KR20230108467A - Composition for treating lupus using SSU72 gene - Google Patents

Abstract

The present invention relates to a composition for the treatment of lupus using the SSU72 gene, wherein the SSU72 gene of the present invention inhibits splenomegaly in lupus animal models accompanied by nephritis or thrombosis, and inhibits lupus-related factors IL-17 and IFNγ It was confirmed that the expression was inhibited. In addition, it was confirmed that the expression of total IgG and IgG2a, which are autoantibodies in the blood, was inhibited, and renal damage was protected.

Description

Composition for treating lupus using SSU72 gene {Composition for treating lupus using SSU72 gene}

The present invention relates to a composition for treating lupus using the SSU72 gene.

Diseases caused by immune hypersensitivity reactions are increasing worldwide, but the fundamental causes of the occurrence of these diseases have not been sufficiently identified. Currently, as a treatment method for diseases caused by an excessive immune response, various symptoms caused by the diseases are alleviated or reduced by administering an immunosuppressive agent alone or in combination.

Immunosuppressive agents refer to various substances used to reduce or block the ability of a host to produce antibodies against the action of an antigen (humoral immune response) or to induce a cellular immune response. These immunosuppressants can be usefully used not only in the field of organ transplantation, but also in autoimmune diseases such as lupus and rheumatoid arthritis, and skin hypersensitivity reactions such as atopy and allergy. An excellent immunosuppressive agent should be able to control the imbalance of the immune response, should be safe for the human body, and should have a low frequency of disease recurrence during long-term treatment.

Currently used immunosuppressants include cyclosporin A and FK506, which are compounds derived from natural products with complex chemical structures that are uneconomical due to the high cost in terms of raw material supply and long-term administration, which can cause various side effects. There is a risk that there is Therefore, there is an urgent need for the development of new immunosuppressants capable of economical production with low toxicity and induction of immune tolerance.

On the other hand, T cells are one of the cell groups that play a central role in the immune system as a biological defense system against various pathogens. T cells are produced in the human thymus and differentiate into T cells with unique characteristics through a series of differentiation processes. It is divided into helper cells (Th2). Among them, the main function of Th1 cells is involved in cell-mediated immunity, and Th2 cells are involved in humoral immunity.

Therefore, most of the immune diseases can be seen as being caused by an imbalance between these two immune cells. For example, when the activity of Th1 cells is abnormally increased, autoimmune diseases can occur, and the activity of Th2 cells is abnormally increased. It is known that immune diseases caused by hypersensitivity reactions occur.

On the other hand, according to recent research results on the differentiation of Th1 cells, as the existence of immunoregulatory T cells (Tregs), a new group capable of regulating the activity of Th1 cells, has been known, research on the treatment of immune diseases using them has emerged. , Treg cells have the property of controlling the inflammatory response by suppressing the function of abnormally activated immune cells, and many studies are being conducted to treat immune diseases through the action of increasing the activity of Treg cells.

In addition, there are Th17 cells as another group formed during the differentiation process of T cells other than Treg cells. It is known that Th17 cells are formed during the differentiation process of undifferentiated T cells through a process similar to that of Treg cells. That is, the differentiation of Treg cells and Th17 cells is commonly achieved in the presence of TGF-β, but IL-6 is not required for Treg cells, whereas IL-6 is present together with TGF-β for Th17 cells. differentiate in the circumstances. Also, differentiated Th17 cells are characterized by secreting IL-17.

It has been revealed that Th17 cells, unlike Treg cells, are involved in the forefront of the inflammatory response seen in immune diseases, maximizing the signal of the inflammatory response and accelerating the progression of the disease. Therefore, in the case of autoimmune diseases that are not controlled by Treg cells among autoimmune diseases, the development of a therapeutic agent for autoimmune diseases targeting inhibition of Th17 cell activity has been greatly highlighted.

On the other hand, systemic lupus erythematosus (hereinafter "SLE" or "lupus") is a chronic autoimmune disease in which healthy "autologous" tissue is mistaken for antigenic. SLE, most severely, is manifested by periodic bouts of symptomatic inflammation, known as “flares,” which are characterized by joint pain, joint swelling, chest pain, fever, mouth sores, swollen lymph nodes, fatigue, and/or most commonly may show findings such as a scarlet skin rash on the face. Indicators of SLE pathogenesis include immunocomplex deposition in the kidneys, skin, brain, heart, and lungs, which cause inflammation in these tissues. The kidneys most often relax, and glomerulonephritis typically develops in human SLE patients. Another indicator of SLE is the presence of anti-double-stranded DNA IgG autoantibodies (the pathogenic role of these autoantibodies in SLE, if any, is indicative, although it remains to be studied) (Isenberg et al. . Am. Soc. Nehprol. 11:1912-27, 2010; Niewold et al., J. Biomed Biotechnol.

Current treatments for SLE include NSAIDs, immunosuppressants, methotrexate, azathioprine, cyclophosphamide, hydroxychloroquine, and corticosteroids. However, current SLE therapy frequently involves long-term use of one or more of these drugs, which may exhibit varying degrees of efficacy, which may be accompanied by severe side effects. Therefore, there is a great need in the art for new therapies for SLE.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating lupus disease accompanied by nephritis or thrombosis, comprising SSU72 or a gene encoding the same as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating lupus disease accompanied by nephritis or thrombosis, comprising a vector expressing the SSU72 gene as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving lupus disease accompanied by nephritis or thrombosis, comprising a vector expressing the SSU72 gene as an active ingredient.

Another object of the present invention is to provide a therapeutic pharmaceutical composition for inhibiting nephritis or thrombosis of lupus disease, comprising SSU72 or a gene encoding the same as an active ingredient.

Another object of the present invention is a pharmaceutical composition for preventing or treating lupus disease accompanied by nephritis or thrombosis, comprising SSU72 or a gene encoding it as an active ingredient,

1) inhibits spleen enlargement;

2) protect against kidney damage;

3) suppress the expression of lupus-related factors IL-17 and IFNγ,

4) To provide a pharmaceutical composition for inhibiting the expression of total TgG and IgG2a, which are autoantibodies in blood.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating lupus disease accompanied by nephritis or thrombosis, comprising SSU72 or a gene encoding the same as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating lupus disease, which includes a vector expressing the SSU72 gene as an active ingredient, accompanied by nephritis or thrombosis.

In addition, the present invention provides a food composition for preventing or improving lupus disease accompanied by nephritis or thrombosis, comprising a vector expressing the SSU72 gene as an active ingredient.

In addition, the present invention provides a therapeutic pharmaceutical composition for inhibiting nephritis or thrombosis of lupus disease, comprising SU72 or a gene encoding the same as an active ingredient.

In addition, the present invention is a pharmaceutical composition for preventing or treating lupus disease accompanied by nephritis or thrombosis, comprising SSU72 or a gene encoding it as an active ingredient,

1) inhibits spleen enlargement;

2) protect against kidney damage;

3) suppress the expression of lupus-related factors IL-17 and IFNγ,

4) A pharmaceutical composition for suppressing the expression of total TgG and IgG2a, which are autoantibodies in blood, is provided.

It was confirmed that the SSU72 gene of the present invention suppresses splenomegaly and suppresses the expression of lupus-related factors IL-17 and IFNγ in lupus animal models accompanied by nephritis or thrombosis. In addition, it suppresses the expression of total IgG and IgG2a, which are autoantibodies in the blood, and it is confirmed that it protects the kidney damage, so it is effective in treating lupus, so it can be usefully used in related industries.

Figure 1 is a diagram confirming spleen enlargement according to SSU72 expression vector treatment in a lupus animal model (A: visual observation of spleen enlargement, B: quantification of spleen size and weight).
Figure 2 is a diagram confirming the expression of IL-17 and IFNγ according to the SSU72 expression vector treatment in lupus animal model.
Figure 3 quantifies the amount of total IgG and IgG2a in the blood according to the SSU72 expression vector treatment in lupus animal models.
Figure 4 is a diagram confirming the renal tissue protective effect according to the SSU72 expression vector treatment in lupus animal models by H&E staining (A: staining result, B: histological index quantification).

The present invention provides a pharmaceutical composition for preventing or treating lupus disease accompanied by nephritis or thrombosis, comprising SSU72 or a gene encoding the same as an active ingredient.

SSU72 of the present invention is RNA Polymerase II CTD Phosphatase, which is known as a molecule involved in transcription, but little is known about the function and role of Ssu72 in vivo.

However, according to the present invention, SSU72 can inhibit the expression of lupus-specific autoantibodies, Total IgG and IgG2a, and effectively inhibit the expression of lupus-related factors IL-17 and IFNγ in lupus-induced mice. It is characterized by the fact that it has been identified that it can effectively prevent and treat lupus by inhibiting damage.

"Nephritis" of the present invention preferably means lupus nephritis (nephritis), and lupus can induce inflammation in all tissues and organs from head to toe by immune cells in the body and cytokines and autoantibodies they secrete. In particular, it is known that the main principle of action is that immune cells and autoantibodies circulate in the blood and induce inflammation in the blood vessel wall. At this time, inflammation and damage can often occur in the kidney, which has a relatively large distribution of blood vessels, and this is called 'lupus nephritis (nephritis)'. Renal involvement in lupus patients is found in approximately 25 to 75%, and renal involvement is recognized as a poor prognostic factor in lupus-related studies in all lupus-related studies. Lupus nephritis uses the classification method revised by the International Society of Nephrology (IPS) and the Society of Renal Pathology (RPS), and is classified into six types from type 1 to type 6. Among the six forms, type 1 lupus nephritis is defined as mild mesangial lupus nephritis in which immunocomplexes accumulate in the mesangium. Type 2 lupus nephritis is defined as glomerular mesangial proliferative lupus nephritis characterized by glomerular mesangial cell proliferation to some degree, in which three or more mesangial cells are present in the mesangium in 3 micron-thick slices. Defined when observed. Type 3 lupus nephritis is defined as focal proliferative lupus nephritis that affects less than 50% of all glomeruli, and the affected glomeruli usually appear as segmental intracapillary proliferative lesions or as inactive glomerular scars, capillary It is defined as presenting mostly segmental distributed subendothelial deposits with or without necrosis and crescent formation. Type 4 lupus nephritis is defined as diffuse proliferative lupus nephritis in which 50% or more of the glomeruli have been involved in histological examination, and if the following lesions are segmental, at least half of the glomerular tufts are sparing. A total case is defined as a case in which more than half of the glomerular capillaries are involved. Item 4 is diffuse segmental lupus nephritis (Type 4-S) when more than 50% of the affected glomeruli have segmental lesions, and diffuse global lupus nephritis when more than 50% of the affected glomeruli have total lesions ( It is subdivided into type 4-G). Type 5 lupus nephritis is defined as membranous lupus nephritis in which total or segmental continuous granular subepithelial immune complexes are present along the capillary wall, often accompanied by immune deposition in the glomerular interstitium. Type 6 lupus nephritis is advanced lupus nephritis, meaning a biopsy showing glomerulosclerosis of 90% or more of the glomeruli, and when there is clinical or pathological evidence that the induration is caused by lupus nephritis.

"Thrombosis" of the present invention refers to thrombosis accompanied by various autoantibodies and immune complexes caused by systemic lupus erythematosus, and due to antiphospholipid antibodies, which are autoantibodies of lupus, thrombosis easily occurs, habitual miscarriage occurs, and thrombocytopenia disease that causes it.

The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. As long as the adjuvant is known in the art, any one may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase the effect.

The pharmaceutical composition according to the present invention may be prepared in the form of incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers usable in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods, respectively. .

When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, and gelatin in addition to active ingredients. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. can Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, witepsol, tween 61, cacao paper, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration are contemplated, eg oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the subject. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be variously selected according to the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 μg/ml, pharmacological activity may not appear, and if the concentration exceeds 5,000 μg/ml, toxicity to the human body may be exhibited.

According to one embodiment of the present invention, the SSU72 may include the amino acid sequence of SEQ ID NO: 1.

SSU72 of the present invention may consist of the amino acid sequence of SEQ ID NO: 1, and includes a functional equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

The "functional equivalent" is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more of the amino acid sequence of SEQ ID NO: 1 as a result of addition, substitution or deletion of amino acids. It refers to a protein having a sequence homology of % or more and exhibiting substantially the same physiological activity as the protein consisting of the amino acid sequence of SEQ ID NO: 1. The "substantially homogeneous activity" means the activity of the SSU72. The functional equivalent may include, for example, an amino acid sequence variant in which some of the amino acids of the amino acid sequence according to the present invention are substituted, deleted or added. Substitutions of amino acids may preferably be conservative substitutions, and examples of conservative substitutions of naturally occurring amino acids are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) and sulfur-containing amino acids (Cys, Met). Deletion of amino acids may preferably be located in a part not directly involved in the activity of the recombinant peptide of the present invention.

According to one embodiment of the present invention, the SSU72 gene may include the nucleotide sequence of SEQ ID NO: 2.

According to one embodiment of the present invention, the composition may inhibit splenomegaly.

According to one embodiment of the present invention, the composition may reduce the expression of IL-17 and IFNγ.

According to one embodiment of the present invention, the composition may reduce the expression of autoantibodies.

According to one embodiment of the present invention, the composition may reduce the expression of autoantibodies, and the autoantibodies may be total IgG or IgG2a, but is not limited thereto.

According to one embodiment of the present invention, it may be to protect kidney tissue.

In addition, the present invention provides a pharmaceutical composition for preventing or treating lupus disease accompanied by nephritis or thrombosis, comprising a vector expressing the SSU72 gene as an active ingredient.

The expression vector of the present invention can be introduced into target cells in an expression form by transduction or transfection by various methods known in the art.

An expression vector is an FDA-approved gene delivery method that can be used in humans, and is a method of directly delivering plasmid DNA to human cells (Nabel, E G et al, Science, 249:1285-1288, 1990). Plasmid DNA is a virus Unlike vectors, it has the advantage of being homogeneously purified. As a plasmid expression vector that can be used in the present invention, mammalian expression plasmids known in the art can be used.

The expression vector containing the nucleic acid according to the present invention can be prepared by methods known in the art, for example, transient transfection, microinjection, transduction, cell fusion, calcium Phosphate precipitation method, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation (electropora tion), gene gun, and other known methods for introducing DNA into cells can be introduced into target cells, but are not limited thereto (Wu et al, J Bio Chem, 267:963 -967, 1992; Wu et al, Bio Chem, 263:14621-14624, 1988).

In addition, the vector may be administered to cells, tissues, or the body by known methods, for example, topically, parenterally, intranasally, intravenously, intramuscularly, subcutaneously, or other suitable means. there is. In particular, the vector can be directly injected in an effective amount to treat a target tissue or target cell.

According to one embodiment of the present invention, the vector may include the nucleotide sequence of SEQ ID NO: 2.

In addition, the present invention provides a food composition for preventing or improving lupus disease accompanied by nephritis or thrombosis, comprising a vector expressing the SSU72 gene as an active ingredient.

In addition to containing the active ingredient of the present invention, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional food compositions.

Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agents described above, natural flavoring agents (thaumatin), stevia extracts (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. The food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements, etc. there is

In addition, the food composition, in addition to the active ingredient extract, various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, Alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like may be contained. In addition, the food composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages.

The functional food composition of the present invention may be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or treating lupus disease. In the present invention, 'health functional food composition' refers to a food manufactured and processed using raw materials or ingredients having useful functionality for the human body according to Health Functional Food Act No. 6727, and the structure and function of the human body It refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological functions. The health functional food of the present invention may contain ordinary food additives, and the suitability as a food additive is determined according to the general rules of the Food Additive Code and General Test Methods approved by the Food and Drug Administration, unless otherwise specified. It is judged according to standards and standards. Examples of the items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations. For example, a health functional food in the form of a tablet is obtained by granulating a mixture obtained by mixing the active ingredient of the present invention with an excipient, a binder, a disintegrant, and other additives in a conventional manner, and then adding a lubricant or the like to compression molding, or as described above. The mixture can be directly compression molded. In addition, the health functional food in the form of a tablet may contain a flavoring agent and the like as needed. Among health functional foods in the form of capsules, hard capsules can be prepared by filling a mixture in which the active ingredient of the present invention is mixed with additives such as excipients in a normal hard capsule. It can be prepared by filling the mixture mixed with gelatin in a capsule base. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary. The health functional food in the form of a pill can be prepared by molding a mixture of the active ingredient of the present invention mixed with an excipient, a binder, a disintegrant, etc. by a conventionally known method, and can be coated with sucrose or other coating agent if necessary, Alternatively, the surface may be coated with a material such as starch or talc. Health functional food in the form of granules can be prepared in granular form by a conventionally known method of mixing the active ingredient of the present invention with excipients, binders, disintegrants, etc., and, if necessary, flavoring agents, flavoring agents, etc. can

In addition, the present invention provides a therapeutic pharmaceutical composition for inhibiting nephritis or thrombosis of lupus disease, comprising SSU72 or a gene encoding the same as an active ingredient.

In addition, the present invention is a pharmaceutical composition for preventing or treating lupus disease accompanied by nephritis or thrombosis, comprising SSU72 or a gene encoding it as an active ingredient,

1) inhibits spleen enlargement;

2) protect against kidney damage;

3) suppress the expression of lupus-related factors IL-17 and IFNγ,

4) A pharmaceutical composition for suppressing the expression of total TgG and IgG2a, which are autoantibodies in blood, is provided.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited to these examples.

Preparation Example 1. Production of lupus animal model accompanied by nephritis or thrombosis

In order to confirm the effect of the SSU72 gene of the present invention on improving lupus accompanied by nephritis or thrombosis, an animal model of lupus accompanied by nephritis or thrombosis was prepared. Specifically, B6 mice were treated with the R848 compound three times a week to induce lupus disease. Then, vectors expressing SSU72 (SSU72 group) were injected intravenously and intramuscularly every 5 days. As a control group, a normal group that was not treated with anything; A (Mock) group injected with the R484 compound and mock vector was used. The amino acid sequence of the SSU72 protein of the present invention is shown in SEQ ID NO: 1, and the base sequence encoding it is shown in SEQ ID NO: 2.

Example 1. Confirmation of suppression of spleen enlargement according to SSU72 expression

In order to confirm whether SSU72 expression suppresses splenomegaly due to induction of lupus accompanied by nephritis or thrombosis, the animal model of Preparation Example 1 was humanely sacrificed at the end of the experiment. Thereafter, the spleen of each group was removed, and the size and weight of the spleen were checked to determine whether spleen enlargement was suppressed.

As a result, as shown in Figure 1, it was confirmed that the size and weight of the spleen increased in the Mock group in which nephritis or lupus accompanied by thrombosis was induced compared to the Normal group, and in the SSU72 group compared to the Mock group, the spleen It was confirmed that the size and weight of were significantly reduced.

Example 2. Confirmation of lupus factor inhibitory effect according to SSU72 expression

We wanted to confirm whether the expression of SSU72 suppresses factors related to lupus accompanied by nephritis or thrombosis. Specifically, splenocytes were obtained from the spleen excised in Example 1, and the expression of IL-17 and IFNγ was analyzed. Specifically, splenocytes were obtained from the spleen, stimulated with PMA+Ionomycin for 4 hours, and then subjected to CD4 surface staining. After that, IL-17 and IFNg intracellular staining was performed to confirm the expression of IL-17 and IFNγ.

As a result, as shown in FIG. 2, the expression of IL-17 and IFNγ increased in the Mock group compared to the Normal group, but in the SSU72 group, the expression of IL-17 and IFNγ was significantly higher than that of the Mock group. decreased.

Example 3. Confirmation of inhibition of autoantibody expression

We wanted to confirm whether the expression of SSU72 suppresses autoantibodies, which are the main cause of lupus. Specifically, whole blood was collected from the eyes of the mouse of Example 1 (orbital blood collection), and the amounts of total IgG and IgG2a, which are autoantibodies, were measured. Specifically, blood collected from the eyes was centrifuged to separate serum, and the levels of total IgG and IgG2a in serum were measured using an ELISA kit.

As a result, as shown in FIG. 3, the amount of total IgG and IgG2a, which are autoantibodies, increased in the Mock group compared to the Normal group, but in the SSU72 group, compared to the Mock group, Total IgG and IgG2a significantly decreased. confirmed that.

Example 4. Confirmation of renal protective effect

It was attempted to confirm whether the SSU72 expression vector of the present invention protects renal cells. Specifically, the kidney tissue of the mouse sacrificed in Example 1 was obtained and sectioned, and confirmed by Hematoxylin & Eosin (H&E) staining. Specifically, Haris Hematoxylin (Youngdong Pharmaceutical, Cat#. S2-1), Eosin Y 1% sol. (Asan Pharmaceutical, Cat#. 2000) was used, and paraffin was removed from the sections using xylene and washed with ethanol. After that, tap water washing was performed 4-5 times, followed by nuclear staining using Haris Hematoxylin (5 minutes), washing 5 times, and cytoplasmic staining using 1% HCL once and Eosin (1 minute). did After that, it was washed 5 times and checked after mouting, and the degree of kidney damage was confirmed by histologic score.

As a result, as shown in FIG. 4, in the Mock group, damage to the kidney tissue was significantly increased compared to the Normal group, but in the SSU72 group, compared to the Mock group, it was confirmed that the kidney damage was significantly suppressed, resulting in lupus. It was confirmed that it is effective for induced nephritis.

Accordingly, it was confirmed that the SSU72 gene of the present invention inhibits splenomegaly and suppresses the expression of lupus-related factors IL-17 and IFNγ in lupus animal models accompanied by nephritis or thrombosis. In addition, it was confirmed that the expression of total IgG and IgG2a, which are autoantibodies in the blood, was inhibited, and renal damage was protected.

<110> Catholic University Industry-Academic Cooperation Foundation <120> Composition for treating lupus using SSU72 gene <130> PN2111-568 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 194 <212> PRT <213> artificial sequence <220> <223> Homo sapiens SSU72 homolog Amino acid sequence <400> 1 Met Pro Ser Ser Pro Leu Arg Val Ala Val Val Cys Ser Ser Asn Gln 1 5 10 15 Asn Arg Ser Met Glu Ala His Asn Ile Leu Ser Lys Arg Gly Phe Ser 20 25 30 Val Arg Ser Phe Gly Thr Gly Thr His Val Lys Leu Pro Gly Pro Ala 35 40 45 Pro Asp Lys Pro Asn Val Tyr Asp Phe Lys Thr Thr Tyr Asp Gln Met 50 55 60 Tyr Asn Asp Leu Leu Arg Lys Asp Lys Glu Leu Tyr Thr Gln Asn Gly 65 70 75 80 Ile Leu His Met Leu Asp Arg Asn Lys Arg Ile Lys Pro Arg Pro Glu 85 90 95 Arg Phe Gln Asn Cys Lys Asp Leu Phe Asp Leu Ile Leu Thr Cys Glu 100 105 110 Glu Arg Val Tyr Asp Gln Val Val Glu Asp Leu Asn Ser Arg Glu Gln 115 120 125 Glu Thr Cys Gln Pro Val His Val Val Asn Val Asp Ile Gln Asp Asn 130 135 140 His Glu Glu Ala Thr Leu Gly Ala Phe Leu Ile Cys Glu Leu Cys Gln 145 150 155 160 Cys Ile Gln His Thr Glu Asp Met Glu Asn Glu Ile Asp Glu Leu Leu 165 170 175 Gln Glu Phe Glu Glu Lys Ser Gly Arg Thr Phe Leu His Thr Val Cys 180 185 190 Phe Tyr <210> 2 <211> 585 <212> DNA <213> artificial sequence <220> <223> Homo sapiens SSU72 homolog Nucleotide sequence <400> 2 atgccgtcgt ccccgctgcg ggtggcggtg gtgtgctcga gcaaccagaa ccggagcatg 60 gaggcgcaca acatcctcag caaacgggga ttcagcgtcc gatcctttgg aacagggact 120 cacgtgaagc ttccaggacc agctcccgac aagcccaatg tttatgattt caaaaccaca 180 tatgaccaga tgtacaatga tcttcttagg aaagacaaag aactctatac acagaatggg 240 attttacata tgctggacag aaataagaga atcaagcccc ggccagaaag attccagaac 300 tgcaaagacc tgtttgatct gatcctcact tgcgaagaga gagtgtatga ccaggtggtg 360 gaagatctga attccagaga acaggagacc tgccagcctg tgcacgtggt caatgtggac 420 atccaggaca accacgagga ggccaccctg ggggcgtttc tcatctgtga gctctgccag 480 tgtatccagc acacggaaga catggagaac gagatcgacg agctgctgca ggaggttcgag 540 gagaagagtg gccgcacctt tctgcacacc gtctgcttct actga 585

Claims (13)

A pharmaceutical composition for preventing or treating lupus disease accompanied by nephritis or thrombosis, comprising SSU72 or a gene encoding the same as an active ingredient. According to claim 1,
Wherein the SSU72 comprises the amino acid sequence of SEQ ID NO: 1. According to claim 1,
The composition, wherein the SSU72 gene comprises the nucleotide sequence of SEQ ID NO: 2. According to claim 1,
Wherein the composition inhibits splenomegaly (splenomegaly). According to claim 1,
Wherein the composition reduces the expression of IL-17 and IFNγ. According to claim 1,
Wherein the composition reduces the expression of autoantibodies. According to claim 6,
Wherein the autoantibody is Total IgG or IgG2a. According to claim 1,
Wherein the composition protects renal tissue. A pharmaceutical composition for preventing or treating lupus disease accompanied by nephritis or thrombosis, comprising a vector expressing the SSU72 gene as an active ingredient. According to claim 9,
Wherein the vector comprises the nucleotide sequence of SEQ ID NO: 2. A food composition for preventing or improving lupus disease accompanied by nephritis or thrombosis, comprising a vector expressing the SSU72 gene as an active ingredient. A therapeutic pharmaceutical composition for inhibiting nephritis or thrombosis of lupus disease, comprising SSU72 or a gene encoding the same as an active ingredient. In the pharmaceutical composition for preventing or treating lupus disease accompanied by nephritis or thrombosis, comprising SSU72 or a gene encoding it as an active ingredient,
1) inhibits spleen enlargement;
2) protect against kidney damage;
3) suppress the expression of lupus-related factors IL-17 and IFNγ,
4) A pharmaceutical composition for suppressing the expression of total TgG and IgG2a, which are autoantibodies in blood.