KR20130105545A - A USE OF RORα FOR CONTROLLING ACTIVITY OF TH17 CELL - Google Patents

Abstract

PURPOSE: An ROR alpha is provided to suppress the activation of Th17 cells and to regulate immune response, thereby effectively treating and preventing various allergic diseases or autoimmune diseases. CONSTITUTION: A method for reducing the activation of Th17 cells comprises the step of promoting the expression or activation of ROR alpha by transducing a recombinant expression vector containing ROR alpha genes into Th17 cells. ROR alpha gene has a sequence of sequence number 11 or 12. The recombinant expression vector contains a promoter or an enhancer which promotes the expression or activation of ROR alpha. A composition for preventing and treating Th17 cell activation-related diseases contains an agent for promoting the expression or activation of ROR alpha; or Th17 cells in which the agent is transduced, as an active ingredient.

Description

A Use of RORα for Controlling Activity of Th17 Cell}

The present invention relates to the interaction between RORα (retinoic acid binding receptor α) -Th17 (T helper type 17) and its use. More specifically, the method of immune regulation using the interaction between RORα-Th17 cells and related molecular mechanisms And it relates to the development of cell therapy using the same.

The immune system can be largely divided into innate immunity and acquired immunity. Acquired immunity is an immune system that exists only in vertebrates, in which several cells are intertwined like a network, and antigen presenting cells, B cells, and T cells are involved. Effector cells include B cells and T cells, which are involved in humoral immunity secreting antibodies, and T cells are mainly responsible for cellular immunity. Cells are classified into cytotoxic T cells and helper T cells.

Cytotoxic T cells protect the host by directly killing infected cells, and participate in host defense by mobilizing and regulating different types of cells, and helper T cells are epithelial cells that are involved in other lymphocytes, innate immunity, and non-immune cells. It also acts on cells to defend the host. One important concept of acquired immunity is diversity. Each T cell has a specific antigen receptor (T cell receptor, TCR) that responds specifically to a variety of pathogens. In order for helper T cells to differentiate, dendritic cells, which are antigen-providing cells, must find pathogens and move to lymph nodes to meet them. When the dendritic cells present the antigen, undifferentiated naive T cells are divided into several times and are sufficient to correspond to the pathogen. Because pathogens also have a variety of lifestyles and immune evasion mechanisms, dendritic cells provide T-cells with information about the pathogen's appearance and location through various mechanisms, such that the pathogen is intracellular, extracellular, bacterial, viral, or eukaryotic. Make sure you know what the creature is and what kind of tissue it is invading. Therefore, T cells specific for pathogens express genes according to the life cycle of the pathogen to be removed and determine which helper T cells are generated more and which cells are recruited according to the types of cytokines secreted.

The change in immune response by specific site by the action of T cells provides clues about whether certain antigens can cause various diseases, such as rhinitis, asthma and food allergies. The diversity can also be explained. For example, mucous production is Th2, Th17 cells, matrix production is Th2 cells, matrix removal is the immune response is different depending on the extent of the specific T cells involved, such as Th17 cells involved, the disease pattern may vary it means.

The most important signal for the differentiation of helper T cells is the cytokine. In T cell differentiation, cytokines induce or inhibit growth factors as well as transcription factors essential for differentiation into Th1 or Th2 cells, and are secreted from differentiated helper T cells themselves to cause growth and differentiation of other immune cells. It also serves as a medium. Representative cytokines secreted by Th1 cells are interferon-γ, and representative cytokines secreted by Th2 cells are IL-4, IL-5, IL-13. IL-17 is a representative cytokine of Th17 cells, a type of helper T cell recently discovered. Cytokines secreted by each helper T cell promote differentiation of the helper cell and inhibit the differentiation of another helper T cell.

Until recently, T-cells were classified as a dichotomy of Th1-Th2 cells. Recently, new evidence suggests that new allies exist, suggesting that new T-cells (Th17) could reverse the existing Th1-Th2 hypothesis. Cells have been discovered, and more and more evidence has been found to prove the existence of Th17 cells. The role of Th17 cells in autoimmunity and host cell defense has been elucidated, and the transcription factors of Th17 cells called RORγt have been revealed to prove the specificity of Th17 cells, and how helper T cells work and overreact. Many facts have been identified and much progress has been made in how immune responses occur at the cellular level through signaling and transcription.

By understanding the molecular mechanisms of the newly discovered Th17 cells, it is thought that they will be helpful in understanding and treating diseases caused by abnormalities in immune response including allergy. However, there are still many unknown molecular mechanisms such as the characteristics and regulation of T17 cells in relation to Th17 cells.

Therefore, the present inventors have confirmed the relationship between the expression of RORα related to the activity of Th17 cells and their molecular mechanisms. As a result, we can control the activity of Th17 cells through RORα expression or activity promotion. The present invention was completed by newly confirming the molecular mechanism between RORα-Treg-Th17 cells.

Republic of Korea Patent Application No. 2009-0001519

It is a main object of the present invention to provide a method of controlling the activity of Th17 cells through the regulation of RORα expression or activity. In particular, it provides a method and system for inhibiting the activity of Th17 cells by promoting the expression or activity of RORα.

Another object of the present invention to provide a composition for regulating Th17 cell immune activity, comprising RORα expression or activity promoter as an active ingredient.

Another object of the present invention to provide a method for regulating immunoregulatory T cells using a molecular mechanism between RORα-Treg-Th17 cells and a cell therapeutic agent using the same.

In order to solve the above object, the present invention provides a method for reducing the activity of Th17 cells by promoting the expression or activity of RORα.

At this time, the RORα expression or activity promotion may be performed by introducing a recombinant expression vector containing the RORα gene into Th17 cells. Preferably the recombinant expression vector comprises a promoter or enhancer that promotes the expression or activity of RORα. The RORα gene may be composed of a nucleic acid sequence represented by SEQ ID NO: 11 or an amino acid sequence represented by SEQ ID NO: 12.

In addition, promoting the activity of the RORα can be carried out in combination with promoting Treg cell activity.

 The present invention, in another aspect,

Promoters for the expression or activity of RORα; or

Provided are compositions for treating and preventing diseases related to the activity of Th17 cells, including Th17 cells into which an RORα expression or activity promoter has been introduced as an active ingredient.

The RORα expression or activity promoter is as described above. The composition may further comprise a Treg cell activity promoter.

In the present invention, diseases related to the activity of the target Th17 cells may be autoimmune diseases and allergic diseases, for example, inflammatory skin diseases including psoriasis and atopic dermatitis; Systemic scleroderma and sclerosis; Inflammatory bowel disease (IBD); Crohn's disease and ulcerative colitis; Surgical tissue reperfusion injury, myocardial ischemic diseases such as myocardial infarction, cardiac arrest, reperfusion after cardiac surgery, and stenosis after percutaneous transluminal coronary angioplasty, stroke Ischemic reperfusion disorders, including abdominal aortic aneurysms; Secondary cerebral edema following seizures; Cranial trauma, hypovolemic shock; asphyxia; Adult respiratory distress syndrome; Acute-lunginjury; Behcet's Disease; Dermatomyositis; Polymyositis; Multiple sclerosis (MS); Dermatitis; Meningitis; Encephalitis; Uveitis; Osteoarthritis; Lupus nephritis; Autoimmune diseases such as rheumatoid arthritis (RA), Sjorgen's syndrome, vasculitis; Diseases associated with leukocyte diapedesis; Multiple organ damage syndrome secondary to central nervous system (CNS) inflammatory disorder, sepicaemia or trauma; Alcoholic hepatitis; Bacterial pneumonia; Antigen-antibody complex mediated diseases, including glomerulonephritis; Sepsis; Sarcoidosis; Immunopathological response to tissue / organ transplantation; Pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis fibrosis: inflammation of the lung, including IPF) and cystic fibrosis; Psoriatic arthritis; At least one disease selected from the group consisting of optic nephritis, Guillain-Barré syndrome (GBS), COPD, and type 1 diabetes mellitus, characterized in that the disease is affected by Th17 activity due to inflammatory cytokines involved.

As such, the present invention can provide general information on the treatment or prevention of Th17 cell activity-related diseases by regulating the immune response of Th17 cells through RORα expression and activity regulation based on the Th17-RORα interaction mechanism. have.

The present invention relates to the use of RORα that can regulate the activity of Th17 cells and cell therapy using the same, by inhibiting the activity of Th17 cells by promoting the RORα expression and activity by controlling the immune response, various allergic diseases or autoimmunity Effective treatment and prevention of the disease will be possible.

1 is a diagram schematically showing the relationship between the types of various cytokines and T cells.
Figure 2 is the result of analyzing the mRNA amount of RORα, RORc, Foxp3 expressed on various T cells.
3 is a graph comparing RORc and RORα expression after introducing RORα siRNA into Th17 cells.
Figure 4 shows the results of observing the arthritis index over time after injection of RORα overexpressing recombinant vector and MOCK vector (control vector without RORα gene) in the arthritis mouse model.
Figure 5 shows the amount of each immunoglobulin contained by separating serum from blood obtained by performing orbital blood collection from the group injected with RORα overexpressing recombinant vector and MOCK vector (control vector without RORα gene) in the arthritis mouse model It shows the result of measuring.
6 is isolated splenocytes from the spleen of the group injected with RORα overexpressing recombinant vector and MOCK vector (control vector without RORα gene) in the arthritis mouse model, RNA was extracted, and cDNA was synthesized to synthesize RORα, RORγT, The expression levels of the IL-17 and Foxp3 genes were analyzed by PCR.
7 is a bone marrow-derived monocytes isolated from the group injected with RORα overexpressing recombinant vector and MOCK vector (control vector without RORα gene) in osteoarthritis mouse model and differentiated into osteoclasts, followed by TRAP staining under a microscope The results of analysis of osteoclast differentiation ability by examining the number of TRAP positive cells are shown.

The terms used in the present invention are defined as follows.

"Nucleic acid" is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role in protein coding or transcription or in the regulation of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

"Primer" refers to an oligonucleotide sequence that hybridizes to a complementary RNA or DNA-targeted polynucleotide and serves as a starting point for the stepwise synthesis of a polynucleotide from a mononucleotide by the action of, for example, a nucleotidyltransferase that occurs in the polymerase chain reaction .

"Coding region" or "coding sequence" refers to a nucleic acid sequence, a complement thereof, or a portion thereof, which encodes a particular gene product or fragment thereof that is required to be expressed, according to a common base pairing and codon usage relationship. Coding sequences include exons in genomic DNA or immature primary RNA transcripts that are joined together by a biochemical machinery of cells to provide mature mRNA. The antisense strand is a complement of the nucleic acid, and the coding sequence can be deduced therefrom. The coding sequence is placed in the relationship of transcriptional regulatory elements and translation initiation and termination codons such that transcripts of appropriate length are generated and translated in the appropriate reading frame to produce the desired functional product

"Transformation or transfection" refers to the process by which extracellular DNA enters a host cell in the presence or absence of an accompanying substance. "Transfected cell" refers to a cell in which extracellular DNA is introduced into a cell and contains extracellular DNA. DNA can be introduced into the cell and the nucleic acid can be inserted into the chromosome or replicated as an extrachromosomal material.

The term vector refers to any nucleic acid comprising a competent nucleotide sequence that is inserted into a host cell and recombined with, inserted into, or spontaneously replicates as a host. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and the like. In one embodiment of the present invention, a viral vector is used.

"Host cell" refers to a eukaryotic or prokaryotic cell into which one or more DNA or vectors are introduced, and is understood to refer not only to a particular target cell but also to its progeny or potential progeny. In fact, the offspring are not identical to the parent cell, but are still included within the scope of the term, as used herein, since certain modifications may occur in subsequent generations due to mutations or environmental influences.

A "polypeptide" is intended to include not only a single polypeptide, but also a plurality of polypeptides and includes a chain or chains of one or more amino acids joined to each other by peptide bonds. The term also includes polypeptides having post-translational modifications.

A "protein" is also intended to include fragments, analogs and derivatives of a protein that retain essentially the same biological activity or function as the reference protein.

"Promoter" means a substance that promotes, enhances or increases the expression or activity of a gene of interest. The activation mechanism of the promoter is not particularly limited. Examples include organic or inorganic compounds, proteins, carbohydrates, polymer compounds such as lipids, composites of various compounds. For example, the 'RORα promoter' may include a substance that promotes, enhances or increases the expression or activity of the RORα protein.

"Inhibitor or inhibitor" means a substance that inhibits, blocks or reduces the expression or activity of a gene of interest. The mechanism of activation of the inhibitor is not particularly limited. Examples include organic or inorganic compounds, proteins, carbohydrates, polymer compounds such as lipids, composites of various compounds.

"Antibody" is used in its broadest sense and specifically refers to intact monoclonal (monoclonal) antibodies, polyclonal antibodies, multispecific antibodies (eg bispecific antibodies) formed from at least two intact antibodies and Antibody fragments that exhibit the desired biological activity.

"RNAi" refers to RNA Interference, which means "interference in RNA". RNA interference is a specific gene suppression phenomenon that is well conserved among most organisms. It is thought to be a type of gene monitoring mechanism used by cells to defend against viral infections, to suppress transposons, or to remove abnormal mRNAs. In particular, gene suppression by small RNA is called RNA interference in a broad sense, and RNA interference in a narrow sense means mRNA degradation by siRNA. In addition, RNA interference may refer to a technique for inhibiting genes using siRNA.

'small RNA' refers to ribonucleic acid, about 17-25 nucleotides in length, that regulates gene expression in vivo. Small RNAs are classified into microRNA (abbreviated as miRNA) and small interfering RNA (abbreviated as siRNA) depending on how they are produced. miRNAs are generated partially from RNA (hairpin RNA), which is derived from long double-stranded RNA (dsRNA). In general terms, small RNA, which plays an important role in various regulation processes in vivo, is classified as microRNA, and small RNA, which is used to control the expression of a specific gene in an experimental technique, is classified as siRNA. miRNAs are naturally produced in cells and specifically inhibit the synthesis of proteins from mRNA by binding to specific mRNAs. siRNAs are small RNAs that are artificially introduced into cells and bind to specific mRNAs with complementary sequences to decompose the mRNAs.

As used herein, an "effective amount" is an appropriate amount that affects a beneficial or desirable clinical or biochemical result. An effective amount may be administered one or more times. For purposes of the present invention, an effective amount of an accelerator is an amount sufficient to temporarily alleviate, ameliorate, stabilize, reverse, slow down, or delay the progression of the relevant disease state.

If the recipient animal is capable of enduring the administration of the composition, or the administration of the composition to the animal is suitable, the composition will be "pharmaceutically or physiologically acceptable ". If the dose administered is physiologically significant, it can be said that the formulation is administered in a "therapeutically effective amount ". The formulation is physiologically relevant if the presence of the formulation results in a physiologically detectable change in the recipient.

"Treatment" means an approach to obtaining beneficial or desired clinical results.

For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (ie, not worsening), delay or slowing of disease progression, disease state Improvement or temporary mitigation and alleviation (partially or wholly), detectable or not detected.

Also, "treatment" may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented. "Palliating" a disease may reduce the extent of the disease state and / or undesirable clinical signs and / or slow or lengthen the time course of progression as compared to untreated treatment. It means losing.

"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to a correlation between RORα and Th17 cells.

The present inventors found that RORα overpromotes the activity of Treg cells and inhibits Th17 cell activity, thereby identifying for the first time that RORα is a transcription factor controlling Th17 cell activity.

RORα (retinoic acid binding receptor α) is a protein also called RAR-related orphan receptor alpha, and splicing variants such as RORα1 and RORα4 have been identified. RORα is a member of the orphanage family that has not yet been identified ligands (Giguere et al., 1994; Gold et al., 2003; Lau et al., 1994). Given that it functions as a potential modulator in the pathology of orphans, orphanage receptors can functionally interact with potential cancer development systems, such as, for example, the Wnt and PKC signaling pathways (Peifer and Polakis, 2000). . And this interaction can lead to changes in cancer development and cell adhesion (Polakis, 2000; van de Wetering et al., 2002). However, as compared to other classes of nuclear receptors, the function and associated signaling pathways of orphan nuclear receptor RORα have not been extensively studied.

Th17 cells were named because the T-cells induced by IL-23 secrete the cytokine IL-17, and recently, Th17 cells are known to secrete IL-22 in addition to IL-17. Subsequent studies have shown that IL-6 and TGF-β are important for the differentiation of Th17 cells in the maintenance of Th17 cells. Th17 cells are involved in various autoimmune diseases and are associated with arthritis, multiple sclerosis, psoriasis and inflammatory bowel disease. Like other immune cells, Th17 cells have a protective function in addition to disease induction.The protective functions of Th17 cells promote the movement of eosinophils in acute inflammatory reactions and increase the barrier function by helping epithelial cells grow and differentiate. There is this. Th17 cells interact with other T cells to affect non-immune cells, resulting in nonspecific immunizations such as apoptosis, mucus secretion, and remodeling, as well as immune regulation that secretes chemokines and inflammatory cytokines. It is involved in the action. Modulation of immune action refers to the regulation of immune responses to maintain homeostasis of certain organs as well as to defend against germs.

On the other hand, Foxp3 + regulatory T cells, or 'Tregs' are essential for the regulation of various immune responses in that they rapidly inhibit the activity of other immune cells. In particular, Tregs are important for maintaining resistance by inhibiting unwanted immune responses to autologous and non-autologous antigens, which constitute about 2-10% of CD4 + T cells in humans and rodents, and CD25, CTLA- 4 and GITR, as well as the transcription factor Foxp3 associated with their development and function are constantly expressed. RORα is highly expressed in Tregs expressing Foxp3, but is very low in human Th17, Th1, and Th2 cells.

The present invention is based on the discovery of a mechanism by which Th17 cell activity is regulated depending on the expression of RORα. Specifically, (i) when RORα is overexpressed (promoted), Treg cell activity is amplified to increase Th17 cell activity. Inhibition of immunomodulation and (ii) inhibition of RORα amplified RORc, an important transcription factor that induces Th17 cell activity, and identified a molecular mechanism that further promotes Th17 cell activity.

And, using this molecular mechanism, the present inventors conceived the use of regulating the immune response of T cells by regulating the activity of Th17 cells by controlling the expression or activity of RORα.

In particular, in the treatment of diseases associated with the expression of Th17 cells, it can be said that the use of the immunomodulatory method of the present invention can provide an effective cell therapy because it has greater utility. For example, cell therapy using the regulation of RORα and Treg cells can be utilized.

The present invention extends to a genetic approach to upregulation or downregulation of expression of genes associated with expression of RORα.

In general, the general techniques used to upregulate expression may use methods to increase gene duplication or antagonize inhibitors of gene expression, and downregulating expression may result in gene silencing such as before or after transcription. Genetic means to induce silencing can be used.

In one aspect, the present invention relates to a method for inhibiting Th17 cell activity and amplification of Treg cell activity through RORα expression or activity promotion and a system using the same.

In order to promote the expression or activity of RORα, it is preferable to use one selected from the group consisting of a substance that promotes transcription of the RORα gene, a substance that promotes translation of the transcribed RORα, or a substance that promotes the function of the RORα protein. It doesn't work.

As a specific example, a recombinant polynucleotide construct (collectively referred to as an "expression vector"), such as a recombinant expression vector containing an RORα gene, an expression promoter or activity promoter, for example, the RORα gene, a microorganism transformed with the expression vector. And Th17 cell activity regulation system, which can be implemented as an aspect of a composition for treating a Th17 cell activity-related disease comprising one of these.

To overexpress RORα, a suitable promoter can be operably linked to the DNA / polynucleotide encoding it, for example, promoters for expression in mammalian cell systems include viral promoters such as polyomas, poultry (fowlpox) and adenoviruses (e.g. adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (especially immediate early gene promoter), retrovirus, hepatitis B virus RNA polymerase II promoters, including actin, rous sarcoma virus (RSV) promoter and early or late Simian virus 40 and nonviral promoters such as EF-1alpha. (Mizushima and Nagata Nucleic Acids Res 1990 18 (17): 5322). The selection of such promoters can be made on the basis of appropriate compatibility with the host cell used for expression.

In addition, if desired, additional 'enhancer elements' may be included instead of or in addition to the elements present at the promoter positions described above to promote expression or activity of RORα and amplify the activity of Treg cells.

In addition, Th17 activity can be controlled by amplifying the activity of these Treg cells. In the regulation of Th17 cell activity of the present invention, it may be performed in combination with promoting Treg cell activity. That is, Th17 cells can be more effectively controlled by using a method applicable in the art that promotes the expression or activity of RORα and at the same time promotes the activity of Treg cells.

Further, in one embodiment of the present invention, according to such a mechanism, a vector encoded so that RORα is overexpressed and thus transformed cells can be provided.

On the other hand, in another aspect, the present invention provides a method for promoting the amplification of RORc and the activity of Th17 through the inhibition or expression of RORα.

As a specific example, recombinant polynucleotide constructs (collectively referred to as "expression vectors"), such as recombinant expression vectors containing expression or activity inhibitors (eg siRNA, shRNA, etc.) of the RORα gene, are transfected with the expression vector. Th17 cell activity regulation system, which is feasible as an aspect of a converted microorganism, a composition for treating ischemic disease comprising one of them.

Likewise, in order to inhibit the expression or activity of RORα, it is preferable to use one selected from the group consisting of a substance that inhibits the transcription of the RORα gene, a substance that inhibits the translation of the transcribed RORα, or a substance that inhibits the function of the RORα protein. It is not limited.

In the present invention, as a substance that can be used to inhibit the expression or activity of the RORα gene, it will be described in detail as follows:

"RNA interference" is a post-transcriptional gene silencing mechanism in which degradation of the corresponding mRNA occurs by introducing two stranded chain RNAs (dsRNA) corresponding to the gene into a cell or organism. The construction may comprise modified ribonnucleotide residues, as oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or analogs thereof. RNAi includes both single-stranded and double-stranded nucleic acid molecules. Examples for RNAi types are, for example, siRNA or miRNA (microRNA) or small hairpin RNA (shRNA). In one embodiment of the present invention was used RORα siRNA.

Alternatively, a nucleic acid molecule that is "antisense" to a nucleic acid encoding RORα can be used as an inhibitor. Antisense nucleic acids include nucleic acid sequences that are complementary to the 'sense' nucleic acid encoding the gene, eg, complementary to the coding strand of a two stranded chain cDNA molecule or complementary to the mRNA sequence. If desired, the antisense nucleic acid can be produced biologically using an expression vector.

The term “antibody” is used in its broadest sense and is specifically a monoclonal antibody, polyclonal antibody, multispecific antibody (eg, bispecific antibody) formed from at least two intact antibodies, and desired As long as it exhibits biological activity, antibody fragments are included. Antibodies can be modified to increase stability and / or half-life.

Alternatively, peptide mimetics that inhibit the protein binding domain of the RORα polypeptide can be prepared to inhibit the original RORα polypeptide from binding to Th17.

In the present invention, as a method of introducing a RORα expression or activity promoter (for example, promoter, enhancer, etc.) or RORα expression or activity inhibitor (siRNA, shRNA, RNAi, antisense, antibody, etc.) described above in Th17 cells, Gene delivery systems known in the art may be useful in the performance of genetic engineering. These include virus and non-viral delivery methods.

To introduce genetic material that modulates RORα expression or activity in Th17 cells, DNA-calcium precipitation, liposomes, polyamines, electroporation, retroviruses, adenoviruses Intracellular transduction technology methods known in the art such as using a method may be used.

In addition, the nucleic acid encoding the RORα expression or activity promoter and the like can be prepared by a gene recombination method known in the art. For example, there are techniques for PCR amplification, chemical synthesis, or cDNA sequence production to amplify nucleic acid from a genome. In addition, a nucleic acid encoding the RORα expression or activity promoter may be operably linked to an expression control sequence and inserted into an expression vector. 'Operably linked' refers to the binding of one nucleic acid fragment to another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment. And Th17 cells according to the present invention can be proliferated and cultured according to methods known in the art.

On the other hand, the present invention provides a use for the recovery of Th17-activity-related diseases, using the association with RORα-Th17 cells, the association with Treg-Th17 cells, the association with RORα-Treg cells in another aspect. In particular, it provides therapeutic use for Th17-activity related allergic diseases or autoimmune diseases.

As the therapeutic use, the present invention provides a composition for the treatment and prevention of diseases related to the activity of Th17, which contains, for example, the expression or activity promoter of RORα. Most preferably, the present invention relates to a Th17-activity-related allergic disease or an autoimmune disease treatment composition or a cell therapeutic agent containing Th1 cells into which RORα expression or activity promoter has been introduced as an active ingredient.

Amplifying the activity of Treg cells through promoting expression or activity of RORα; Or by promoting the expression or activity of RORα and activating Treg cells, ultimately reducing Th17-activity and further affecting the secretion of related cytokines resulting in the treatment of Th17-activity related allergic or autoimmune diseases. will be.

Such a therapeutic composition or cell therapy agent of the present invention may be prepared in a suitable formulation including an acceptable carrier depending on the mode of administration. Formulations suitable for the mode of administration are known and may comprise agents that facilitate movement, typically through the membrane.

In addition, the therapeutic composition of the present invention can be used in the form of a general pharmaceutical formulation. Parenteral preparations may be prepared in the form of sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions or lyophilized preparations, oral administration in the form of tablets, troches, capsules, elixirs, suspensions, syrups or wafers. It can be prepared in unit dosage ampoules or in multiple dosage forms. In addition, the therapeutic composition of the present invention may be administered with a pharmaceutically acceptable carrier. For example, in the case of oral administration, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a coloring agent, or a flavoring agent may be used, and in the case of an injection, a buffer, a preservative, a painless agent, a soluble agent may be used. Topical agents, isotonic agents, stabilizers and the like can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used.

In addition, the method for treating Th17-activity related allergic disease or autoimmune disease using the therapeutic composition of the present invention may include administering through a general route of introducing a predetermined substance to the patient in an appropriate manner. The administration method may include, but is not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, and rectal administration. However, upon oral administration, the cells may be digested so that the oral composition is preferably formulated to coat the active agent or protect it from degradation in the stomach.

In addition, the pharmaceutical composition may be administered by any device capable of transferring the active substance to the target cell. Preferred modes of administration and preparations are intravenous, subcutaneous, intradermal, intramuscular or injectable. Injections include non-aqueous solvents such as aqueous solvents such as physiological saline solution or ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, or glycerin). Stabilizers (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, to prevent microbial development Pharmaceutical carriers such as preservatives (eg, mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, and the like). Preferably, the method for treating brain or nervous system diseases using the therapeutic composition of the present invention comprises administering a therapeutically effective amount of the therapeutic composition of the present invention. The pharmaceutically effective amount is well known in the medical field such as the type of disease, the age, weight, health, sex of the patient, sensitivity to the drug of the patient, the route of administration, the method of administration, the frequency of administration, the duration of treatment, the combination or drug used It can be easily determined by those skilled in the art depending on the factors.

In the present invention, the disease to be treated is a disease related to the activity of Th17, for example, a Th17-activity related allergy disease or an autoimmune disease.

In particular, inflammatory cytokines such as IL-6, IL-8, IL-17, etc. are involved in relation to the Th17-activity, and some studies have been conducted on the association between Th17 and allergic diseases. In addition, an increase in IL-17 mRNA or protein in the lung, sputum, or bronchoalveolar lavage fluid (BALF) of asthma patients was observed, and IL-17 was reported to be associated with bronchial hypersensitivity in asthma patients. It is known that it is associated with the pathogenesis of asthma. In addition, IL-17 is an IL-6, IL-8, IL-11, CXCL1 / Groα in bronchial fibroblast, β-defensin-2, ICAM-1, IL-8, CXCL1, CCL20, G-CSF, MUC5B, MUC5AC and smooth muscle cells activate the cells by increasing the secretion of IL-6 and IL-8

Allergic diseases and autoimmune diseases targeted in the present invention are closely related to these cytokines and include, for example, inflammatory skin diseases including psoriasis and atopic dermatitis; Systemic scleroderma and sclerosis; Inflammatory bowel disease (IBD); Crohn's disease and ulcerative colitis; Surgical tissue reperfusion injury, myocardial ischemic diseases such as myocardial infarction, cardiac arrest, reperfusion after cardiac surgery, and stenosis after percutaneous transluminal coronary angioplasty, stroke Ischemic reperfusion disorders, including abdominal aortic aneurysms; Secondary cerebral edema following seizures; Cranial trauma, hypovolemic shock; asphyxia; Adult respiratory distress syndrome; Acute-lunginjury; Behcet's Disease; Dermatomyositis; Polymyositis; Multiple sclerosis (MS); Dermatitis; Meningitis; Encephalitis; Uveitis; Osteoarthritis; Lupus nephritis; Autoimmune diseases such as rheumatoid arthritis (RA), Sjorgen's syndrome, vasculitis; Diseases associated with leukocyte diapedesis; Multiple organ damage syndrome secondary to central nervous system (CNS) inflammatory disorder, sepicaemia or trauma; Alcoholic hepatitis; Bacterial pneumonia; Antigen-antibody complex mediated diseases, including glomerulonephritis; Sepsis; Sarcoidosis; Immunopathological response to tissue / organ transplantation; Pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis fibrosis: inflammation of the lung, including IPF) and cystic fibrosis; Psoriatic arthritis; Optic neuromyelitis, Guillain-Barré syndrome (GBS), COPD, type 1 diabetes and the like.

< Example >

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

The techniques and procedures used in the experiments are generally well understood by those skilled in the art and can be carried out according to conventional methods, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, the process involving the use of commercially available kits and reagents is generally performed according to protocols and / or parameters defined by the manufacturer, unless otherwise noted.

Cell culture

CD4 T cells 1 × 10 ⁶ from Peripheral blood mononuclear cells (PBMC) isolated from human peripheral blood were dispensed into 24-well plates coated with 1 μg / ml of anti-CD3 antibody, and anti-CD28 antibodies. Stimulated at 1 ug / ml. Conditions for stimulating Th17 cells were stimulated with IL-6 20ng / ml, IL-1 20ng / ml, IL-23 20ng / ml, anti-IL-4 10μg / ml, anti-IFNr 10μg / ml, and Th1 In order to stimulate the cells, IL-12 10ng / ml and anti-IL-4 10μg / ml were added, and for differentiation of Th2 cells, IL-4 10ng / ml and anti-IFNr 10μg / ml were added and incubated for 3 days. .

< Example  1>

In T cells ROR Expression of genes such as α

mRNA expression of RORα was investigated in human Th17, Th1 and Th2 cells.

RNAs were extracted from the cells according to methods known in the art to obtain cDNA products. The polymerase chain reaction was performed using primers shown below to amplify RORα, Foxp3, RORrt, and ß-actin as a control using the cDNA product thus obtained. The reaction compound for RT-PCR was brought to a total of 25 μl and the composition of the solution was used for each primer pair having 2.5 μl of 10 × reaction buffer 0.5 mM dNTP and the sequence described below. For the amplification, the reaction was repeated 25 times for 94 ° C. 30 seconds in the denaturation step, 60 ° C. 30 seconds in the annealing step, and 72 ° C. 30 seconds in the extension step, using a dual-bay thermal cycler system.

RORc

Sense: AGT CGG AAG GCA AGA TCA GA (SEQ ID NO: 1)

Anti-sense: CAA GAG AGG TTC TGG GCA AG (SEQ ID NO: 2)

Foxp3

Sense: CAC TGC CCC TAG TCA TGG T (SEQ ID NO: 3)

Anti-sense: GGA GGA GTG CCT GTA AGT GG (SEQ ID NO: 4)

RORα

Sense: AAA TCG CAT CTG GAA ACC TG (SEQ ID NO: 5)

Anti-sense: TTG GCA AAC TCC ACC ACA TA (SEQ ID NO: 6)

β-actin

Sense: GGA CTT CGA GCA AGA GAT GG (SEQ ID NO: 7)

Anti-sense: TGT GTT GGC GTA CAG GTC TTT G (SEQ ID NO: 8)

As a result, as shown in Figure 2, RORα expression was very low in human Th17 and Th1, Th2 cells, while it was confirmed that the expression of RORα is very high in Trep expressing Foxp3.

Controlled T lymphocytes, or Treg (regulatory T lymphocytes) cells, are known to play a key role in the maintenance of immune tolerance (Nat. Rev. Immunol. 2009, 7: 305) and are produced primarily in the thymus and are transcription factors Expression of Foxp3 is required and Foxp3 is known to play a role in inhibiting the production of inflammatory cytokines.

From the mRNA analysis results, it was found that promoting the expression of RORα amplifies the activity of Treg cells, whereas the activity of Th17 cells is inhibited.

< Example  2>

Th17 Cell and ROR expression regulation mechanism of α

In consideration of the results of Example 1, in order to identify the mechanism of expression regulation of RORα related to Th17 activity, the present inventors were injected into Th17 cells using siRNA against RORα that can inhibit the expression of RORα. RORα siRNA was purchased from Dharmacon RNAi Techologies.

The results are shown in Fig.

Inhibiting RORα expression by introducing siRNA of RORα into Th17 cells confirmed that amplification of RORc, an important transcription factor inducing Th17 cell activity, was confirmed. That is, RORc amplification suggests that Th17 cell activity, which is an inflammatory cell, is further promoted. Through these results, it was confirmed that RORα is a transcription factor that controls the activity of Th17 cells.

< Example  3>

mouse ROR α and human RORc Creation of overexpression vectors

In order to reevaluate that RORa is an important transcription factor for Th17 cell activity control, a recombinant expression vector capable of expressing mouse RORα and human RORα genes was constructed. The nucleic acid sequence and protein sequence of the mouse RORα gene used at this time are shown in SEQ ID NOs: 9 and 10, respectively, and the nucleic acid sequence and protein sequence of the human RORα gene are shown in SEQ ID NOs: 11 and 12, respectively. At this time, a MOCK vector to which the RORa gene was not introduced was used as a control.

Each of the recombinant overexpression vectors prepared above was transduced with a selected plasmid in the NIH3T3 cell line and confirmed the expression amount of each of RORα in the transduced cells. As a result, all of the recombinant vectors were well prepared so that the protein of RORα was overexpressed. It could be confirmed.

< Example  4>

Target mouse model ROR Analysis of Arthritis Treatment Effect

The inventors of the present invention Mock prepared in Example 3 using a hydrodynamic method 8 days after inducing arthritis in mice to determine whether RORα is effective in treating arthritis, one of immune diseases Vector or RORα overexpression vector (mouse RORα transduction vector) were injected intravenously, and 8 days after vector injection through injection, Mock vector or RORα overexpression vector was added to each mouse foot by electroporation. Injected. The symptoms of arthritis were then observed.

As a result, as shown in FIG. 4, the arthritis index was decreased in the group injected with the RORα overexpression vector compared to the Mock vector injection group.

Therefore, these results showed that RORα has the effect of preventing and treating arthritis.

< Example  5>

ROR Analysis of the Regulatory Effect of α on Immune Responses

In the mouse model tested in Example 4, blood was obtained from each mouse group through orbital blood collection, and serum was separated to measure the amount of IgG, IgG1, and IgG2a.

As a result, as shown in Fig. 5, in the group injected with the RORα overexpression vector compared to the group injected with the mock vector, the amount of each immunoglobulin (total IgG, IgG1, IgG2) was reduced in the arthritis animal model. Appeared.

Therefore, through these results, it can be seen that the RORα of the present invention has an immunomodulatory function that suppresses an excessive immune response.

< Example  6>

In an arthritis animal model ROR Analysis of the Regulatory Effect of Immune-related Genes by Overexpression of α

After inducing arthritis in mice as in Example 3, three weeks later, the spleen was extracted at the expense of the Mock vector injection group and the RORα overexpression vector injection group mice, and the splenocytes isolated from the spleen were treated with RNA to treat TriZol. After separation, cDNA was synthesized using this as a sample. Thereafter, the expression of the RORα, RORγT, IL-17 and Foxp3 genes was confirmed by real-time PCR using the synthesized cDNA.

As a result, the expression of RORα was slightly increased in splenocytes injected with the RORα overexpression vector in a mouse model of arthritis, whereas RORγT, a factor related to Th17 cell development, was slightly decreased. In addition, the expression of inflammatory cytokine IL-17 was decreased in the arthritis mice group injected with RORα overexpression vector, and the expression of Foxp3, a factor expressed in immunoregulatory T cells, was increased (see FIG. 6).

< Example  7>

In an arthritis animal model ROR Analysis on the Inhibitory Effect of Osteoclast Differentiation by Overexpression of α

Arthritis induced mice were sacrificed three weeks after arthritis-induced mice, respectively, and mice were isolated from bone marrow-derived monocytes. These cells were cultured for 3 days after treatment with M-CSF to induce osteoclast precursor production. After 3 days, the medium of the generated osteoclast precursors was exchanged, and then cultured for 3 days by stimulating M-CSF and RANKL. After the osteoclasts were differentiated with 10% formaldehyde, TRAP staining was performed, and the degree of osteoclast differentiation was analyzed by microscopic observation.

As a result, as shown in Figure 7, under the same RANKL stimulation concentration compared to the Mock vector injection group, the RORα overexpression vector injection group, it was confirmed that the differentiation ability of osteoclasts causing joint destruction was significantly reduced.

Through these results, the present inventors can control the activity of Th17 cells through the action of RORα inhibiting the production of inflammatory cytokines, but promoting the activity of Treg cells, which are immunoregulatory T cells, thereby preventing the immune diseases associated with Th17 cells. It can be seen that it can be used as a therapeutic agent.

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> A Use of ROR alpha for Controlling Activity of Th17 Cell <130> pn1303-092 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RORc sense primer <400> 1 agtcggaagg caagatcaga 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RORc anti-sense primer <400> 2 caagagaggt tctgggcaag 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Foxp3 sense primer <400> 3 cactgcccct agtcatggt 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> Foxp3 anti-sense sequence <400> 4 ggaggagtgc ctgtaagtgg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ROR alpha sense primer <400> 5 aaatcgcatc tggaaacctg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ROR alpha anti-sense primer <400> 6 ttggcaaact ccaccacata 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta actin sense primer <400> 7 ggacttcgag caagagatgg 20 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> beta actin anti-sense primer <400> 8 tgtgttggcg tacaggtctt tg 22 <210> 9 <211> 1608 <212> DNA <213> Artificial Sequence <220> <223> mouse ROR-alpha DNA sequence <400> 9 aagcttgaat tcgcccggat ggagtcagct ccggcagccc ccgaccccgc cgccagcgaa 60 ccgggcagca gcggctcgga ggcggccgcg ggctccaggg agaccccgct gacccaggac 120 acgggccgca agagcgaggc accgggcgcg gggcgcaggc agagctatgc gagctccagc 180 cgaggtatct cagtcacgaa gaagacacac acatctcaaa ttgaaattat tccatgcaag 240 atctgtggag acaaatcgtc aggaatccat tatggtgtca ttacgtgtga aggctgcaag 300 ggctttttca ggagaagtca gcagagcaat gccacctact cctgtcctcg tcagaagaac 360 tgtttgattg atcggaccag cagaaaccgc tgccagcatt gtcggctgca gaaatgcctg 420 gccgtgggga tgtcccgaga tgctgtcaag tttggtcgga tgtccaagaa gcagagagac 480 agcttgtacg ccgaggtgca gaagcaccgg atgcagcagc agcagcgaga ccaccagcag 540 cagcctgggg aggcggagcc gctgacgccc acctacaaca tctcagccaa tgggctgacg 600 gaactgcatg atgacctcag cacctatatg gatgggcaca cccccgaggg cagcaaggcc 660 gactcagccg tcagcagctt ctacctggac atccagccct ccccagacca gtcgggattg 720 gacatcaatg ggatcaaacc cgaacccata tgtgactaca caccagcatc tggcttcttc 780 ccctactgtt ccttcaccaa cggagagact tccccaaccg tgtccatggc agaactagaa 840 caccttgccc agaacatatc caaatcccac ctggaaacct gccagtactt gcgggaagag 900 ctccagcaga taacgtggca gaccttcctg caggaggaga ttgaaaacta ccagaacaag 960 cagagagagg tgatgtggca gctgtgtgcc atcaagatta cagaagctat ccagtatgtg 1020 gtggagtttg ccaaacgcat tgatggattt atggagctgt gtcaaaatga tcaaattgtg 1080 cttctaaaag caggctcgct agaggtggtg tttattagga tgtgccgtgc ctttgactct 1140 cagaacaaca ccgtgtactt tgacgggaag tatgcgagcc ccgatgtctt caagtcccta 1200 ggttgtgaag acttcatcag ctttgtgttt gaatttggga agagtttgtg ttctatgcac 1260 ctgaccgaag acgaaatcgc gttattttct gcattcgtac tgatgtcagc ggatcgctcg 1320 tggcttcagg aaaaggtaaa aatagaaaag ctgcaacaga aaattcagct ggcccttcag 1380 cacgtcctac agaagaacca ccgagaagat ggcattctaa ccaagctaat atgcaaggtg 1440 tctacgttaa gagccctatg tggacgacat acggaaaagc taatggcatt taaagcaata 1500 tacccagaca ttgtgcgact ccattttcct ccattataca aggaattgtt cacttcagaa 1560 tttgagccag ccatgcaaat cgatgggtaa gagccttcta gactcgag 1608 <210> 10 <211> 535 <212> PRT <213> Artificial Sequence <220> Mouse ROR-alpha amino acids sequence <400> 10 Lys Leu Glu Phe Ala Arg Met Glu Ser Ala Pro Ala Ala Pro Asp Pro   1 5 10 15 Ala Ala Ser Glu Pro Gly Ser Ser Gly Ser Glu Ala Ala Ala Gly Ser              20 25 30 Arg Glu Thr Pro Leu Thr Gln Asp Thr Gly Arg Lys Ser Glu Ala Pro          35 40 45 Gly Ala Gly Arg Arg Gln Ser Tyr Ala Ser Ser Ser Arg Gly Ile Ser      50 55 60 Val Thr Lys Lys Thr His Thr Ser Gln Ile Glu Ile Ile Pro Cys Lys  65 70 75 80 Ile Cys Gly Asp Lys Ser Ser Gly Ile His Tyr Gly Val Ile Thr Cys                  85 90 95 Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Gln Gln Ser Asn Ala Thr             100 105 110 Tyr Ser Cys Pro Arg Gln Lys Asn Cys Leu Ile Asp Arg Thr Ser Arg         115 120 125 Asn Arg Cys Gln His Cys Arg Leu Gln Lys Cys Leu Ala Val Gly Met     130 135 140 Ser Arg Asp Ala Val Lys Phe Gly Arg Met Ser Lys Lys Gln Arg Asp 145 150 155 160 Ser Leu Tyr Ala Glu Val Gln Lys His Arg Met Gln Gln Gln Gln Arg                 165 170 175 Asp His Gln Gln Gln Pro Gly Glu Ala Glu Pro Leu Thr Pro Thr Tyr             180 185 190 Asn Ile Ser Ala Asn Gly Leu Thr Glu Leu His Asp Asp Leu Ser Thr         195 200 205 Tyr Met Asp Gly His Thr Pro Glu Gly Ser Lys Ala Asp Ser Ala Val     210 215 220 Ser Ser Phe Tyr Leu Asp Ile Gln Pro Ser Pro Asp Gln Ser Gly Leu 225 230 235 240 Asp Ile Asn Gly Ile Lys Pro Glu Pro Ile Cys Asp Tyr Thr Pro Ala                 245 250 255 Ser Gly Phe Phe Pro Tyr Cys Ser Phe Thr Asn Gly Glu Thr Ser Pro             260 265 270 Thr Val Ser Met Ala Glu Leu Glu His Leu Ala Gln Asn Ile Ser Lys         275 280 285 Ser His Leu Glu Thr Cys Gln Tyr Leu Arg Glu Glu Leu Gln Gln Ile     290 295 300 Thr Trp Gln Thr Phe Leu Gln Glu Glu Ile Glu Asn Tyr Gln Asn Lys 305 310 315 320 Gln Arg Glu Val Met Trp Gln Leu Cys Ala Ile Lys Ile Thr Glu Ala                 325 330 335 Ile Gln Tyr Val Val Glu Phe Ala Lys Arg Ile Asp Gly Phe Met Glu             340 345 350 Leu Cys Gln Asn Asp Gln Ile Val Leu Leu Lys Ala Gly Ser Leu Glu         355 360 365 Val Val Phe Ile Arg Met Cys Arg Ala Phe Asp Ser Gln Asn Asn Thr     370 375 380 Val Tyr Phe Asp Gly Lys Tyr Ala Ser Pro Asp Val Phe Lys Ser Leu 385 390 395 400 Gly Cys Glu Asp Phe Ile Ser Phe Val Phe Glu Phe Gly Lys Ser Leu                 405 410 415 Cys Ser Met His Leu Thr Glu Asp Glu Ile Ala Leu Phe Ser Ala Phe             420 425 430 Val Leu Met Ser Ala Asp Arg Ser Trp Leu Gln Glu Lys Val Lys Ile         435 440 445 Glu Lys Leu Gln Gln Lys Ile Gln Leu Ala Leu Gln His Val Leu Gln     450 455 460 Lys Asn His Arg Glu Asp Gly Ile Leu Thr Lys Leu Ile Cys Lys Val 465 470 475 480 Ser Thr Leu Arg Ala Leu Cys Gly Arg His Thr Glu Lys Leu Met Ala                 485 490 495 Phe Lys Ala Ile Tyr Pro Asp Ile Val Arg Leu His Phe Pro Pro Leu             500 505 510 Tyr Lys Glu Leu Phe Thr Ser Glu Phe Glu Pro Ala Met Gln Ile Asp         515 520 525 Gly Glu Pro Ser Arg Leu Glu     530 535 <210> 11 <211> 1608 <212> DNA <213> Artificial Sequence <220> <223> human ROR-alpha DNA sequence <400> 11 aagcttgaat tcgcccggat ggagtcagct ccggcagccc ccgaccccgc cgccagcgag 60 ccaggcagca gcggcgcgga cgcggccgcc ggctccaggg agaccccgct gaaccaggaa 120 tccgcccgca agagcgagcc gcctgccccg gtgcgcagac agagctattc cagcaccagc 180 agaggtatct cagtaacgaa gaagacacat acatctcaaa ttgaaattat tccatgcaag 240 atctgtggag acaaatcatc aggaatccat tatggtgtca ttacatgtga aggctgcaag 300 ggctttttca ggagaagtca gcaaagcaat gccacctact cctgtcctcg tcagaagaac 360 tgtttgattg atcgaaccag tagaaaccgc tgccaacact gtcgattaca gaaatgcctt 420 gccgtaggga tgtcccgaga tgctgtaaaa tttggccgaa tgtcaaaaaa gcagagagac 480 agcttgtatg cagaagtaca gaaacaccgg atgcagcagc agcagcgcga ccaccagcag 540 cagcctggag aggctgagcc gctgacgccc acctacaaca tctcggccaa cgggctgacg 600 gaacttcacg acgacctcag taactacatt gacgggcaca cccctgaggg gagtaaggca 660 gactccgccg tcagcagctt ctacctggac atacagcctt ccccagacca gtcaggtctt 720 gatatcaatg gaatcaaacc agaaccaata tgtgactaca caccagcatc aggcttcttt 780 ccctactgtt cgttcaccaa cggcgagact tccccaactg tgtctatggc agaattagaa 840 caccttgcac agaatatatc taaatcgcat ctggaaacct gccaatactt gagagaagag 900 ctccagcaga taacgtggca gaccttttta caggaagaaa ttgagaacta tcaaaacaag 960 cagcgggagg tgatgtggca attgtgtgcc atcaaaatta cagaagctat acagtatgtg 1020 gtggagtttg ccaaacgcat tgatggattt atggaactgt gtcaaaatga tcaaattgtg 1080 cttctaaaag caggttctct agaggtggtg tttatcagaa tgtgccgtgc ctttgactcc 1140 cagaacaaca ccgtgtactt tgatgggaag tatgccagcc ccgacgtctt caaatcctta 1200 ggttgtgaag actttattag ctttgtgttt gaatttggaa agagtttatg ttctatgcac 1260 ctgactgaag atgaaattgc attattttct gcatttgtac tgatgtcagc agatcgctca 1320 tggctgcaag aaaaggtaaa aattgaaaaa ctgcaacaga aaattcagct agctcttcaa 1380 cacgtcctac agaagaatca ccgagaagat ggaatactaa caaagttaat atgcaaggtg 1440 tctacattaa gagccttatg tggacgacat acagaaaagc taatggcatt taaagcaata 1500 tacccagaca ttgtgcgact tcattttcct ccattataca aggagttgtt cacttcagaa 1560 tttgagccag caatgcaaat tgatgggtaa gagcctctcg aggaattc 1608 <210> 12 <211> 535 <212> PRT <213> Artificial Sequence <220> <223> human ROR-alpha amino acids sequence <400> 12 Lys Leu Glu Phe Ala Arg Met Glu Ser Ala Pro Ala Ala Pro Asp Pro   1 5 10 15 Ala Ala Ser Glu Pro Gly Ser Ser Gly Ala Asp Ala Ala Ala Gly Ser              20 25 30 Arg Glu Thr Pro Leu Asn Gln Glu Ser Ala Arg Lys Ser Glu Pro Pro          35 40 45 Ala Pro Val Arg Arg Gln Ser Tyr Ser Ser Thr Ser Arg Gly Ile Ser      50 55 60 Val Thr Lys Lys Thr His Thr Ser Gln Ile Glu Ile Ile Pro Cys Lys  65 70 75 80 Ile Cys Gly Asp Lys Ser Ser Gly Ile His Tyr Gly Val Ile Thr Cys                  85 90 95 Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Gln Gln Ser Asn Ala Thr             100 105 110 Tyr Ser Cys Pro Arg Gln Lys Asn Cys Leu Ile Asp Arg Thr Ser Arg         115 120 125 Asn Arg Cys Gln His Cys Arg Leu Gln Lys Cys Leu Ala Val Gly Met     130 135 140 Ser Arg Asp Ala Val Lys Phe Gly Arg Met Ser Lys Lys Gln Arg Asp 145 150 155 160 Ser Leu Tyr Ala Glu Val Gln Lys His Arg Met Gln Gln Gln Gln Arg                 165 170 175 Asp His Gln Gln Gln Pro Gly Glu Ala Glu Pro Leu Thr Pro Thr Tyr             180 185 190 Asn Ile Ser Ala Asn Gly Leu Thr Glu Leu His Asp Asp Leu Ser Asn         195 200 205 Tyr Ile Asp Gly His Thr Pro Glu Gly Ser Lys Ala Asp Ser Ala Val     210 215 220 Ser Ser Phe Tyr Leu Asp Ile Gln Pro Ser Pro Asp Gln Ser Gly Leu 225 230 235 240 Asp Ile Asn Gly Ile Lys Pro Glu Pro Ile Cys Asp Tyr Thr Pro Ala                 245 250 255 Ser Gly Phe Phe Pro Tyr Cys Ser Phe Thr Asn Gly Glu Thr Ser Pro             260 265 270 Thr Val Ser Met Ala Glu Leu Glu His Leu Ala Gln Asn Ile Ser Lys         275 280 285 Ser His Leu Glu Thr Cys Gln Tyr Leu Arg Glu Glu Leu Gln Gln Ile     290 295 300 Thr Trp Gln Thr Phe Leu Gln Glu Glu Ile Glu Asn Tyr Gln Asn Lys 305 310 315 320 Gln Arg Glu Val Met Trp Gln Leu Cys Ala Ile Lys Ile Thr Glu Ala                 325 330 335 Ile Gln Tyr Val Val Glu Phe Ala Lys Arg Ile Asp Gly Phe Met Glu             340 345 350 Leu Cys Gln Asn Asp Gln Ile Val Leu Leu Lys Ala Gly Ser Leu Glu         355 360 365 Val Val Phe Ile Arg Met Cys Arg Ala Phe Asp Ser Gln Asn Asn Thr     370 375 380 Val Tyr Phe Asp Gly Lys Tyr Ala Ser Pro Asp Val Phe Lys Ser Leu 385 390 395 400 Gly Cys Glu Asp Phe Ile Ser Phe Val Phe Glu Phe Gly Lys Ser Leu                 405 410 415 Cys Ser Met His Leu Thr Glu Asp Glu Ile Ala Leu Phe Ser Ala Phe             420 425 430 Val Leu Met Ser Ala Asp Arg Ser Trp Leu Gln Glu Lys Val Lys Ile         435 440 445 Glu Lys Leu Gln Gln Lys Ile Gln Leu Ala Leu Gln His Val Leu Gln     450 455 460 Lys Asn His Arg Glu Asp Gly Ile Leu Thr Lys Leu Ile Cys Lys Val 465 470 475 480 Ser Thr Leu Arg Ala Leu Cys Gly Arg His Thr Glu Lys Leu Met Ala                 485 490 495 Phe Lys Ala Ile Tyr Pro Asp Ile Val Arg Leu His Phe Pro Pro Leu             500 505 510 Tyr Lys Glu Leu Phe Thr Ser Glu Phe Glu Pro Ala Met Gln Ile Asp         515 520 525 Gly Glu Pro Leu Glu Glu Phe     530 535

Claims (12)

A method of reducing the activity of Th17 cells by promoting the expression or activity of RORα. The method of claim 1,
Said RORα expression or activity promotion is carried out by introducing a recombinant expression vector comprising a RORα gene into Th17 cells. 3. The method of claim 2,
The RORα gene is characterized in that consisting of the nucleic acid sequence represented by SEQ ID NO: 11 or the amino acid sequence represented by SEQ ID NO: 12. 3. The method of claim 2,
The recombinant expression vector comprises a promoter or enhancer for promoting the expression or activity of RORα. The method of claim 1,
The method can be carried out in combination with promoting Treg cell activity. Promoters for the expression or activity of RORα; or
Th17 cells incorporating RORα expression or activity promoter
Comprising as an active ingredient, a composition for treating and preventing diseases related to the activity of Th17 cells. The method according to claim 6,
The RORα expression or activity promoter is a composition characterized in that the recombinant expression vector containing the RORα gene. The method of claim 7, wherein
The RORα gene is a composition comprising a nucleic acid sequence represented by SEQ ID NO: 11 or an amino acid sequence represented by SEQ ID NO: 12. The method of claim 7, wherein
The recombinant expression vector is a composition, characterized in that it comprises a promoter or enhancer that promotes RORα expression or activity. The method according to claim 6,
The composition is characterized in that it further comprises a Treg cell activity promoter. The method according to claim 6,
The disease associated with the activity of the Th17 cells, characterized in that the autoimmune diseases and allergic diseases. The method of claim 11, wherein the autoimmune disease and allergic disease,
Inflammatory skin diseases including psoriasis and atopic dermatitis; Systemic scleroderma and sclerosis; Inflammatory bowel disease (IBD); Crohn's disease and ulcerative colitis; Surgical tissue reperfusion injury, myocardial ischemic diseases such as myocardial infarction, cardiac arrest, reperfusion after cardiac surgery, and stenosis after percutaneous transluminal coronary angioplasty, stroke Ischemic reperfusion disorders, including abdominal aortic aneurysms; Secondary cerebral edema following seizures; Cranial trauma, hypovolemic shock; asphyxia; Adult respiratory distress syndrome; Acute-lunginjury; Behcet's Disease; Dermatomyositis; Polymyositis; Multiple sclerosis (MS); Dermatitis; Meningitis; Encephalitis; Uveitis; Osteoarthritis; Lupus nephritis; Rheumatoid arthritis (RA), Sjorgen's syndrome, vasculitis; Diseases associated with leukocyte diapedesis; Multiple organ damage syndrome secondary to central nervous system (CNS) inflammatory disorder, sepicaemia or trauma; Alcoholic hepatitis; Bacterial pneumonia; Antigen-antibody complex mediated diseases, including glomerulonephritis; Sepsis; Sarcoidosis; Immunopathological response to tissue / organ transplantation; Pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis fibrosis: inflammation of the lung, including IPF) and cystic fibrosis; Psoriatic arthritis; At least one disease selected from the group consisting of optic nephritis, Guillain-Barré syndrome (GBS), COPD, and type 1 diabetes.

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