KR101805771B1 - A pharmaceutical composition for preventing or treating blood dyscrasia comprising TMEM100 protein - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating a hematopoietic disorder comprising a TMEM100 protein, and more particularly, to a pharmaceutical composition for preventing or treating a hematopoietic disorder comprising a TMEM100 protein or a polynucleotide encoding the TMEM100 protein, , A food composition for improving hematopoietic disorders comprising a TMEM100 protein or a polynucleotide encoding the TMEM100 protein, and a feed composition.
The TMEM100 protein of the present invention promotes the proliferation or differentiation of hematopoietic stem cells, promotes the development of red blood cells or lymphocyte cells, and helps the development of the liver, spleen or bone marrow, which controls the hematopoiesis, so that the hematopoietic process normally occurs. Therefore, the TMEM100 protein can be usefully used as a therapeutic agent for hematopoietic disorders related diseases.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating a hematopoietic disorder comprising a TMEM100 protein,

The present invention relates to a pharmaceutical composition for preventing or treating a hematopoietic disorder comprising a TMEM100 (Transmembrane protein 100) protein, and more particularly to a pharmaceutical composition for preventing or treating a hematopoietic disorder comprising a TMEM100 protein or a polynucleotide encoding the same, A method for preventing or treating a hematopoietic disorder comprising administering a composition to a subject, a food composition for improving hematopoietic disorders comprising a TMEM100 protein or a polynucleotide encoding the TMEM100 protein, and a feed composition.

Hematopoiesis is the process of making all of the hematopoietic cells in our body from the multipotential stem cells hematopoietic stem cells. The key organ of the hematopoietic process changes several times during the development of the mammal, which occurs mainly in the yolk sac in the early pregnancy, in the liver in the middle pregnancy, in the spleen just after birth, and in the adult bone marrow. These hematopoietic processes are important not only in hematopoietic stem cells themselves, but also in interaction with surrounding cells such as liver or spleen (hematopoietic stem cell niche).

In the maintenance and regulation of stem cells, the "Niche hypothesis," which claims to be important not only stem cells themselves, but also the local environment around them, called "Niche", was proposed by Ray Schofield in 1978 , And is supported by many researchers. Studies on hematopoietic stem cell transplantation have been under way and studies have shown that hematopoietic stem cells collected around the blood vessels remain in specific niches for several weeks and undergo the effects of endothelial cells and perivascular cells to divide and differentiate hematopoietic stem cells have. These blood vessels are rich in CXCL12 (or SDF-1) and glycoprotein-E-selectin, cytokines that play an important role in the location of hematopoietic stem cells.

The myelodysplastic syndrome, which is one of various diseases caused by abnormal hematopoiesis, is a malignant hematologic disease which was known as a pre-leukemia pre-hematopoietic system. It is a hematopoietic hematopoietic organ that makes blood cells, Disease. In 2009, 680 cases of myelodysplastic syndrome were reported to occur, accounting for 0.35% of total cancer incidence. However, in 2011, 840 cases were reported to occur in the year. Thus, the number of patients suffering from hematopoietic disorders including myelodysplastic syndrome (Ministry of Health and Welfare, Central Cancer Registration Division).

Allogeneic hematopoietic stem cell transplantation is the only method that can be expected to cure hematopoietic disorders, but only about 5-10% of the patients are diagnosed because of side effects. In addition, hematopoietic disorders require various treatment modalities for each subtype because there are various subtypes with different disease progression patterns. However, not only the kinds of medicines vary but also the therapeutic effects of the medicines currently used for treatment are unsatisfactory, and it is urgent to develop a therapeutic agent for improving or treating hematopoietic disorders.

On the other hand, TMEM100 (Transmembrane protein 100) gene exists in various vertebrates from zebrafish to human. However, recent studies have shown that TMEM100 is a sub-gene of the Alk1 signaling pathway (Proc Natl Acad Sci USA 2012 Jul 24; 109 (30): 12064- 9). Alk1 signaling system abnormalities cause vascular anomalies called "hereditary full blood capillary dilation", capillary hemorrhage in the skin or mucosa; Or arteriovenous malformations in which blood vessels easily rupture and bleed in various internal organs including the brain, lung, and liver. Furthermore, there is a report that TMEM100 is involved not only in angiogenesis but also in neuronal cell development (Neuroscience. 2013 Jun 14; 240: 117-28). However, the function of TMEM100 in relation to proliferation and differentiation of hematopoietic stem cells has not been known to date.

Under these circumstances, the present inventors have made intensive efforts to develop a novel therapeutic agent capable of regulating hematopoiesis. As a result, when the TMEM100 gene is deleted, the development of red blood cells or lymphocytes is inhibited by inhibition of hematopoietic stem cell differentiation, Liver, spleen, or bone marrow. Thus, the present inventors have completed the present invention by confirming that the TMEM100 protein can be effectively used as a therapeutic agent for hematopoietic disorders-related diseases.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating hematopoietic disorders comprising a TMEM100 (Transmembrane protein 100) protein or a polynucleotide encoding the same.

Another object of the present invention is to provide a method for preventing or treating hematopoietic disorders comprising administering the composition to an individual.

It is another object of the present invention to provide a food composition for improving hematopoietic disorders comprising a TMEM100 protein or a polynucleotide encoding the same.

It is another object of the present invention to provide a feed composition for improving hematopoietic disorders comprising a TMEM100 protein or a polynucleotide encoding the TMEM100 protein.

One aspect of the present invention provides a pharmaceutical composition for preventing or treating a hematopoietic disorder comprising a TMEM100 (Transmembrane protein 100) protein or a polynucleotide encoding the same.

When the TMEM100 gene of the present invention is deleted, the differentiation of hematopoietic stem cells is inhibited and the normal hematopoietic process does not occur as the development of the liver, spleen or bone marrow, which controls the hematopoiesis, is inhibited. Therefore, the TMEM100 protein can be usefully used as a therapeutic agent for hematopoietic disorders related diseases.

The term " TMEM100 (Transmembrane protein 100) "of the present invention means a protein that functions as a lower regulator of the ALK1 signal transduction system. The function of the TMEM 100 with respect to the proliferation or differentiation of hematopoietic stem cells is not known at all and was first identified by the present inventors. The specific nucleotide sequence and protein information of the gene encoding the TMEM100 protein are known in NCBI (GenBank: Accession NP_001093110.1, NP_060756.2, etc.).

TMEM100 protein of the present invention is one as a fusion protein, the other amino acids, peptides, etc. can be, and the amino acid sequence comprising the amino acid sequence of SEQ ID NO: 2 (MTEEPIKEILGAPKAHMAATMEKSPKSEVVITTVPLVSEIQLMAATGGTELSCYRCIIPFAVVVFIAGIVVTAVAYSFNSHGSIISIFGLVVLSSGLFLLASSALCWKVRQRSKKAKRRESQTALVANQRSLFA, SEQ ID NO: 2) indicating the prevention or treatment of hematopoietic disorders , And are included in the scope of the present invention. The amino acid sequence of SEQ ID NO: 2 may be TMEM100-derived, and may be specifically derived from human TMEM100, but is not limited thereto.

The protein can be produced by a known protein synthesis method or by culturing a transformed host cell. When the protein of the present invention is produced by culturing transformed host cells, a recombinant vector comprising a polynucleotide encoding the protein of the present invention is introduced into a host cell, transformed, and then the transformant is cultured to produce . The method for culturing the transformant may be suitably selected from any method known in the art for producing the protein of the present invention.

The amino acid sequence can be easily modified by substitution, deletion, insertion, or a combination thereof of one or more amino acids. Therefore, peptides and proteins having high homology with SEQ ID NO: 2, for example, peptides and proteins having a homology of not less than 70%, particularly not less than 80%, are interpreted as being included in the scope of the present invention do.

The term "homology " in the present invention is intended to indicate a degree of similarity to the amino acid sequence of a wild-type protein, and includes a sequence having the same sequence as the amino acid sequence of the present invention, . This homology can be determined by comparing the two sequences visually, but can be determined using a bioinformatic algorithm that aligns the sequences to be compared and analyzes the degree of homology. The homology between the two amino acid sequences can be expressed as a percentage. Useful automated algorithms are available in the GAP, BESTFIT, FASTA and TFASTA computer software modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, Wis. USA). The automated array algorithms in this module include Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman sequence alignment algorithms. Algorithm and homology determination for other useful arrays is automated in software including FASTP, BLAST, BLAST2, PSIBLAST and CLUSTAL W.

The term "polynucleotide" of the present invention means a polymer of a nucleotide in which a nucleotide unit is extended in a chain form by covalent bonds. A polynucleotide of the present invention is not meant a polynucleotide encoding a TMEM100 protein of the invention and, specifically, may be composed of the nucleotide sequence of SEQ ID NO: 1 (ATGACTGAAGAGCCCATCAAGGAGATCCTGGGAGCCCCAAAGGCTCACATGGCAGCGACGATGGAGAAGAGCCCCAAGAGTGAAGTTGTGATCACCACAGTCCCTCTGGTCAGTGAGATTCAGTTGATGGCTGCTACAGGGGGTACCGAGCTCTCCTGCTACCGCTGCATCATCCCCTTTGCTGTGGTTGTCTTCATCGCCGGCATCGTGGTCACCGCGGTGGCTTACAGCTTCAATTCCCATGGGTCTATTATCTCCATCTTTGGCCTGGTTGTTCTGTCATCTGGACTTTTTTTACTAGCCTCCAGTGCCTTGTGCTGGAAAGTGAGACAAAGGAGCAAGAAAGCCAAGAGACGGGAGAGTCAAACAGCTCTCGTGGCAAATCAGAGAAGCTTGTTTGCTTGA, SEQ ID NO: 1), but limited.

In addition, the polynucleotide can be modified in various ways in the coding region within a range that does not change the amino acid sequence of the protein expressed from the coding region in consideration of the codon preference in the organism to which the protein is to be expressed, Various modifications or modifications can be made within a range that does not affect the expression of the gene in the excluded part. That is, as long as the polynucleotide of the present invention encodes a protein having equivalent activity, one or more nucleotide bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention.

As a means for introducing the recombinant vector into cells and expressing the protein of the present invention, known expression vectors such as plasmid vectors, cosmid vectors, and bacteriophage vectors may be used. The vectors may be any known methods using DNA recombinant techniques Can be easily manufactured by those skilled in the art.

In the present invention, the TMEM100 protein exhibits an effect of promoting the differentiation of hematopoietic stem cells .

The term "hematopoietic stem cell" as used herein means a hematopoietic stem cell, which carries out the hematopoietic function through self-proliferation and differentiation. The hematopoietic stem cells mature into the final cells through the intermediary process of the various hemocytes. The hematopoietic stem cells are transformed into various endocrine cells such as red blood cells, platelets, neutrophils, eosinophils, basophils, monocytes, T cells, B cells, The development of red blood cells or lymphocyte cells can be promoted by the differentiation of the hematopoietic stem cells.

In a specific example of the present invention, the TMEM100 gene was examined for the effect on the differentiation of hematopoietic stem cells into red blood cells. As a result, it was confirmed that the number of undifferentiated erythrocytes including the nucleus was increased when the TMEM100 gene was deleted (FIG. 1), specifically confirming the increase in the number of hemocytes in the undifferentiated stage (FIG. 2). This suggests that the TMEM100 gene plays an essential role in erythrocyte differentiation, so that the TMEM100 protein or a polynucleotide encoding the TMEM100 protein may be useful for preventing or treating hematopoietic disorders.

In another specific example of the present invention, the TMEM100 gene was examined for the effect on the differentiation of hematopoietic stem cells into lymphocytes. When the TMEM100 gene was deleted, the number of B cells in the undifferentiated stage was increased 3). This suggests that the TMEM100 gene plays an essential role in the differentiation of lymphoid cells, suggesting that the TMEM100 protein or a polynucleotide encoding the TMEM100 protein may be useful for preventing or treating hematopoietic disorders.

In addition, in the present invention, the TMEM100 protein exhibits an effect of promoting the development of liver, spleen or bone marrow .

The terms "liver "," spleen "and" bone marrow "of the present invention refer to hematopoietic regulatory organs involved in making or removing blood cells. Hematopoiesis are mainly produced in the liver in infancy, but gradually grow in proportion to the rate of bone marrow formation.

In a specific embodiment of the present invention, the TMEM100 gene was examined for its effect on the development of each organ involved in hematopoiesis. As a result, when the TMEM100 gene was deleted, the number of cells of the liver, spleen or bone marrow was significantly decreased, And the weight decreased (FIG. 4). This suggests that the TMEM100 gene plays an essential role in the growth and development of each organ involved in the hematopoiesis process, so that the TMEM100 protein or a polynucleotide encoding the TMEM100 gene can be useful for preventing or treating hematopoietic disorders.

The term "hematopoietic disorder" of the present invention means a state in which hematopoiesis does not normally occur due to a defect in the hematopoietic effect. Hematopoietic disorders can cause a variety of diseases, such as aplastic anemia, malignant lymphoma or leukemia, chronic liver failure, renal failure, severe infection, myelodysplastic thrombocytopenia, idiopathic thrombocytopenic purpura (ITP) Platelet insensitivity, lymphocytopenia, neutropenia, mononucleosis, granulocytopenia, myelodysplastic syndrome or myeloproliferative disease.

The term "prevention" of the present invention means all actions that inhibit or delay the onset of hematopoietic disorders by administration of a pharmaceutical composition comprising the TMEM100 protein or a polynucleotide encoding the TMEM100 protein as an active ingredient.

The term "treatment" of the present invention means any suspicion of a hematopoietic disorder and all the actions that alleviate or alleviate symptoms of an onset subject by the administration of the pharmaceutical composition.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient or diluent conventionally used in the manufacture of a pharmaceutical composition, and the carrier may comprise a non-naturally occuring carrier.

The term "pharmaceutically acceptable" of the present invention means that the composition is free of toxicity to cells or humans exposed to the composition.

Specifically, the pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . In the present invention, the carrier, excipient and diluent which may be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension, liquid solutions, emulsions and syrups have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

Another embodiment provides a method for preventing or treating a hematopoietic disorder comprising the step of administering the composition to a subject other than a human having a hematopoietic disorder.

Here, the definition of hematopoietic disorder, prevention and treatment is as described above.

The term "administering" of the present invention means introducing a given substance into an individual in a suitable manner.

The term "individual" of the present invention means all animals such as mice, mice, livestock, etc., including humans who have developed or are capable of developing hematopoietic disorders. Specific examples include, but are not limited to, mammals including humans.

The method for preventing or treating hematopoietic disorders according to the present invention specifically comprises administering a pharmaceutically effective amount of a pharmaceutical composition for preventing or treating a hematopoietic disorder comprising, as an active ingredient, a TMEM100 protein or a polynucleotide encoding the same, .

The term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects. The effective dose level is determined by the sex, age And other medical fields, including drugs used in combination or concurrently, with respect to body weight, health status, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, administration time, route of administration, Can be readily determined by those skilled in the art according to well known factors.

Specifically, the composition of the present invention may be administered at a dose of 0.0001 to 100 mg / kg body weight per day, more specifically 0.001 to 100 mg / kg body weight, based on the solid content. The administration may be such that the recommended dose is administered once a day or divided into several doses.

In the method for preventing or treating hematopoietic disorders of the present invention, the administration route and method of administering the composition are not particularly limited, and any route of administration may be used as long as the composition containing the composition can reach the desired site Depending on the mode of administration. Specifically, the composition may be administered orally or parenterally through various routes. Non-limiting examples of routes of administration include oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, Intramuscularly or through inhalation or the like.

Another embodiment provides a food composition for improving hematopoietic disorders comprising a TMEM100 protein or a polynucleotide encoding the TMEM100 protein.

The definition of TMEM100, protein, polynucleotide and hematopoietic disorder is as described above.

The term "improvement" of the present invention means all the actions that improve or ameliorate the hematopoietic disorder by the administration of the composition.

The term "food" of the present invention is intended to encompass all kinds of foods, such as meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice creams, Vitamin complex, health functional food, and health food, all of which include foods in a conventional sense.

The term "functional food" as used herein means the same term as "food for special health use" (FoSHU). In addition to nutrition, It means food. Here, the term "function (surname)" means that the structure and function of the human body have a beneficial effect for health use such as controlling nutrients or physiological action. The food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients which are conventionally added in the art. In addition, the formulations of the foods can also be produced without restrictions as long as they are formulations recognized as food. The composition for food of the present invention can be manufactured in various forms, and unlike general drugs, it has advantages of being free from side effects that may occur when a food is used as a raw material for a long period of time, and is excellent in portability, Can be ingested as an adjuvant to improve the hematopoietic disorder.

The health food refers to a food having an active health promotion or promotion effect compared with a general food, and a health supplement food refers to a food for health assistance. In some cases, the terms health functional foods, health foods, and health supplements may be used interchangeably. Specifically, the health functional food is a food prepared by adding the TMEM100 protein of the present invention or a polynucleotide encoding the TMEM100 protein to a food material such as a beverage, a tea, a spice, a gum, or a confection, or by encapsulating, This means that it takes a certain effect on health when consumed, but unlike general medicine, there is an advantage that there is no side effect that may occur when a drug is taken for a long time by using food as a raw material.

Since the food composition of the present invention can be ingested on a daily basis, a high effect can be expected for improvement of hematopoietic disorder, so that it can be very usefully used.

The food composition may further comprise a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier conventionally used in the art can be used.

In addition, the food composition may contain additional components that are commonly used in food compositions and can improve odor, taste, visual appearance, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid and the like. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu) It may also include amino acids such as lysine, tryptophan, cysteine, valine, and the like.

In addition, the food composition may further contain antiseptic agents (such as potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate), bactericides (Sodium nitrite), bleach (sodium sulfite), seasoning (sodium MSG glutamate, etc.), sweeteners (dicin, cyclamate, saccharin, etc.), coloring agents , Sodium, etc.), perfume (vanillin, lactones, etc.), swelling agents (alum, potassium hydrogen D-tartrate), emulsifiers, thickeners (foams), encapsulating agents, gum bases, foam inhibitors, solvents, And may include food additives. The additives may be selected and used in appropriate amounts depending on the type of food.

The TMEM100 protein of the present invention or a polynucleotide encoding the TMEM100 protein can be used as it is or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the food composition of the present invention may be added in an amount of not more than 50 parts by weight, specifically not more than 20 parts by weight, based on the food or beverage, when the food or drink is prepared. However, in case of long-term ingestion for health and hygiene purposes, the active ingredient may be contained in an amount not exceeding the above range and there is no problem in terms of safety.

As an example of the food composition of the present invention, it can be used as a health beverage composition. In this case, various flavors or natural carbohydrates can be added as an additional ingredient like ordinary beverages. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose; Polysaccharides such as dextrin, cyclodextrin; Xylitol, sorbitol, erythritol, and the like. Sweeteners include natural sweeteners such as tau Martin and stevia extract; Synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 mL of the health beverage composition of the present invention.

In addition to the above, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acid, protective colloid thickener, pH adjuster, stabilizer, Alcohols or carbonating agents, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks, or vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

The food composition of the present invention may contain various weight percentages as long as it can exhibit an improvement effect of hematopoietic disorder. Specifically, the TMEM100 protein of the present invention may be contained in an amount of 0.00001 to 100% by weight or 0.01 to 80% by weight But are not limited thereto.

Another embodiment provides a feed composition for improving hematopoietic disorders comprising a TMEM100 protein or a polynucleotide encoding the same.

The definition of TMEM100, protein, polynucleotide, hematopoietic disorder and improvement is as described above.

The term "feed " of the present invention means any natural or artificial diet, single meal, or the like ingredients for feeding, ingesting, digesting or suitable for the livestock.

The feed may comprise a feed additive or supplementary feed.

The kind of the feed is not particularly limited, and feeds conventionally used in the art can be used. Non-limiting examples of such feeds include vegetable feeds such as cereals, muscle roots, food processing busines logistics, algae, fibers, pharmaceutical buses, oils, fats, pastes or grain by-products; Animal feeds such as proteins, inorganic substances, fats, oils, fats, oils, monocellular proteins, animal plankton or foods. These may be used alone or in combination of two or more.

The TMEM100 protein of the present invention promotes the proliferation or differentiation of hematopoietic stem cells, promotes the development of red blood cells or lymphocyte cells, and helps the development of the liver, spleen or bone marrow, which controls the hematopoiesis, so that the hematopoietic process normally occurs. Therefore, the TMEM100 protein can be usefully used as a therapeutic agent for hematopoietic disorders related diseases.

Figure 1 shows the degree of erythrocyte differentiation according to the deletion of TMEM100 gene. A is a normal control (control), B is an image showing nucleated red blood cells in an experimental group (mutant) in which TMEM100 gene is not expressed, and C Is a graph showing the number of red blood cells. The number of undifferentiated red blood cells stained with nuclei was relatively higher in the experimental group than in the control group.
Fig. 2 shows the degree of erythrocyte differentiation according to deletion of the TMEM100 gene. Fig. 2 shows the results of analysis of the erythrocyte differentiation according to the deletion of the TMEM100 gene, wherein A is the liver of the embryo, B is the spleen of newborn mice, Graph. It can be seen that the ratio of cells belonging to stem cells in which differentiation has not started in all of the above A, B and C is increased, while the ratio of cells in progression is remarkably reduced.
FIG. 3 shows the degree of differentiation of lymphocytes according to deletion of the TMEM100 gene, wherein A is a B cell, B is a B cell lineage, and C is a T cell analysis result. The cells isolated from the adult bone marrow were labeled with respective antibodies and analyzed by gating only the lymphocytes. The number of T cells was not significantly different between the control and experimental groups, but the number of pre-B cells was decreased in B cells.
Figure 4 shows the degree of development of each organ according to the deletion of the TMEM100 gene through appearance, weight or number of whole cells. A is liver, B is spleen and C is adult bone BM. And a graph showing the results of the analysis in FIG. The development of the above-mentioned organs is inhibited in the test group (mutant) in which the TMEM100 gene is not expressed, as compared with the normal control (control).
FIG. 5 is an image showing the cell death of each organ due to the deletion of the TMEM100 gene. Double staining was performed on activated caspase 3 (red) and markers representing each cell type (Dlk: hepatocyte, F4 / 80: macrophage, Ter119: erythroid lineage, all brown). It is shown that cell death is induced in Dlk-positive stem cells and some macrophages that are F4 / 80-positive cells, whereas cell death does not occur in Ter119-positive cells in hematopoietic processes.
FIG. 6 shows the ability of each cell to differentiate according to deletion of the TMEM100 gene. FIG. 6 is a graph showing the results of analysis of cells of embryo and B in adult cells. Specifically, the results of the CFU assay show no significant difference between the normal control (control) and the experimental group (mutant) in which the TMEM100 gene is not expressed.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Example  1. For red blood cell development TMEM100  Analysis of gene effects

Example  1-1. Blood test

In order to analyze the effect of TMEM100 gene on erythroid cells among hematopoietic agents, we examined the effect of the gene on the process of red blood cell differentiation in hematopoietic stem cells.

Specifically, the presence of nuclei in the red blood cells of the normal control (control) and the experimental group (mutant) in which the TMEM100 gene was not expressed was analyzed. The blood isolated from the control and experimental groups was plated on a slide glass using Cytospin 4 (Thermo Scientific), and Microscopy Hemacolor (MERCK) was used to stain nucleated undifferentiated red blood cells with deep blue to distinguish them from non-nucleated differentiated red blood cells ) Kit.

As a result, as shown in FIG. 1, it was confirmed that the number of nucleus-free red blood cells in the cells of the test embryo in which the TMEM100 gene was not expressed decreased in the experimental group compared to the control group.

From the above results, it was confirmed that the TMEM100 gene plays an essential role in the process of red blood cell differentiation in hematopoietic stem cells, so that the gene is essential for the hematopoietic process.

Example  1-2. FACS  analysis

The erythrocyte undifferentiation following deletion of the TMEM100 gene identified in Example 1-1 was confirmed by FACS analysis.

Specifically, in order to investigate the abnormality of erythropoiesis, FACS analysis using Ter119 and CD71 antibodies was performed on the spleen and adult mouse bone marrow of embryo liver, newborn mouse, The ratio of erythrocytes was analyzed using the ratio. In the case of embryos and infants, the SO-S5 population was analyzed according to the degree of differentiation. In the case of adult, the proerythroblast (PEB, Ter119 ^- / CD71 ^high ) (EBC), Ter119 ⁺ / CD71 ^high ), chromophilic erythroblast (CEB, Ter119 ⁺ / CD71 ^med ) and orthochromatophilic erythroblast (OCEB, Ter119 ⁺ / CD7 ^low ) .

As a result, as shown in FIG. 2, it was confirmed that embryo and newborn mice had a larger number of cells belonging to the SO or S1 group that did not undergo differentiation in the experimental group (mutant) as compared with the control group. In particular, the number of cells in the S3 group that underwent differentiation was significantly reduced in the experimental group compared to the control group. Specifically, the number of embryos decreased from 65.7% to 27.1%, and that of the infant mice decreased from 58.7% to 1.8% , It was once again confirmed that erythrocyte differentiation is inhibited when the TMEM100 gene is deleted.

In addition, the number of PEBs or BEBs that did not undergo differentiation similar to that of the embryo and infant mice was not significantly different between the control and experimental groups, but the number of differentiated CEB or OCEB was significantly By confirming that it was significantly reduced in the experimental group, it was once again confirmed that the deletion of the TMEM100 gene inhibited erythrocyte differentiation.

From the above results, it was confirmed that the TMEM100 gene plays an essential role in the process of red blood cell differentiation in hematopoietic stem cells, so that the gene is essential for the hematopoietic process.

Example  2. Lymphocyte system  For cell development TMEM100  Analysis of gene effects

To analyze the effect of TMEM100 gene on the process of hematopoietic stem cell differentiation from lymphocytes, the number of B cells and T cells was measured by FACS analysis in a normal control (control) and an experiment group (mutant) in which TMEM100 gene was not expressed Respectively.

Specifically, among the whole lymphocytes, the cells belonging to the B cell lineage were singly labeled with B220 antibody or complexed with B220, CD43 and IgM antibodies, and cells belonging to the T cell line were labeled with CD3e antibody Respectively. More specifically, the adult femur was disrupted and the bone marrow was collected and made into a single cell using a syringe with a 26G needle. The cells were washed with staining buffer (PBS / 0.2% BSA / 5 mM glucose) and reacted with B220, B220 / CD43 / IgM, or CD3e antibody. The cells were washed with the staining buffer and FACS analysis was performed using FACSAria II from BD. Herein, the B220 antibody recognizes a 220 kDa isoform surface maker of CD45, which is generally expressed in B cell lineage ranging from early Pro-B cells to mature B cells; CD43 antibody recognizes CD43, an early B cell marker that expresses up to Pro-B cells during B cell development and does not express from pre-B cell stage; IgM antibodies recognize Immunoglobulin IgM expressed after the immature B cell stage but not to the Pre-B cells during B cell development; The CD3e antibody recognizes the 25-kDa ε chain of the T-cell receptor-associated CD3 complex expressed in Thymocyte, mature T lymphocyte, and NK-T cells.

As a result, as shown in FIG. 3, the number of B cells labeled with B220 was significantly decreased in the experimental group than in the control group. B cells were stained with progenitor B cells (B220 ⁺ / CD43 ⁺ ; Pro-B) and precursor B cells (B220 ⁺ CD43 ⁺ / CD43 ^- / lgM ^-; After a review by dividing the Pre-B), the number of Pro-B cells in the control group and test group is I did not have a difference, compared with the control group, the experimental group it was confirmed that the number of Pre-B cells decreased in the experimental group.

In addition, the number of CD3e-labeled T cells was not different between the control and experimental groups.

As a result, it was confirmed that when the TMEM100 gene is deleted, the differentiation of B cells is inhibited in the lymphoid cells, and that the TMEM100 gene plays an essential role in the process of lymphocyte differentiation from hematopoietic stem cells This gene was found to be essential for the hematopoietic process.

Example  3. For organ development TMEM100  Analysis of gene effects

Example  3-1. External analysis

To investigate the effect of the TMEM100 gene on the development of the organs involved in hematopoiesis, we examined the development of each organ, including the liver, spleen and bone marrow, in the normal control (con) and in the mutant in which the TMEM100 gene was not expressed Respectively.

Specifically, E14.5 embryo liver, spleen of 8-day-old baby spleen and adult bone marrow were collected and liver and spleen were first weighed. Each tissue was then disrupted with a syringe with a 26G needle, and the number of cells per tissue was measured with a Bio-Rad TC-10 cell counter.

As a result, as shown in FIG. 4, the embryo in which the TMEM100 gene was not expressed showed smaller parenchyma than the control group, and the weight and number of cells were significantly decreased. In addition, the spleen was small and pale, and the weight and number of cells were significantly reduced. In addition, it was confirmed that the total number of cells was decreased in the bone marrow, but no abnormal abnormality was found, but it was confirmed that the bone marrow of the control group was redder than the experimental group.

As a result, it was confirmed that the development of liver, spleen and bone marrow involved in hematopoiesis was not normally performed when the TMEM100 gene was deleted, and thus it was found that the gene is essential for hematopoiesis.

Example  3-2. Molecular mechanism analysis

In addition to analyzing the mechanism of inhibition of development of each organ by the deletion of the TMEM100 gene, the present inventor's previous study found suspicious cell death in the liver of an experimental embryo in which the TMEM100 gene was not expressed, The cell death was examined.

Concretely, double-staining was performed on Activated caspase3 (red), which is a marker for cell death, and markers (Dlk: hepatocyte, F4 / 80: macrophage, Ter119: erythroid lineage, Respectively. More specifically, the liver of E14.5 embryos was extracted, fixed with 4% paraformaldehyde, and made into paraffin blocks. The block was cut to a thickness of 5 μm and attached to a slide glass. Paraffin was removed by xylene, and the particles were subjected to hydration using 100%, 95%, 70% ethanol and PBS, followed by antigen retrieval. Activated caspase 3 antibody produced by Rabbit and antibody of each cell marker prepared in rat were mixed with the tissue on the slide glass for 30 minutes and double immunostaining was performed using Polink DS-RRt-Hu / Ms A Kit from GBI Respectively.

As a result, as shown in FIG. 5, most of the D1k-positive cells were activated caspase 3-positive cells in the experimental group (mutant) and the activated caspase 3-positive cells were partially activated in the F4 / 80- , It was confirmed that apoptosis was induced in Dlk-positive cells of hepatocytes and F4 / 80-positive cells of some macrophages. In contrast, in Terl 19-positive cells, almost no activated caspase 3-positive cells were found, indicating that apoptosis did not occur in cells belonging to Ter119-positive cells.

As a result, when the TMEM100 gene was deleted, it was confirmed that apoptosis was induced in the liver and spleen, which are involved in the hematopoiesis process, so that the gene is essential for the hematopoiesis process. In addition, it was found that the abnormalities of hematopoiesis due to the deletion of TMEM100 gene are due to apoptosis of peripheral cells, hepatocytes and / or macrophages, rather than apoptosis of cells during hematopoiesis.

Example  4. TMEM100  Causal analysis of abnormalities of hematopoiesis due to gene deletion

Through the above Example 3-2, it was confirmed that the TMEM100 gene deletion resulted in undifferentiation into red blood cells or lymphoid cells In order to analyze more specific causes, we analyzed the intrinsic differentiation potential of hematopoietic stem cells, which is a higher level of red blood cells or lymphoid cells, although it was found that the phenomenon is due to apoptosis of surrounding cells which regulate the hematopoiesis process.

Specifically, the ability of the cells belonging to the erythroid lineage to undergo colony forming cell (CFC) assay using CFU-E medium. More specifically, the liver and adult bone marrow of E13.5 embryos were collected and disrupted with a syringe with a 26G needle into single cells. 0.3 ml of Iscove's MDM / 2% FBS medium containing the above cells and 3 ml of Stemcell Technologies' MethoCult M3334 medium were mixed, and finally the cells were seeded at 35 mm to have 2x10 ⁵ bone marrow cells or 5x10 ⁵ liver cells per each culture dish And 1.1 ml each was dispensed into the culture dish. After incubation for ₂ days in a CO ₂ incubator, colonies were counted.

As a result, as shown in FIG. 6, it was confirmed that the ability of the hematopoietic stem cells to differentiate in the embryo did not show any significant difference between the experimental group and the control group, and the results of the analysis of adult cells also showed the above tendency Respectively.

These results indicate that abnormalities of hematopoiesis due to deletion of TMEM100 gene are due to abnormalities of surrounding cells rather than the inherent differentiation potential of hematopoietic stem cells.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> Gachon University of Industry-Academic cooperation Foundation <120> A pharmaceutical composition for preventing or treating blood          dyscrasia comprising TMEM100 protein <130> KPA150980-KR <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 405 <212> DNA <213> Artificial Sequence <220> <223> cDNA of TMEM100 <400> 1 atgactgaag agcccatcaa ggagatcctg ggagccccaa aggctcacat ggcagcgacg 60 atggagaaga gccccaagag tgaagttgtg atcaccacag tccctctggt cagtgagatt 120 cagttgatgg ctgctacagg gggtaccgag ctctcctgct accgctgcat catccccttt 180 gctgtggttg tcttcatcgc cggcatcgtg gtcaccgcgg tggcttacag cttcaattcc 240 catgggtcta ttatctccat ctttggcctg gttgttctgt catctggact ttttttacta 300 gcctccagtg ccttgtgctg gaaagtgaga caaaggagca agaaagccaa gagacgggag 360 agtcaaacag ctctcgtggc aaatcagaga agcttgtttg cttga 405 <210> 2 <211> 134 <212> PRT <213> Artificial Sequence <220> <223> Recombinant protein of TMEM100 <400> 2 Met Thr Glu Glu Pro Ile Lys Glu Ile Leu Gly Ala Pro Lys Ala His   1 5 10 15 Met Ala Ala Thr Met Glu Lys Ser Pro Lys Ser Glu Val Val Ile Thr              20 25 30 Thr Val Pro Leu Val Ser Glu Ile Gln Leu Met Ala Ala Thr Gly Gly          35 40 45 Thr Glu Leu Ser Cys Tyr Arg Cys Ile Pro Phe Ala Val Val Val      50 55 60 Phe Ile Ala Gly Ile Val Val Thr Ala Val Ala Tyr Ser Phe Asn Ser  65 70 75 80 His Gly Ser Ile Ile Ser Ile Phe Gly Leu Val Val Leu Ser Ser Gly                  85 90 95 Leu Phe Leu Leu Ala Ser Ser Ala Leu Cys Trp Lys Val Arg Gln Arg             100 105 110 Ser Lys Lys Ala Lys Arg Arg Glu Ser Gln Thr Ala Leu Val Ala Asn         115 120 125 Gln Arg Ser Leu Phe Ala     130

Claims (11)

Prevention or prophylaxis of one or more hematopoietic disorders selected from the group consisting of aplastic anemia, malignant lymphoma, leukemia, lymphocytopenia, myelodysplastic syndrome and myeloproliferative diseases comprising a TMEM100 (Transmembrane protein 100) protein or a polynucleotide encoding the same. A pharmaceutical composition for therapeutic use.
The method according to claim 1,
Wherein said TMEM100 protein promotes differentiation of hematopoietic stem cells.
3. The method of claim 2,
Wherein the differentiation of said hematopoietic stem cells promotes the development of red blood cells or lymphocyte cells.
The method according to claim 1,
Wherein the TMEM100 protein promotes the development of liver, spleen or bone marrow.
The method according to claim 1,
Wherein the protein consists of the amino acid sequence of SEQ ID NO: 2.
The method according to claim 1,
Wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1.
The method according to claim 1,
Wherein the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.
delete A method for treating a hematopoietic disorder, comprising administering a composition according to any one of claims 1 to 7 to a subject other than a human who has or is likely to develop hematopoietic disorders, a malignant lymphoma, leukemia, lymphopenia, myelodysplastic syndrome, Myeloproliferative disorder, myelodysplastic syndrome or myeloproliferative disorder.
For the improvement of one or more hematopoietic disorders selected from the group consisting of aplastic anemia, malignant lymphoma, leukemia, lymphocytopenia, myelodysplastic syndrome and myeloproliferative diseases including TMEM100 (Transmembrane protein 100) protein or a polynucleotide encoding the same Food composition.
For the improvement of one or more hematopoietic disorders selected from the group consisting of aplastic anemia, malignant lymphoma, leukemia, lymphocytopenia, myelodysplastic syndrome and myeloproliferative diseases including TMEM100 (Transmembrane protein 100) protein or a polynucleotide encoding the same Feed composition.

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