KR20130088690A - Sirna against mdc for treatment of asthma and rhinitis, and composition of therapeutic agent comprising the same - Google Patents

Abstract

PURPOSE: A composition for treating asthma or rhinitis using siRNA is provided to be used in a medical industry as an agent for preventing and treating asthma and rhinitis. CONSTITUTION: A composition for treating asthma and rhinitis contains siRNA as an active ingredient, which is specific to a target sequence of macrophage-derived chemokine (MDC) gene. The siRNA has a base sequence of sequence number 2. A composition for treating asthma and rhinitis contains bacterial cells which are transformed by a recombinant vector containing a gene which transcribes the siRNA. The siRNA suppresses the expression of MDC gene. The bacterial cell is Salmonella typhimurium.

Description

SiRNA against MDC gene having a therapeutic effect on asthma and rhinitis diseases and composition containing same {siRNA against MDC for treatment of asthma and rhinitis, and composition of therapeutic agent comprising the same}

The present invention relates to siRNA of MDC gene having a therapeutic effect of asthma and rhinitis diseases and compositions containing the same. More specifically, the present invention is directed to a composition for treating asthma and rhinitis, comprising bacterial cells transformed with recombinant vectors expressing small interfering RNA (siRNA) that inhibit the expression of the macrophage-derived chemokine (MDC) gene as an active ingredient. It is about.

Asthma is an inflammatory airway obstruction that causes dyspnea. Patients with asthma often complain of symptoms such as wheezing, breathing difficulties, coughing, etc., due to the closure of the airway, and symptoms tend to get worse at night than during the day. In addition, excessive sputum formation (breathing, breathing) when you have trouble.

Asthma is a growing trend worldwide. Therefore, asthma population is increasing in Korea. Even in Korea, the official prevalence of asthma in Korea is around 6%, which is equivalent to the prevalence of diabetes mellitus. According to the recent National Health and Nutrition Survey, asthma is a common disease with 17.45% of the national population.

The causes and mechanisms of asthma are diverse and complex, and despite many studies, it has not been clear yet. Recent studies have suggested that the basic mechanism of asthma is airflow obstruction and airway hyperresponsiveness due to chronic inflammation of the airways. Until recently, asthma immunity has been described as a 'Hygiene hypothesis' that focuses on the Th2 immune response and that the Th2 immune response predominates over the Th1 immune response, resulting in a balance between the two immune responses.

Half of pediatric asthma continues to adulthood and continues to adult asthma. People with asthma can have acute airway obstruction due to upper respiratory tract infections and, in severe cases, may die. In the acute phase, low-dose oxygen (2 L / m 2) or less, steroids, aminophylline, or epinephrine injection may be used. Asthma is a kind of allergic disease. Currently, there are no medicines that give more than 50% therapeutic effect. Avoiding asthma-inducing antigens is the best prevention.

Currently, asthma treatment uses a medication that suppresses asthma by blocking the process of bronchoconstriction and allergic inflammation reaction by eliminating or avoiding inducing factors such as ticks and mold dandruff which are known seizure substances in allergy test. However, existing therapies do not have an efficacy of more than 50%, and asthmatic diseases that are controlled through the proper use of drugs and combined with repeated or frequent recurrences can not be cured at present.

The currently used asthma medications are anti-inflammatory, anti-inflammatory, bronchodilator, and antihistamines. The main remedy is anti-inflammatory, or steroid, treatment for airway inflammation. Bronchodilators can be used for symptom relief if needed.

Currently the most effective remedy is a steroid preparation used to sedate the inflammatory response. However, eating long-term use of steroids causes severe systemic side effects such as hypertension, cataracts, osteoporosis, and decreased immune function. Oral use for more than two weeks is just about side effects.

Inhalation steroids are recommended as top priority drugs, and patients with inhaled steroids who do not control asthma are advised to use inhaled steroids and long-acting beta-2 (b-2).

It is known that the combination of long-acting b-2 agonists and inhaled steroids, which are currently the most prevalent asthma medication, is said to be most effective. The combination of these two drugs is a drug such as seretide, and currently the world's largest prescription for asthma treatment. However, even in the case of inhaled steroids, long-term inhalation of high-dose steroids may result in systemic side effects such as adrenal suppression, decreased bone density, growth disturbances, and eye and skin complications in proportion to dose and duration of use.

On the other hand, chronic rhinitis is a chronic chronic complaint of runny nose, sneezing, and nasal congestion (Dykewicz MS, et al. Ann Allergy Asthma Immunol 1998; 81: 463-468). In most countries, about 10-20% of the population is a common disease (Sly RM. Ann Allergy Asthma Immunol 1999; 82: 233-48.).

In addition, there is a reported relationship between bronchial asthma and chronic rhinitis. Usually, the majority of patients with bronchial asthma (80-99%) have chronic rhinitis and about 40% of patients with chronic rhinitis It is known to be accompanied by asthma (Simons FE. J Allergy Clin Immunol 1999; 104: 534-540). In addition, as a result of chronic nasal mucosal biopsy, it is known that pathological findings similar to those of bronchial asthma have been observed. Recently, bronchial asthma and chronic rhinitis are caused by the same cause. ) And chronic inflammatory diseases that invade the lower part (bronchial mucosa) (Simons FE., J. Allergy Clin. Immunol. 1999; 104: 534-540).

As a background technology of the present invention, EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 43, No. 2, 63-70 (February 2011), describes the therapeutic effect of atopic dermatitis, and US Patent Publication 2006 / 0057112A1 (2006.03.16) describes MDC, MDC analogues, MDC inhibitor substances and their use. have. However, there is no disclosure of asthma and rhinitis treatment using small interfering RNAs of MDC.

United States Patent Application Publication 2006 / 0057112A1 (2006.03.16)

EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 43, No. 2, 63-70 (February 2011)

Accordingly, the present inventors have made diligent research to overcome the problems of the prior art, and asthma was induced using Salmonella as a carrier of RNAi (RNA interference) of siRNA, which is a single nucleotide strand composed of 21 to 25 nucleotides. Oral administration to the experimental group can inhibit Th-2 immunity, increase Th-1 immunity, inhibit inflammatory cells in the lung, inhibit ROS expression and inhibit airway hyperresponsiveness to alleviate asthma and rhinitis. It has been confirmed that the present invention has been completed.

Accordingly, it is an object of the present invention to provide a composition for treating asthma and rhinitis comprising siRNA specific to the target sequence of the MDC gene as an active ingredient.

Another object of the present invention is to provide a composition for treating oral asthma and rhinitis using bacterial cells transformed with recombinant vectors expressing small interfering RNA (siRNA) that inhibits the expression of the MDC gene.

It is another object of the present invention to reduce the expression of IL-4 by inhibiting the expression of the MDC gene, to increase the expression of IFN-γ, to inhibit the expression of inflammatory cells, ROS and airway hyperresponsiveness in lungs to prevent asthma and rhinitis. It is to provide a small interfering RNA (siRNA) having a therapeutic effect.

Still other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the invention, the present invention is to provide a composition for treating asthma and rhinitis comprising siRNA specific to the target sequence of the macrophage-derived chemokine (MDC) gene as an active ingredient.

According to another aspect of the present invention, the present invention contains a bacterial cell transformed with a recombinant vector comprising a gene for transcribing a small interfering RNA (siRNA) that inhibits the expression of a macrophage-derived chemokine (MDC) gene as an active ingredient. It is to provide a composition for treating asthma and rhinitis.

According to another aspect of the present invention, the present invention provides a kit for diagnosing asthma and rhinitis comprising an antibody or aptamer specifically binding to Macrophage-derived chemokine (MDC) or fragments thereof.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

According to one aspect of the invention, the present invention provides a composition for treating asthma and rhinitis comprising siRNA specific to the target sequence of the MDC gene as an active ingredient.

In the present invention, the MDC target sequence is characterized in that SEQ ID NO: 1.

In the present invention, 'siRNA (small interfering RNA)' refers to a short double-stranded RNA that can induce RNA interference through cleavage of specific mRNA. siRNAs are not limited to pairs of double-stranded RNA portions that are paired with each other, but are paired by mismatch (corresponding bases are not complementary), bulge (no bases corresponding to one chain) May be included. The paired base length is 10 to 40 bases, preferably 15 to 30 bases. The siRNA terminal structure can be either a smooth end or a protruding end if it can inhibit the expression of the target gene by RNA interference. The sticky end structure can have both a 3 'end protruding structure and a 5' end protruding structure.

The siRNA of the human MDC gene of the present invention may preferably be a double-stranded siRNA comprising a nucleotide sequence of SEQ ID NO: 2 and a sequence complementary thereto.

SiRNAs of the invention include variants having one or more substitutions, insertions, deletions, and combinations thereof that do not degrade their activity. Such modifications may have a sequence homology of 80% or more, preferably 90%, more preferably 95% or more, with the sequence of the above siRNA.

The siRNA of the present invention can be synthesized directly (Sui G et al., (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells, Proc Natl Acad Sci USA 99: 5515-5520) (Brummelkamp TR et al., (2002) A system for stable expression of short interfering RNAs in mammalian cells, Science 296: 550-553) using a transcription transcription method .

The siRNA of the present invention is basically a form in which two strands of RNA are paired to form a double-stranded siRNA, but a structure having a short hairpin so that it can be used for transfection by a plasmid-based shRNA vector and a PCR expression cassette It may also be in the form of

According to another aspect of the present invention, the present invention provides oral asthma and rhinitis containing bacterial cells transformed with a recombinant vector comprising a gene for transcription of small interfering RNA (siRNA) that inhibits the expression of MDC genes as an active ingredient. A therapeutic composition is provided.

In the composition for treating asthma and rhinitis of the present invention, the bacterial cells are characterized in that the Salmonella typhimurium (salmonella typhimurium). The Salmonella strain belongs to the group D Salmonella which causes typhoid fever. In the present invention, an attenuated Salmonella strain was used to eliminate the pathogenicity of the Salmonella strain. The attenuated Salmonella strain is characterized in that it is an attenuated vaccine for typhoid prevention. The term " attenuation "refers to artificially weakening the toxicity of a living pathogen. It means mutating a gene involved in essential metabolism of a pathogen and inducing the immune system by stimulating only the immune system without causing disease in the body .

The Salmonella strain used in the specific examples of the present invention is salmonella typhimurium strain BRD509, which is an attenuated strain without pathogenicity.

The siRNA of the present invention is characterized by being an siRNA having the nucleotide sequence of SEQ ID NO: 2. The siRNA is a nucleotide sequence complementary to the mRNA of the MDC gene of 21 to 25 nucleotides, preferably, may be a double-stranded siRNA comprising the nucleotide sequence of SEQ ID NO: 2 and a sequence complementary thereto. In the present invention, the double stranded oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2 (top and bottom strand) were synthesized and used. The siRNA inhibits gene function through a process that interferes with the translation by complementarily binding to the mRNA of the MDC gene. Table 1 below shows the target sequence of the MDC gene and siRNA sequence used for siRNA.

MDC TARGET SEQUENCE SEQ ID NO: 1: SENSE SEQUENCE 5`-TGC TGT TAT GGA GTA GCT TCT TCA CCG TTT TGG CCA CTG ACT GAC GGT GAA GAC TAC TCC ATA A-3`, SEQ ID NO: 2: ANTISENSE SEQUENCE 5`-CCT GTT ATG GAG TAG TCT TCA CCG TCA GTC AGT GGC CAA AAC GGT GAA GAA GCT ACT CCA TAA C-3`

The siRNA polynucleotide sequence according to the present invention may be operably linked to an expression control sequence, wherein the operably linked gene sequence and expression control sequence are one expression containing a selection marker and a replication origin together. Can be included in a vector. Said "operably linked" may be a gene and expression control sequence linked in such a way as to enable gene expression when a suitable molecule is coupled to an expression control sequence. The expression control sequence refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked to a particular host cell. Such regulatory sequences include promoters for carrying out transcription, any operator sequence for regulating transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control termination of transcription and translation.

In a specific embodiment of the present invention, by introducing a recombinant vector expressing an siRNA that inhibits the MDC gene into attenuated Salmonella strains, the Salmonella of the present invention is produced, and the transduced Salmonella is administered to a mouse model inducing asthma. As a result, the effect of preventing and treating asthma disease caused by the control Salmonella itself, which was not transduced, was compared and confirmed. As a result of the analysis, as shown in FIGS. 3 to 4, only Salmonella without transfection increased asthma disease by oral administration, but when salmonella transfected with a recombinant vector expressing siRNA was orally administered, the effect of treating asthma Respectively.

In the present invention, the recombinant vector may be any expression vector having a promoter capable of expressing the siRNA and a multicloning site for cloning, but preferably a cleavage map of pcDNA-MDCmiRNA shown in FIG. 1. It is characterized by.

Salmonella strains expressing siRNA that induces asthma and rhinitis therapeutic effects in the present invention can be prepared by a transduction method commonly used in the art, and when the prepared Salmonella is introduced into the body, the siRNA may be overexpressed. .

According to another aspect of the present invention, the present invention provides a small interfering RNA (siRNA) that suppresses the expression of the MDC gene, characterized in that the siRNA having the nucleotide sequence of SEQ ID NO: 2.

The present invention provides a recombinant vector comprising a gene that transcribes said siRNA and a bacterial cell transformed with said recombinant vector. The bacterial cell of the present invention is characterized in that Salmonella typhimurium.

The composition for treating asthma and rhinitis of the present invention may be prepared by a method known in the pharmaceutical field for use as a medicament, and may be mixed with a pharmaceutically acceptable carrier, excipient, diluent, etc. to powder, granule, tablet, capsule Or in the form of an injection or the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995). They may also be administered parenterally (e. G., Intravenously, subcutaneously, intraperitoneally or topically) or orally. Preferably orally.

The therapeutic composition of the present invention is administered in a therapeutically effective amount. The term "therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and includes the age, sex, weight, , The time of administration, and the method of administration, and preferably 0.01 to 100 mg per day on an adult basis may be administered.

According to another aspect of the invention, the present invention provides a kit for diagnosing asthma and rhinitis comprising an antibody or aptamer specifically binding to Macrophage-derived chemokine (MDC) or fragments thereof.

MDC according to the present invention has increased expression in asthma and rhinitis patients. Therefore, the MDC or fragment thereof according to the present invention can be usefully used as a diagnostic marker of asthma and rhinitis diseases.

In addition, a kit for diagnosing asthma and rhinitis diseases comprising an antibody or aptamer specifically binding to the MDC or fragments thereof of the present invention can be easily prepared by a manufacturing method commonly used in the art using the marker. Can be. The aptamer is a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) having a stable tertiary structure and capable of binding to a target molecule with high affinity and specificity.

The kit for diagnosing asthma and rhinitis diseases is a secondary antibody conjugate conjugated with an antibody that specifically binds to MDC or a fragment thereof, a label that is developed by reaction with a substrate, and a color substrate solution that will react with the label. It may include a washing solution and an enzyme stopping solution.

The marker of the secondary antibody conjugate is preferably a conventional coloring agent that performs a color reaction, and is preferably selected from the group consisting of horseradish peroxidase (HRP), alkaline phosphatase, coloid gold, poly-L-lysine-fluoresceinisothiocyanate ), Fluorescein such as rhodamine-B-isothiocyanate (RITC), and dye can be used.

The coloring substrate solution is preferably used according to the markers, and TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid )], OPD (o-phenylenediamine) and the like can be used. At this time, it is more preferable that the chromogenic substrate is provided in a dissolved state in a buffer solution (0.1 M NaOAc, pH 5.5).

The washing solution preferably includes a phosphate buffer solution, NaCl and Tween 20, and more preferably a buffer solution (PBST) composed of 0.02M phosphate buffer solution, 0.13M NaCl, and 0.05% Tween 20. After the antigen-antibody binding reaction, the washing solution is reacted with the secondary antibody to the antigen-antibody conjugate, and then an appropriate amount of the antibody is added to the solid body and washed three to six times. The reaction stop solution may be a sulfuric acid solution.

In addition, the present invention can detect the MDC or its fragments through an antigen-antibody binding reaction using an antibody that specifically binds to the MDC or a fragment thereof in a biological sample, thereby predicting the diagnosis or prognosis of asthma and rhinitis diseases early. . The biological sample includes, but is not limited to, tissue, cells, blood, serum, peritoneal fluid, synovial fluid, saliva, urine or feces. Specifically, an MDC or a fragment thereof in a biological sample is electrophoresed on an SDS-PAGE, fractionated, transferred to a fixed body, immobilized, and then an antigen-antibody binding reaction is performed by adding an antibody that specifically binds to the immobilized MDC or a fragment thereof. The expression level of MDC or fragments thereof is measured. In other words, if the expression level of MDC or fragment thereof in a biological sample is high, it is predicted to be diagnosed as having asthma and rhinitis diseases or to have a possibility of asthma and rhinitis diseases.

As a fixture for the antigen-antibody conjugation reaction, a nitrocellulose membrane, a PVDF membrane (polyvinylidene difluoride membrane), a 96 well plate synthesized from polyvinyl resin or polystyrene resin, and a glass slide glass can be used.

The antigen-antibody binding reaction can be carried out by conventional enzyme immunoassay (ELISA), radioimmunoassay (RIA), sandwich ELISA, Northern blot, Western blot, immunoprecipitation, immunohistochemistry, fluorescent immunoassay, (Surface plasmon resonance), biochip (DNA, RNA), electrophoresis, PCR, RT-PCR, and the like.

As described above, according to the present invention, a bacterial cell transformed with a recombinant vector expressing a small interfering RNA (siRNA) that inhibits the expression of the MDC gene can be prepared, and the bacterial cell can be used to treat asthma and rhinitis diseases. Induction can be suppressed. When a Salmonella BRD509 strain transduced with a recombinant vector expressing the siRNA (small interfering RNA) specific for the MDC gene is administered to mice induced with inflammation and asthma disease, one of the active factors of asthma disease is IL-4. Inhibition of expression, increased the expression of the inhibitory factor IFN-γ and at the same time it was confirmed that the effect of suppressing the immune response of inflammatory cells, which is an indicator of asthma disease. In addition, the inhibition of MDC gene induced the inhibition of MDC expression in the blood and the inhibition of ROS, an inflammation-inducing factor, and the relief of airway hyperresponsiveness, a symptom of asthma disease, in the asthma model. Therefore, the therapeutic composition using small interfering RNA (siRNA) having a therapeutic effect on asthma and rhinitis diseases can be very useful in the pharmaceutical industry as a treatment and prevention of asthma and rhinitis diseases in the absence of a suitable treatment for intractable diseases. .

1 is a diagram of a plasmid capable of expressing a gene for transcription of an siRNA of an MDC gene according to the present invention.
FIG. 2 is a diagram confirming that recombinant vector pcDNA-MDCmiRNA is expressed through GFP expression in a mouse macrophage cell line (raw cell). Control is PBS, miRCV is a miRNA-specific vector not inserted into the MDC gene, miR-MDC is a recombinant vector expressing the miRNA specific to the MDC gene.
Figure 3 is a measure of the change in inflammatory cytokines including MDC after administration of the Salmonella expressing MDC gene to the splenocytes isolated from the mouse by the composition according to the present invention. Cell lines administered with Salmonella (ST-miR-MDC) expressing siRNA that inhibits MDC induced inhibition of IL-4 in addition to inhibition of MDC and increased genes of IFN-γ, a Th1 cytokine.
4 is a diagram showing that the amount of MDC in the blood is suppressed 1 week after the administration of Salmonella (ST-miR-MDC) expressing siRNA that inhibits MDC according to the present invention.
5 is a week after administration of Salmonella (ST-miR-MDC) expressing the siRNA that suppresses MDC according to the present invention in an asthma mouse animal model is suppressed inflammatory cells (eosinophils: red arrow) of the lung to asthma The figure shows that it can be relaxed.
FIG. 6 shows that ROS expression is inhibited by one week after administration of Salmonella (ST-miR-MDC) expressing the siRNA that suppresses MDC according to the present invention in an asthma mouse animal model to relieve asthma. to be.
FIG. 7 is a diagram showing that airway hyperresponsiveness can be relieved 1 week after administration of Salmonella (ST-miR-MDC) expressing an siRNA that suppresses MDC according to the present invention in an asthma mouse animal model. .
8 is a result of serological analysis of asthma patient group. Analysis of the amount of MDC expression in asthma and control patients confirms that MDC expression is increased in asthma patients.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example  One. MDC  Gene-specific miRNA Confirmation of

In order to determine the target sequence complementary to miRNA in the MDC gene sequence (NCBI Accession No. NM_002990), the secondary structure, GC contents, cis-acting sequences, etc., which affect the efficiency of miRNA activity are determined. In consideration of the miRNA target nucleotide sequence by putting MDC gene sequence into an algorithm program that can design functional miRNA, miRNA oligonucleotide of 21-nucleotide was synthesized.

Each single strand oligo has a linker sequence (TGCT, CCTG), a sense strand and an anti-sense strand that can be linked to a plasmid vector based on the miRNA sequence. The top and bottom strands of each 64-nucleotide designed by inserting a loop nucleotide sequence (TTGGCCACTGACT) connecting the strands were synthesized by double strand oligos, and then pcDNA6.2 through T4 DNA ligase. Recombinant vector pcDNA-MDC (FIG. 1) was prepared by inserting between 5 ′ and 3 ′ miR flanking regions of the −GW / ± EmGFP-miR vector (INVITROGEN, USA). The synthesized top and bottom strand base sequences are shown below.

MDC TARGET SEQUENCE SEQ ID NO: 1: SENSE SEQUENCE
(Top strand) 5`-TGC TGT TAT GGA GTA GCT TCT TCA CCG TTT TGG CCA CTG ACT GAC GGT GAA GAC TAC TCC ATA A-3`, SEQ ID NO: 2: ANTISENSE SEQUENCE (Bottom strand) 5`-CCT GTT ATG GAG TAG TCT TCA CCG TCA GTC AGT GGC CAA AAC GGT GAA GAA GCT ACT CCA TAA C-3`

Expression of MDC-miRNA prepared as described above was transduced into RAW 264.7 Cells and the expression pattern was confirmed by GFP confirmation (Fig. 2).

Example  2. Built MDC  Gene specific miRNA  vector( vector ) Transformation

Salmonella strain BRD509 (J Infect Dis. 1994 Dec; 170 (6): 1508-), which is an attenuated Salmonella strain miRNA vector (pcDNA-MDCmiRNA) specific to the MDC gene constructed as in Example 1 17, Medeva, available from London) was transduced via electrophoration shock. Specific examples of the present invention are shown below.

First, the attenuated Salmonella strain BRD509 ( Salmonella typhimurium BRD509) were cultured at 37 占 폚 in Luria Bertani medium. The cultured Salmonella strains were transduced with mouse pcDNA-MDC miRNA vectors. More specifically, the Salmonella strain was cultivated again in an amount of 100 ml for 24 hours, and the optical absorbance (OD600) was measured at 600 nm. Thereafter, the bacteria were centrifuged at a rate of 5500 rpm at a culture concentration of 0.7. After centrifugation, only the bacteria sinking to the bottom were collected and diluted with cold distilled water of the same volume as the culture volume. The diluted bacteria were again centrifuged in the same manner as described above, diluted with 2 ml of cold 20% glycerol distilled water, and centrifuged again. Thereafter, the obtained bacteria were diluted with 100 [mu] l of 20% glycerol. 30 μl (2 μg) of the mixed solution obtained by the above procedure was placed in a minicentrifuge container, and then 3 μl (2 μg) of pcDNA-MDCmiRNA vector was added to the vessel, followed by induction of transduction at 275-325V. It was.

Example  3. In animal cells MDC  Measurement of inhibitory effect

5-week-old ICR mice were divided into two groups, control and experimental groups. Five groups of mice were injected with 100 μl of 1% DNCB, an inflammation inducing substance, for 1 week to induce inflammation in the mice. Then, the mouse was dissected and the spleen tissue was extracted and then separated into single cells using a homogenizer in 10 ml of PBS solution. Separated spleen cells were cultured in RPMI 1640 medium (100 units / ml penicillin, 100 mg / ml streptomycin and 10% FBS) by RBC lysing buffer (Sigma USA). 5.0 x 10 ⁷ cells were cultured with Lectin (10 μg / ml, Sigma; USA) and IL-4 (20 ng / ml, Invitrogen, Carlsbad, Calif.), Followed by Salmonella (ST) expressing siRNA that inhibits MDC. -miR-MDC) was treated to cells at the level of 100MOI to measure the expression of MDC, IL-4, IFN-gamma using RT-PCR (Fig. 3). Each used probe was used according to the usual technique.

Example  4. In vivo ( in vivo ) Through experiment MDC  Measurement of inhibitory effect

5-week-old ICR mice were divided into two groups of control and experimental groups, and each mouse was coated with 100 μl of 1% DNCB as an inflammation inducer for 1 week. Subsequently, Salmonella (ST-miR-MDC) expressing siRNA that inhibits MDC was administered orally to the experimental rats at a concentration of 1.6 × 10 ⁸ . At one week after the administration, ELISA experiments were performed to determine the serum concentrations of MDC by extracting the serum of each rat using orbital sampling.

As a result, as shown in Figure 4, the blood expression level of MDC was reduced in the experimental group administered with Salmonella (ST-miR-MDC) expressing the siRNA that inhibits MDC.

Example  5. Measurement of asthma treatment effect through asthma model

Ovalbumin treatment in mice induced asthma, an allergic inflammatory response, and oral administration of Salmonella (ST-miR-MDC), which expresses siRNA that suppresses MDC, and changes in the total number of cells and eosinophils in bronchoalveolar lavage fluid (FIG. 5), the change in the production of cytoactive substances in the splenocytes and lung tissue (Fig. 6) was measured. In addition, airway hypersensitivity (Penh) was analyzed for methacholine stimulation using a brometric plethysmographic chamber (FIG. 7).

Example  6. Through serum analysis of clinical patients MDC Measurement of

About 20 asthma patients at Anam Hospital of Korea University were analyzed and the expression of MDC was analyzed by ELISA for blood serum samples. Test test was analyzed using MDC ELISA KIT of R & D SYSTEM (Fig. 8).

Although specific portions of the present invention have been described in detail, those skilled in the art will appreciate that these specific embodiments are merely examples of preferred embodiments and that the scope of the present invention is not limited thereto. It is therefore intended that the scope of the present invention be defined by the appended claims and their equivalents.

<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> siRNA against MDC for treatment of asthma and rhinitis, and          composition of a therapeutic agent comprising the same <130> P11-111215-01 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 64 <212> DNA <213> Homo sapiens <400> 1 tgctgttatg gagtagcttc ttcaccgttt tggccactga ctgacggtga agactactcc 60 ataa 64 <210> 2 <211> 64 <212> DNA <213> Homo sapiens <400> 2 cctgttatgg agtagtcttc accgtcagtc agtggccaaa acggtgaaga agctactcca 60 taac 64

Claims (6)

Composition for the treatment of asthma and rhinitis comprising siRNA specific to the target sequence of the macrophage-derived chemokine (MDC) gene as an active ingredient. According to claim 1, wherein the siRNA is siRNA having a nucleotide sequence of SEQ ID NO: 2 composition for the treatment of asthma and rhinitis. A composition for the treatment of asthma and rhinitis, comprising bacterial cells transformed with a recombinant vector comprising a gene for transcription of a small interfering RNA (siRNA) that inhibits the expression of a macrophage-derived chemokine (MDC) gene. According to claim 3, wherein the bacterial cells are Salmonella typhimurium (salmonella typhimurium), characterized in that the composition for the treatment of asthma and rhinitis. According to claim 3 or 4, wherein the siRNA is siRNA having a nucleotide sequence of SEQ ID NO: 2 composition for the treatment of asthma and rhinitis. Kit for diagnosing asthma and rhinitis comprising an antibody or aptamer specifically binding to macrophage-derived chemokine (MDC) or fragments thereof.

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