL-2) cultured in Example 2-1 was cultured in 6- well plate for 24 hours under the same condition of Example 2-1, washed with Opti-MEM mediumCGIBCO/Invitrogen, USA), and 500/^ Opti-MEM was aliquoted to each of the wells.
<i23> A solution for transformation"transformation solution") of human uterine cancer cell(HeLa) was prepared as follows: a first solution was prepared by mixing 3.5μJt lipofectamine 2000(Invitrogen, USA) and 250//I Opti- MEM medium, and a second solution was prepared by adding each of siRNAs obtained in Example 1 to 250μi Opti-MEM medium to the final concentration of 200pM(200 fmol/ml.), and the said first and second solutions were mixed with each other and reacted at room temperature for 20min to prepare 500//£ of transformation solution.
<124> Then, 500μJt of transformation solution was aliquoted to each of the wells containing tumor cell and 500μJl Opti-MEM medium, cultured for 6 hrs, and Opti-MEM medium was removed therefrom, and 2ml RPMI medium was aliquoted to each of the wells and cultured at 37°C for 24hrs under an environment of 5%(v/v) CO2 gas.

<126> Example 2-3: Measurement of NF- κ B/RelA mRNA level
<127>
<i28> Total cellular RNAs were extracted from the transformed tumor cells which were contained in the wells and cultured in Example 2-2 using RNeasy mini kit(Qiagen, Germany), the levels of NF-κB/RelA mRNAs in the total cellular RNAs were measured using real-time PCR(11RT-PCR") technique as follows.
<129> 2μg of total cellular RNA of each of the transformed tumor cells was mixed with 0.5/Λ oligo-dTi8(500ng/μ«) and 3.2//« dNTP(2.5mM), reacted at 70°C for 5 min, and cooled on ice for lmin. Then, lfd of reverse transcriptase(Superscript II (200U/μg), Invitrogen) was mixed with Aμi of 5 X reaction buffer and adequate amount of sterile water to reach the final volume of 20//!, and the mixture was reacted at 42°C for lhr and at 70°C for 15min, to prepare each of desired cDNAs. And then, 0.5//4 of each of cDNAs, 10μ£ of SYBR Green Master Mix contained in RT-PCR system(Applied Biosystems Prism 7900 Sequence Detection System, Applied Biosystems, USA), 0.5//£ of each of sense primers(10pmole/μlD and Q.5id of each of antisense primersQOpmol/ id) specific for a gene coding for NF-κB/RelA and a gene coding for glyceraldehyde-3-phosρhate dehydrogenase(GAPDH) to be used to plot a standard curve, were mixed with one another, and RT-PCR was performed at 50°C for 2min and at 95°C for lOmin in the initial reaction, then, 40cycles of reactions at 95°C for 30sec, at 60°C for 30sec, and at 72°C for 30sec. <i30> The sense primer and antisense primer sequences used in RT-PCR were as follows:
<131 >
<i32> Sense pr imer speci f i c for NF- κ B/RelA
<i33> : 5 ' -ctgcagtttgatgatgaaga-3 ' (SEQ. ID . No . 31)
<134> Ant i sense pr imer speci f i c for NF- κ B/RelA
<i35> : 5 ' -taggcgagttatagcctcag-3 ' (SEQ. ID. No . 32)
<136> Sense pr imer speci f ic for GAPDH <137> : 5 ' -tgcaccaccaactgcttagc-3 ' (SEQ . ID . No . 33)
<138> Ant i sense pr imer speci f ic for GAPDH
<i39> : 5 ' -ggcatggactgtggtcatgag-3 ' (SEQ. ID. No . 34)
<140>
<i4i> After completion of RT-PCR, the amounts of NF-κB/RelA PCR product and GAPDH PCR product were measured using cDNA standard curve, and the measured value of NF-κB/RelA was divided by that of GAPDH, to calculate the relative level of NF-κB/RelA mRNA, and the levels of NF-κB/RelA mRNAs were compared with one another(see: Fig. 1).
<i42> Figure 1 is a graph showing the levels of NF-κB/RelA mRNAs in each of HeLa cells transformed with 30 kinds of SiRNAs(SEQ. ID. Nos. 1 to 30). As can be seen in Fig. 1, the levels of NF-κB/RelA mRNAs in each of HeLa cells transformed with 30 kinds of siRNAs were different from one another, implying that the level of NF-κB/RelA mRNA was changed by the treatment of siRNA, and, the level of NF-κB/RelA mRNA was relatively low in each of the cells transformed with siRNAs of SEQ. ID. Nos. 2, 3, 8, 9, 14, 16, 17, 26, 27, 28 and 30, and, among them, the lowest level was examined in the cell transformed with siRNA of SEQ. ID. No. 17.
<143>
<144> Examp Ie 3 : Inhibi t ion of NF- κ B/RelA expression in f ibroblast-l ike synoviocyte (FLS) by using siRNA
<145>
<i46> Fibroblast-like synoviocyte("FLS") was transformed with siRNA of SEQ. ID. No. 17 which was measured to possess the highest inhibiting activity for NF-κB/RelA mRNA expression on HeLa cell transformed with various siRNAs in Example 2, and the mode of NF-κB/RelA expression and the level of inhibiting the expression induction of target genes by the treatment with TNF- α in the transformed FLS were measured, respectively.
<147>
<148> Example 3-1 : Cul t ivat ion of f ibroblast-l ike synoviocyte (FLS) <15O> Synovial membrane collected from an arthritic with arthroplasty replacement, was cut in a size of 2-3mm, submerged in 9ml of PBS, and incubated with collagenase(l% type II collagenase, Sigma Chem. Co., USA) at 37°C for 4 hrs under an environment of 5%(v/v) CO2 gas. Then, 5ml of DMEM medium(GIOBCO/Invitrogen, USA, 10%(v/v) fetal bovine serum, penicillin 100unit/ml, and streptomycin 100μg/ml) was added to inhibit collagenase activity, and centrifuged to harvest the cells. The cells thus collected was added to 15ml of DMEM medium, and cultured at 37°C for 1 day under an environment of 5%(v/v) CO2 gas. Then, the cells attached on the bottom of culture container were collected and cultured again in DMEM medium under the same condition as the above. And then, subculture was performed repetitively to obtain fibroblast-like synoviocyte of the passage of 3-6.
<151>
<i52> Example 3-2: Transformation of fibroblast-like synoviocyte with siRNA

<153>
<i54> 2.5 x 10 FLS cells cultured in Example 3-1 were cultured on 6-well plate under the same condition as Example 3-1, washed with Opti-MEM mediumCGIBCO/Invitrogen, USA), and to each of the wells was aliquoted 500μi Opti-MEM medium.
<155> Transformation solution for FLS was prepared similarly as in Example 2-2, and FLS was cultured in the transformation solution for 6 hrs, and Opti- MEM medium was removed therefrom. 2.0ml of DMEM medium was aliquoted to each of the wells, and FLS was cultured again at 37°C under an environment of 5%(v/v) CO2 gas to obtain transformed FLS.
<156>
<157> Example 3-3 : Measurement of NF- κ B/RelA mRNA level
<158>
<i59> Total cellular RNA was extracted from the FLS cells transformed with siRNA and cultured in Example 3-2, after 24hrs of transformation, using RNeasy mini kit(Qiagen, Germany). The level of NF-κB/RelA mRNA in total cellular RNA was measured similarly as in Example 2-3 using RT-PCR technique(see: Fig. 2). Sense and antisense primers specific for genes coding for NF-κB/RelA and GAPDH were the same as in Example 2-3. Figure 2 is a graph showing the level of NF-κB/RelA mRNA in FLS cells transformed with siRNA of SEQ. ID. No. 17, where NC indicates non-transformed FLS cell(control). As can be seen in Fig. 2, it was examined that the level of NF-κB/RelA mRNA was drastically decreased in FLS cell transformed with siRNA of SEQ. ID. No. 17, which was shown to inhibit NF-κB/RelA mRNA expression in the highest level in HeLa cell.
<160>
<i6i> Example 3-4 : Northern blot analysi s of NF- κ B/RelA mRNA
<162>
<i63> Northern blot analysis was performed on the FLS cells transformed with siRNA and cultured in Example 3-2 using a probe having a partial sequence of NF-κB/RelA gene.
<i64> Total cellular RNA was extracted from each of the transformed FLS cell cultured in Example 3-2 and FLS cell transformed with NC siRNA(5'- ccuacgccaccaauuucgu-3' , SEQ. ID. No. 52)(control) , after 24hrs of transformation, using RNeasy mini kit(Qiagen, Germany). Then, 10//g of the total cellular RNA was electrophoresed on 1% agarose/formaldehyde gel, transferred to Hybond-N+ nylon membrane(Amersham, USA), and UV-crossliking was performed to the membrane using UV crossInker(Amersham, USA). The nylon membrane crossli Inked with RNA was submerged into a hybridization solution, and prehybridization was performed at 65°C for 30 min(see_: Church & Gilbert, Proc. Natl. Acad. Sci . , USA, 81:1991-1995, 1984). Then, heat-denatured radioactive NF-κB/RelA probe was added to the membrane and hybridized at 65 °C for 20hrs.
<165>
<i66> Heat-denatured radioactive NF-κB/RelA probe was prepared using nested PCR technique using labeling kit as follows: NF-κB/RelA cDNA as a template, sense primer(SEQ. ID. No. 35), and antisense primer(SEQ. ID. No. 36) were used to perform PCR to give PCR product. The PCR product as a template, sense primer(SEQ. ID. No. 37), and antisense primer(SEQ. ID. No. 36) as below were used to perform PCR to obtain a DNA fragment(972bp) containing a nucleotide sequence corresponding to position nos. 611 to 1582 of NF-κB/RelA
32
gene. The said DNA fragment and α- P were applied on a labeling kit(Prime- It π random primer, Stratagene, USA) to prepare a heat-denatured radioactive probe.
<167>
<i68> Sense pr imer
<i69> : 5 ' -gactacgacctgaatgctgt-3 ' (SEQ. ID . No . 35)
<17O> Ant i sense pr imer
<i7i> : 5 ' -ctaggcgagttatagcctca-3 ' (SEQ . ID. No . 36)
<172> Sense pr imer
<i73> : 5 ' -ctcatcccatctttgacaat-3 ' (SEQ. ID. No . 37)
<i74> Ant i sense pr imer
<175> : 5 ' -ctaggcgagttatagcctca-3 ' (SEQ. ID. No . 36)
<176>
<177> Meanwhile, β -actin mRNA was used as a control for Northern blot analysis and a probe for the detection of β -actin mRNA was prepared as follows: First, β-actin(NM_001101) cDNA as a template, sense primer(SEQ. ID. No. 38), and antisense primer(SEQ. ID. No. 39) as below were used to perform PCR to prepare PCR product. Then, the PCR product as a template, sense primer(SEQ. ID. No. 38), and antisense primer(SEQ. ID. No. 40) as below were used to perform PCR to obtain DNA fragment(788bρ) containing a nucleotide sequence corresponding to position nos. 433 to 1220 of β -actin
32
gene. Finally, the DNA fragment of 788bp and α- P were applied on a labeling kit(Prime-It II random primer, Stratagene, USA) to prepare a radioactive probe for β -act in mRNA.
<178>
<179> Sense pr imer
<i80> : 5 ' -ccagatcatgtttgagaccg-3 ' (SEQ . ID . No . 38)
<i8i> Ant i sense pr imer
<i82> : 5 ' -aaacaacaatgtgcaatcaa-3 ' (SEQ. ID. No . 39)
<183> Sense pr imer
<184> : 5 ' -ccagatcatgtttgagaccg-3 ' (SEQ . ID. No . 38)
<185> Ant i sense pr imer
<186> : 5 ' -caactaagtcatagtccgcc-3 ' (SEQ. ID . No . 40)
<187>
<188> After hybridization with NF-κB/RelA probe, nylon membrane was washed, exposed to X-ray film for 3days, and developed. Then, the nylon membrane was hybridized with a radioactive probe for β-actin mRNA, washed, exposed to X- ray film for lhr and developed. And then, the said films thus developed were compared with each other(see_: Fig. 3). Figure 3 is photographs of Northern blot analysis results showing the level of NF-κB/RelA mRNA in FLS cells transformed with siRNA of SEQ. ID. No. 17. As can be seen in Fig. 3, the level of mRNA transcribed from NF-κB/RelA gene upon the treatment with the siRNA was decreased, implying that the said siRNA effectively inhibits the expression of NF-κB/RelA.
<189>
<19O> Example 3-5 : Western blot analysi s of NF- κ B/RelA protein
<191>
<i92> Western blot analysis was performed on the FLS cell transformed with siRNA and cultured in Example 3-2 by using an antibody for NF-κB/RelA.
<i93> The FLS cells transformed with siRNA and cultured in Example 3-2, after 72 hrs of transformation, was treated with TNF-αSigma Chem. Co., USA) in a concentration of 50ng/ml and cultured for lhr, and cellular protein was purified from the cells using Protein Purifying KitOntroBioTechnology, Korea). 40μgof the purified cellular protein was treated with SDS-sample bufferCSanta Cruz Biotechnology, USA) and electrophoresed on 4-20% SDS- polyacrylamide gel(SDS-PAGE). Then, Western blot analysis was performed by using anti-NF-κB/RelA-monoclonal antibody(Santa Cruz Biotechnology, USA) as a primary antibody and anti-mouse-antibody(Pierce, USA) bound with horseradish peroxidase as a secondary antibody, respectively, where non- transformed FLS cell was used as a control(see: Fig. 4).
<i94> Figure 4 is a photograph of Western blot analysis results showing the level of NF-κB/RelA in FLS cell transformed with siRNA of SEQ. ID. No. 17. As can be seen in Fig. 4, the expression level of NF-κB/RelA upon the treatment with siRNA of SEQ. ID. No. 17 was drastically decreased.
<195>
<196> Example 3-6 : Measurement of mRNA level of proteins whose expression i s induced by NF- κ B/RelA
<197>
<i98> mRNA levels of proteins which are associated with immune response or inflammatory response, and whose expression is induced by NF~κB/RelA, such as IL-lβ, IL-6, IL-8, RANTES(Regulated upon Activation, Normal T-cell Expressed and Secreted) and BIRC3(Baculoviral IAP repeat-containing 3), were determined, respectively.
<199>
<2oo> The transformed FLS cells obtained in Example 3-2, after 72hrs of transformation, were treated with TNF- α (Sigma Chem. Co., USA) in a concentration of 50ng/ml and subsequently cultured for 12hrs, and total cellular RNA was extracted from the cells using RNeasy mini kit(Qiagen, Germany) .
<201>
<202> mRNA level in the total cellular RNA was measured using RT-PCR technique similarly as in Example 2-3, and compared with that of the non- transformed FLS cell (see: Fig. 5), where FLS cell transformed with NC SiRNA(SEQ. ID. No. 52) was used as a control and sense and antisense primers used were as follows:
<203>
<204> (IL-lβ)
<205> Sense primer
<206> : 5'-cccaactggtacatcagcac-3'(SEQ. ID. No. 41)

<207> Ant i sense primer
<208> : 5'-ggaagacacaaattgcatgg-3'(SEQ. ID. No. 42)

<209>
<2io> (IL-6)
<2ii> Sense primer
<2i2> : 5'-ggcacctcagattgttgttg-3'(SEQ. ID. No. 43)

<2i3> Ant i sense primer
<2i4> : 5'-taagttctgtgcccagtgga-3'(SEQ. ID. No. 44)

<215>
<2i6> (RANTES)
<2i7> Sense primer
<2i8> : 5'-gggttcgggagtacatcaac-3'(SEQ. ID. No. 45)

<2i9> Ant i sense primer
<220> : 5'-cctcccaagctaggacaaga-3' (SEQ. ID. No. 46)

<221>
<222> (BIRC3)
<223> Sense primer
<224> : 5'-cctagctgcagattcgttca-3'(SEQ. ID. No. 47)

<225> Ant i sense primer
<226> : 5'-gagccacggaaatatccact-3'(SEQ. ID. No. 48)

<227>
<228> (IL-8)
<229> Sense primer
<230> : 5'-gcagagggttgtggagaagt-3'(SEQ. ID. No. 49)

<23i> Ant i sense primer
<232> : 5'-ccctacaacagacccacaca-3'(SEQ. ID. NO.50) <233>
<234> Figure 5 is a graph showing the mRNA level of proteins(IL-lβ , IL-6, IL-8, RANTES and BIRC3) in FLS cell transformed with siRNA of SEQ. ID. No. 17 and treated with TNF- α which is measured by RT-PCR technique, where NC indicates a control. As can be seen in Fig. 5, it was examined that, upon the treatment with TNF- α mRNA levels of the proteins were increased in the control, while those of the proteins was not increased in the FLS cells transformed with siRNA of SEQ. ID. No. 17 as much as the former, implying that siRNA of SEQ. ID. No. 17 inhibits the expression of NF-κB/RelA.
<235>
<236> Example 3-7: ELISA(Enzyme-linked Immunosorbent Assay) analysis
<237>
<238> ELISA analysis was performed on the FLS cells transformed with siRNA and cultured in Example 3-2 to measure the level of IL-6 and IL-8 secreted to a culture using ELISA kit(Quantikine HIGH sensitivity human IL-6 and IL-8 kit, R&D Systems, USA) containing antibodies for IL-6 and IL-8.
<239> The FLS cells transformed with siRNA and cultured in Example 3-2, after 72 hours of transformation, were treated with TNF-αSigma Chem. Co., USA) in a concentration of 50ng/ml and cultured for 12hrs to obtain cell culture, which was then diluted at a ratio of 1:5000 to prepare a sample. The standard reagents were prepared by adding a calibrator diluent(Calibrator Diluent RD6-11) to each of IL-6 and IL-8 protein standards contained in the ELISA kit to reach the concentrations of 0pg/ml , 0.156pg/ml, 0.312pg/ml, 0.625pg/ml, 1.25pg/ml, 2.5pg/ml, 5pg/ml and lOpg/ml , respectively.
<240> To each of wells in 96-well plate coated with anti-mouse monoclonal antibody for IL-6 or IL-8, was added 100fd of assay reagent(Assay Diluent RD1-75) contained in the kit, and further added lOOμβ of sample or IL-6 or IL-8 standards diluted as stated above and reacted overnight, and washed with a washing buffer contained in the kit. Then, 200/z£ of IL-6 or IL-8 polyclonal antibody bound with alkaline phosphate was added to each of wells and reacted on a shaking incubator at 450rpm for 2hrs, and washed again as previously described, and added 50μi of substrate solution and reacted for 60min, and added 50μi of amplification solution and reacted for 30min. And then, bOμJL of stop solution was added to each of the wells, and absorbance at 690nm was measured using a plate reader(SPECTRAmaxPLUS384, Molecular Devices, USAXsee: Fig. 6), where FLS cells transformed with NC siRNA(5'- ccuacgccaccaauuucgu-3' , SEQ. ID. No. 52) which can not inhibit the expression of NF-κB/RelA, was employed as a control.
<24i> Figure 6 is a graph of ELISA results showing the expression level of IL-6 and IL-8 in FLS cell which was transformed with siRNA of SEQ. ID. No. 17 and treated with TNF- α where NC indicates a control, (D) IL-6, and (■) IL- 8, respectively. As can be seen in Fig. 6, in case of a control, the levels of IL-6 and IL-8 in the cells treated with TNF- α was dramatically increased, while in case of the cell treated with siRNA of SEQ. ID. No. 17, the levels were not changed so much, implying that siRNA of SEQ. ID. No. 17 could inhibit the expression of IL-6 and IL-8 induced by NF-κB/RelA, and prevent the extracellular secretion of said proteins.
<242>
<243> Example 4: Preparation of modified siRNAs and evaluation of their effects
<244>
<245> Example 4-1: Preparation of modified siRNAs
<246>
<247> Variants of siRNAs represented as SEQ. ID. Nos. 17, 30, 19 and 6, i.e., 17CFM/P) and 17(FM/AS), 30(FM/P), 19(FM/P), and 6(FM/P), which have sense and antisense sequences as Table 1 below were prepared in a similar manner as in Example 1, with an exception of using nucleotides with 2'-0-methyl and phosphorothioate substituents which are known to increase the stability of RNA(Dharmacon, Lafayette, Co., USAXsee: Table 1).
<248>
<249> [Table 1]
<250> The modified sequence of the variants of siRNAs represented as SEQ. ID.

Nos . 17 , 30 , 19 and 6
<251>


<252> In the above Table 1, underlined parts of sense sequences and antisense sequences indicate variants of siRNAs with 2'-0-methyl and phosphorothioate, respectively.
<253>
<254> Example 4-2: Inhibition of NF-κB/RelA mRNA expression by modified siRNA
<255>
<256> Transformed human uterine cancer eel Is(HeLa cells) were obtained similarly as in Example 2-2, with an exception of using variants of siRNAs prepared in Example 4-1. Then, the level of NF-κB/RelA mRNA in HeLa cells transformed with each of siRNA variants were measured in a similar manner as in Example 2-3, and the expression rate of each of NF-κB/RelA mRNAs in the transformed human uterine cancer eel Is(HeLa cells) were measured, respectively, based on the rate of 100% of NF-κB/RelA mRNA expression in HeLa cell transformed with NC siRNA(5'-ccuacgccaccaauuucgu~3' , SEQ. ID. No. 52). And then, they were compared with those of NF-κB/RelA mRNA expression in HeLa cells transformed with non-modified-siRNAs(SEQ. ID. Nos. 17, 30, 19 and 6)(see: Table 2).
<257>
<258> [Table 2]
<259> Expression rates of NF-κB/RelA mRNA in HeLa cells transformed with non-modified- or modified-siRNA(unit : %) <260>



<261> As shown in Table 2 above, it was examined that most of the variants(17(FM/P), 17(FM/AS), 30(FM/P), 19(FM/P) and 6(FM/P)) showed similar or little high expression rate of NF-κB/RelA mRNA, when compared with those of non-modified-siRNAs, implying that each of the variants of siRNA can inhibit the expression of NF-κB/RelA mRNA.
<262>
<263> Example 4-3: Comparison of stabilities of non-modified- and modified- siRNAs in blood
<264>
<265> Blood sample was collected from healthy people and left to stand at room temperature for 3hrs, and centrifuged at 3000rpm for 15min to obtain serum as a supernatant. To 90μi of solution containing serum(10%(v/v)) and water treated with diethylpyrocarbonate(DEPC), a RNase inhibitor was added 9 μgof each of siRNAs of SEQ. ID. Nos. 17, 30, 19 and 6 and variants of the siRNAs prepared in Example 4-1, and incubated at 37°C for 72hrs. After 0, 1, 3, 6, 24, 48 and 72 hrs of incubation, VZμA of the solution was aliquoted, snap frozen and stored at -70°C , 2.5μl of each samples was then applied to 12% SDS-PAGE to examine whether each of siRNAs was maintained in the blood serum(see_: Fig. 7).
<266> Figure 7 is a photograph of gel electrophoresis pattern showing the time courses of the stability of siRNAs of SEQ. ID. Nos. 17, 30, 19 and 6 and their variants in blood serum. As shown in Fig. 7, the variants of siRNAs(17(FM/P), 17(FM/AS), 30(FM/P), 19(FM/P), 6(FM/P)) maintained their stabilities for a longer time than non-modified-siRNAs of SEQ. ID. Nos. 6, 17, 19 and 30.
<267>
<268> Example 4-4: Effects of modified siRNAs on the suppression of immune response.
<269>
<270> It was examined whether non-modified-siRNA or modified siRNAs could induce immune response in the body.
<27i> siRNA of SEQ. ID. No. 17 and each of the variants(17(FM/P) , 17(FM/AS)) prepared in Example 4-1 were bound with in vivo Megafectin liposome(Qbiogene, USA) to obtain siRNA/liposome complexes, each of which was then injected intravenously to caudal vein of a mouse. And, the level of interferon- α ("INF- α") in blood serum was measured by using ELISA kit(Quantikine mouse INF- α kit, R&D Systems, USA) containing an antibody for INF- α .
<272> The siRNA/liposome complexes were obtained as follows: in vivo Megafectin(Qbiogene, USA) was dissolved in lOOμi of Hepes-buffered saline, and mixed with 100fd of 5% dextrose solution in which 50//g of siRNA of SEQ. ID. No. 17, 17CFM/P) or 17(FM/AS) was dissolved, and incubated at room temperature for 15min to obtain 200/^ of a solution containing siRNA/liposome complex. The siRNA/liposome complex solution thus obtained was injected intravenously to caudal vein of SIc/ICR mice(Japan SLC Ic, Hamamatsu, Japan) of 5-6 weeks old, collected blood after 6hrs of injection and centrifuged to obtain blood serum.
<273> The blood serum was diluted with a dilution buffer contained in the said kit at a ratio of 1/100 to obtain a sample, and IFN- α standard contained in the kit was mixed with a dilution buffer to prepare standard reagents of Opg/ml, 12.5pg/ml, 25pg/ml , 50pg/ml , lOOpg/ml , 200pg/ml and 500pg/ml, respectively.
<274> To 96-well plate coated with murine monoclonal antibody for IFN- α contained in the said kit were added 100μ£ of the sample or each of the diluted standard reagents prepared as above, and reacted at room temperature for lhr. Then, each of the wells was washed with a washing solution contained in the kit in a repetitive manner. And then, lOOμJt of antibody concentrate containing anti-IFN- α antibody diluted at a ratio of 1/200 with a dilution buffer contained in the kit was aliquoted to each of wells, and reacted at room temperature for 24hrs, and each of wells was washed with a washing solution contained in the said kit in a repetitive manner. Then, 100 μJL of HRP conjugate concentrate diluted with HRP conjugate diluent contained in the said kit at a ratio of 1/300, was aliquoted to each of wells in a clean bench, and reacted at room temperature for lhr. Then, each of wells was washed with a washing solution contained in the kit in a repetitive manner. Finally, lOOμi of a colorizing reagent, TMB substrate solution contained in the said kit was aliquoted to each of wells, and reacted at room temperature for 15min, and lOOμi of stop solution contained in the kit was aliquoted to each of wells, and absorbance at 650nm was measured by using plate reader(see: Fig. 8).
<275> Figure 8 is a graph showing the level of IFN- α in the blood serum of a mouse injected with siRNA of SEQ. ID. No. 17 and its variants, where Mega and Dextrose indicate each of controls of mice injected with in vivo Megafectin only and 5% dextrose only. As can be seen in Fig. 8, it was examined that the level of IFN- α in the blood serum of a mouse injected with siRNA of SEQ. ID. No. 17 was drastically increased, while those with its variants were not increased and similar to those of controls.
<276> From the above results, it was clearly demonstrated that siRNA variants were effective for the suppression of the immune response caused by siRNA.
<277>
<278> Taken together the results of Examples 4-2 to 4-4, it was clearly demonstrated that siRNA variants which were prepared by introducing 2'-0- methyl or phosphorothioate to siRNAs represented as SEQ. ID. Nos. 17, 30, 19 and 6, inhibit NF-κB/RelA expression, maintain their stabilities in the blood serum for a long time, suppress immune response caused by siRNA, which makes possible its practical application for the preparation of antirheumatic drugs.
<279>
<280> Example 5: Therapeutic effects of two kinds of siRNAs on rheumatoid arthritis
<281>
<282> Since NF- KB family has five NF- KB members, each of which forms a dimmer to have a biological activity, the inventors assumed that the inhibition of expression of NF- KBl, in addition to NF-κB/RelA, may affect on the treatment of RA in a more efficiently, and tried to verify the said assumption.
<283>
<284> Example 5-1: Decrease in the mRNA level of inflammatory cytokine and cellular FLICE-inhibitory protein by the treatment of two kinds of siRNAs

<285>
<286> Each of the FLS cell transformed with siRNA of SEQ. ID. No. 17, FLS cell transformed with siRNA of SEQ. ID. No. 51 and FLS cell cotransformed with siRNA of SEQ. ID. Nos. 17 and 51 was obtained in a similar manner as in Example 3-2, with an exception of using siRNA of SEQ. ID. No. 17 and siRNA(5'-gucacucuaacguaugcaa-3' , SEQ. ID. No. 51) designed to inhibit the expression of NF- KBl, respectively. The transformed FLS cells, after 96hrs of transformation, were treated with TNF- α at a concentration of 50ng/ml, and the mRNA levels of inflammatory cytokines(IL-6 and IL-8), cFLIP(cellular FLICE-inhibitory protein) and BIRC3 whose expression was induced by NF- KB were measured, respectively, in a similar manner as in Example 2-3(see: Fig. 9)
<287> Figure 9 is graphs showing the mRNA levels of proteins(IL-6, IL-8, cFLIP and BIRC3) in FLS cell transformed with siRNAs of SEQ. ID. Nos. 17 and 51 and treated with TNF- α which is measured by RT-PCR technique. As can be seen in Fig. 9, it was examined that the mRNA levels of IL-6, IL-8, cFLIP and BIRC3 were decreased similarly in both cases of inhibiting the expression of NF-κB/RelA by the treatment of siRNAs of SEQ. ID. No. 17 only and inhibiting the expression of NF~κB/RelA and NF- κBl simultaneously.
<288>
<289> Example 5-2: Increase in the level of FLS cell death by the treatment of two kinds of siRNAs
<290>
<29i> Based on the previous knowledge that the reduced activity of NF- KB may lead to cell death(see_: Bai et al . , Arthritis Rheum., 50:3844-3855, 1988; Beg & Baltimore, Science, 274:782-784, 1999), it was assumed that cell death would be accelerated by inhibiting the expression of NF-κB/RelA and NF- κBl as well .
<292> The death of FLS cells transformed with siRNA of SEQ. ID. No. 17, FLS cell transformed with siRNA of SEQ. ID. No. 51 and FLS cell cotransformed with siRNA of SEQ. ID. Nos. 17 and 51, were examined, respectively: that is, FLS cells transformed with siRNA of SEQ. ID. No. 17, FLS cell transformed with siRNA of SEQ. ID. No. 51, FLS cell cotransformed with siRNA of SEQ. ID. Nos. 17 and 51 and FLS cell transformed with NC siRNA of SEQ. ID. No. 52(control), all of which were prepared in Example 5-1 with/without treatment of TNF- α were examined with a microscope(see_: Fig. 10), and each of the said transformed FLS cells was treated with a fluorescence dye, Annexin V conjugated with Alexa 568(Roche Applied Science, GB) after 4hrs from the point of with/without treatment of TNF- α and examined with a fluorescence microscope(see: Fig. 11).
<293> Since normal FLS cells and dead FLS cells have filamentous forms and spherical forms, respectively, microscopic examination of cell structure can be used to determine whether the FLS cells are dead or not. Annexin V is a ligand which can bind with phosphatidyl serine in cellular membrane, cannot bind with a normal cell in which phosphatidyl serine is located within the inner side of cellular membrane. However, if a dead cell whose cellular membrane is destructed is treated with Annexin V bound with Alexa 568, Annexin V is bound with phosphatidyl serine exposed to outer side of cell and show fluorescence at the binding site, by which the dead cells with destructed cellular membrane was examined.
<294> Figure 10 is photomicrographs showing the levels of cell death by the treatment of TNF- α in FLS cells transformed with siRNAs of SEQ. ID. Nos. 17, 51, 17 and 51, respectively, and Figure 11 is fluorescence photomicrographs showing the levels of cell death by the treatment of TNF- α in FLS cells transformed with siRNAs of SEQ. ID. Nos. 17, 51, 17 and 51, respectively, where NC indicates a control.
<295> As can be seen in Figs. 10 and 11, it was examined that: a control and FLS cell transformed with siRNA of SEQ. ID. No. 51 were not dead, irrespective of the treatment of TNF- α ; FLS cell transformed with siRNA of SEQ. ID. No. 17 and treated with TNF- α was dead; and, death of FLS cell cotransformed with siRNAs of SEQ. ID. Nos. 17 and 51 and treated with TNF- α was dramatically increased.
<296>
<297> Taken together the results of Examples 5-1 and 5-2, it was clearly demonstrated that siRNAs of SEQ. ID. Nos. 17 and 51 inhibit the expression of inflammatory cytokines and induce the death of abnormally proliferated FLS cells, implying that rheumatoid arthritis can be treated by using the said siRNAs in a more efficient manner.
<298>
[Industrial Applicability]
<299> As clearly illustrated and demonstrated as the above, the present invention provides siRNA for inhibiting NF-κB/RelA and a pharmaceutical composition for treating rheumatoid arthritis which comprises an active ingrediant of the siRNA. The pharmaceutical composition for treating rheumatoid arthritis of the invention can directly inhibit NF-κB/RelA expression in the gene level as well as induce the death of abnormally proliferated synovial cells, which makes possible its practical application for the treatment of rheumatoid arthritis in a more efficient manner.