KR20180079033A - Cell secreting IGF-1 and IL-10, and use thereof - Google Patents

Abstract

The present invention relates to a cell secreting insulin-like growth factor-1 (IGF-1) and interleukin-10 (IL-10), and uses of the same. More specifically, the present invention relates to a cell secreting IGF-1 and IL-10, transformed to nucleic acids encoding IGF-1 and IL-10; and uses of the cell for suppression of cell or tissue transplant rejection, and prevention or treatment of fibrosis, liver diseases, graft-versus-host disease, inflammatory diseases, and autoimmune diseases. The cell secreting IGF-1 and IL-10, produced by a gene introduction system, have excellent co-expression and co-secretion abilities of IGF-1 and IL-10. Based on the above, the cell of the present invention, by suppressing the activation and proliferation of hepatic stellate cells, shows excellent therapeutic and prophylactic effects for liver diseases such as liver fibrosis and liver cirrhosis, and also may be utilized in therapeutic and prophylactic compositions for other diseases that need suppression of inflammation, immunomodulation, and tissue regeneration.

Description

Cells secreting IGF-1 and IL-10, and their uses {Cell secreting IGF-1 and IL-10, and use thereof}

The present invention relates to cells that secrete insulin like growth factor-1 (IGF-1) and IL-10 (interleukin-10), and uses thereof, and more particularly to a nucleic acid encoding IGF-1 and IL- For the prophylactic or therapeutic use of cells which secrete IGF-1 and IL-10 and which are transformed with the above-mentioned transformed cells, fibrosis of such cells, inhibition of liver disease, cell or tissue transplantation rejection reaction, graft versus host disease, inflammatory diseases and autoimmune diseases It is about.

The liver is traditionally known as an important organ for the absorption and metabolism of substances. Main functions of the liver include carbohydrate metabolism (glucose is stored in the form of glycogen), amino acid and protein metabolism, fat metabolism, bile emission (to help digest fat), bithimine and minerals metabolism, hormone metabolism, detoxification, , And bactericidal action to filter bacteria through the bloodstream flowing into the liver from the large intestine. Damage to these important institutions can cause serious injury to the human body.

Hepatopathy is characterized by liver fat accumulation in liver, alcoholic hepatitis, hepatitis A virus, hepatitis B virus, hepatitis C virus, alcoholic cirrhosis, rare diseases and immune mediated hepatitis and drug toxicity It is caused by a number of factors, including liver disease and unclear causes.

When hepatocytes are damaged by alcohol, virus, cholesterol, autoimmunity, etc., many inflammatory cells gather around the injured hepatocytes, and hepatocytes in the dormant state due to the stimulation of cytokines such as TGF-beta (Transforming growth factor beta) Hepatic stellate cells are activated to secrete a large amount of extracellular matrix such as collagen and accumulate them in the liver resulting in hepatic fibrosis. (liver cirrhosis).

Generally, liver fibrosis is reversible, unlike cirrhosis, is composed of thin fibrils and has no nodule formation. In addition, if the cause of liver damage is lost, normal recovery may be possible. However, if such a hepatic fibrosis mechanism is repeatedly continued, crosslinking between ECM connective tissues increases, thick fibrils accumulate, and the normal structure of hepatic lobules is lost and nodules are formed (Liver cirrhosis, hepatic fibrosis, hepatocellular carcinoma, hepatocellular carcinoma, and hepatocellular carcinoma).

Examples of the substance that inhibits hepatic fibrosis include PDGF activity inhibitors such as TGF-beta inhibitor, HGF promoter, PPARy ligand, Angiotensin inhibitor, PDGF inhibitor (anti-PDGF antibody or the like, RNAi molecule for PDGF, ribozyme, antisense Inhibitors of PDGF production such as nucleic acids, vectors expressing them, and transformed cells thereof), Relaxin or its production-promoting substance, substances inhibiting the production and secretion of extracellular matrix components, collagen such as TIMP Substances were reported. In the past decades, studies on astrocyte cells and studies on drugs that inhibit extracellular matrix secretion have been carried out at a cellular level and in animal experiments. In actual clinical studies, however, There has been a problem.

Recently, tissue regeneration research utilizing stem cells has been actively conducted. Stem cells produce the same cells as themselves and are characterized by proliferation. It also has plasticity that can be differentiated into necessary cells under the influence of the surrounding environment. The types of stem cells are divided into pluripotency cells and multipotency cells depending on the degree of differentiation. The precursor cells are embryonic stem cells (ES) and induced pluripotent stem cells (iPSC), and multipotent cells are mainly mesenchymal stem cells (MSC). do. The mesenchymal stem cells are most likely to be clinically used because they are not susceptible to cancer, have immunoregulatory functions, and can differentiate into fat, bone, cartilage, muscle, nerve, and myocardial cells.

There are increasing studies to use stem cells as a cell therapy agent. Recently, stem cells have been applied to various diseases such as cerebral infarction, traumatic nerve injury, and musculoskeletal diseases, and preclinical and clinical studies have been attempted. However, the current technical level is limited to the extraction and cultivation / proliferation and injection of simple stem cells. The disadvantage of such a single stem cell is that the tissue differentiation and tissue regeneration efficiency is low. In addition, it is known that the efficiency of hepatocyte differentiation using human embryonic stem cells, which are known to have total differentiation potential, is low (National Institute for Health Sciences, Center for Life Science, 2010, pp.1-5)

In order to improve the therapeutic effect, studies using various genetically modified stem cells have been carried out. However, in the introduction of genes into stem cells, the efficiency and effect of expression thereof have been questioned according to specific methods, and in particular, there has been a lack of research on multigen gene expression technology that allows the desired components to exhibit effective functions by introducing multiple genes State.

[Non-Patent Document 1] Diastase, mechanism of liver fibrosis. Korean Society of Hepatology 3rd Postgraduate Course. 2005; 9-19 [Non-Patent Document 2] Use of Stem Cells as a Evaluation Tool for Intractable Disease and New Drug Development, 2010, pp.1-5

Accordingly, the present inventors have studied a method of maximizing the therapeutic effect of a cell therapy agent, and have found that a specific gene that causes IGF-1 and IL-10 to coexpress and co-secrete in an effective amount in a cell The present inventors have developed a cell therapy agent using the same, and confirmed the excellent disease treatment effect of the cell therapy agent, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a cell that secretes IGF-1 and IL-10, which is transformed with a nucleic acid encoding insulin like growth factor-1 (IGF-1) and interleukin-10 will be.

It is another object of the present invention to provide a method for preventing or treating a disease selected from the group consisting of fibrosis comprising the cell as an active ingredient, liver disease, inhibition of cell or tissue transplant rejection reaction, graft versus host disease, inflammatory disease and autoimmune disease And to provide a pharmaceutical composition.

In order to accomplish the above object, the present invention provides a method for producing an insulin-like growth factor-1 (IGF-1) and IL-10 (interleukin-10) ≪ / RTI >

In order to accomplish another object of the present invention, the present invention provides a method for preventing or treating fibrosis, liver disease, cell or tissue transplant rejection inhibition, graft versus host disease, inflammatory disease and autoimmune disease Or a pharmaceutically acceptable salt thereof.

Hereinafter, the present invention will be described in detail.

The present invention provides cells that secrete IGF-1 and IL-10, transformed with a nucleic acid encoding insulin like growth factor-1 (IGF-1) and interleukin-10 (IL-10).

As used herein, the term "protein" may be used interchangeably with "polypeptide" or "peptide" and refers to a polymer of amino acid residues, as is commonly found in natural state proteins.

 In the present invention, "nucleic acid" or "polynucleotide" refers to deoxyribonucleotides or ribonucleotides in the form of single- or double-stranded. Unless otherwise constrained, also includes known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.

As used herein, the term " expression "refers to the production of a protein or nucleic acid in a cell.

The term " cell " used in the present invention means an autologous, allogenic, xenogenic animal cell. The origin of the cell is not particularly limited, and examples thereof include skin, cartilage, Including cells differentiated into various tissues such as blood vessels, brain, liver, heart, ligament, muscle, spinal cord, blood, bone marrow, lung, tooth, nerve, cornea, retina, esophagus, vertebrae, kidney, pancreas or urethra, Stem cells, induced pluripotent stem cells, mesenchymal stem cells, undifferentiated precursor cells. The cells of the present invention may preferably be one or more selected from the group consisting of embryonic stem cells, adult stem cells, induced pluripotent stem cells, mesenchymal stem cells and undifferentiated progenitor cells.

The most preferred cells in the present invention may be mesenchymal stem cells. In addition to the proliferative and differentiating ability of mesenchymal stem cells, the expression of MHC class I is low and the expression of MHC class 2 is not induced. Therefore, since xenograft does not induce in vivo immune response, This is possible. In addition, mesenchymal stem cells have been reported to migrate to damaged tissues or organs in response to SDF-1α, CXCR4 system, immune response-inducing growth factors and cytokines (TGFβ1, IL1β, TNFα, PDGF-AB) In conclusion, it is known that paracrine effects on the regeneration process of injured tissues are produced by secretion of various kinds of nutrients (HGF, FGFs, VEGF, EGF, GDNF, BDNF, PDGF etc.) It is possible to trans-differentiate into damaged cells. At present, molecular mechanisms of regeneration of mesenchymal stem cells are not known, but when cytokines are secreted from damaged tissues, they activate mesenchymal stem cells that remain in niche, It receives additional homing signals from the mouth tissue, promotes mesenchymal stem cells to proliferate and differentiate into mature cells, and newly formed mature cells migrate to the injured tissue and contribute to the regeneration of injured tissue.

The mesenchymal stem cells may be derived from one or more tissues selected from the group consisting of fat, bone marrow, umbilical cord, umbilical cord blood, placenta, synovial membrane, periosteum and chondrocyte, although the origin thereof is not particularly limited.

The IGF-1 (insulin-like growth factor-1) is mainly synthesized in the liver and released into the blood to promote cell growth in vivo and to promote regeneration. The receptor of IGF-1 is known to exist not only in hepatocytes but also in various tissues such as muscle, bone, cartilage, intestin. It has been reported that IGF-1 promotes liver regeneration in an animal model of liver injury and has an anti-fibrotic effect. In particular, inhibition of the activation of smad3 by phosphatidylinositol 3 kinase / Akt signaling may inhibit the response to TGFβ (Song et al. J Biol Chem 278: 38342-38351). This is one of the important mechanisms to inhibit liver fibrosis. It has also been reported that hepatocyte growth and viability are enhanced via MAPK and beta-catenin signaling (Kunda et al. Cell Struct Funct 28: 255-263). IGF-1, which is involved in the maintenance of homeostasis, stimulates hepatic stellate cells and helps secrete HGF (Hepatocyte growth factor), another growth factor that has anti-fibrosis and liver protective functions. It can be said that it is an important cytokine.

In the present invention, the IGF-1 protein is not particularly limited as long as it is known in the art as IGF-1. For example, the IGF-1 protein may have the amino acid sequence shown in SEQ ID NO: The IGF-1 may also be meant to include functional equivalents thereof.

The IL-10 (interleukin-10) is known as a cytokine synthesis inhibitor factor or a cytokine synthesis inhibitory factor (CSIF), and is a strong immunoregulatory factor of hematopoietic cells and immune cells. IL-10 inhibits a number of cell activation including T cells, mononuclear cells and macrophages. In particular, it reduces the expression of MHC class 2 (major histocompatibility complex class 2) in mononuclear cells and inhibits the action of Th1 (helper T cell1). It also inhibits inflammation by inhibiting the chemotaxis of inflammatory cells by inhibiting the secretion of cytokines from macrophages. IL-10, IL-1, Interferon-γ (INF-γ), Tumor Necrosis Factor (TNF), TGF-β, and IL-1, which are inflammatory cytokines of cells such as Th1 cells, natural killer cells, monocytes and macrophages -6, IL-8, and IL-12. In addition, IL-10 inhibits the secretion of cytokines secreted from antigen-presenting cells, thereby exerting its effect and inhibiting antigen presentation of antigen-presenting cells such as monocytes, macrophages or dendritic cells. Thus, IL-10 may play a role as a substantial inhibitor of cellular immune response. And IL-10 inhibits the function of inflammatory cells that invade tissues. The liver is an organ that collects a lot of inflammatory cells and immune cells. Therefore, the onset of liver disease is caused by the imbalance of these inflammatory cells and immune cells and the cells regulating their activity, resulting in the induction of cirrhosis. Thus, inhibition of abnormal inflammatory responses by IL-10 and induction of immune modulation are helpful in the treatment of liver disease.

In the present invention, the IL-10 protein is not particularly limited as long as it is known in the art as IL-10. For example, the IL-10 protein may have the amino acid sequence shown in SEQ ID NO: 14. The IL-10 may also be meant to include functional equivalents thereof.

Said functional equivalent means at least 70%, preferably at least 80%, more preferably at least 90% sequence identity with the amino acid sequence of IGF-1 (particularly SEQ ID NO: 13) or IL-10 (I. E., Identity). For example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% Sequence homology of the nucleotide sequences of the nucleotide sequences of the nucleotide sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, Quot; refers to a polypeptide having substantially the same physiological activity as the polypeptide represented by SEQ ID NO: 13 or SEQ ID NO: 14. Said functional equivalents may result from the addition, substitution or deletion of part of the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14. The substitution of the amino acid is preferably a conservative substitution. Examples of conservative substitutions of amino acids present in nature are: aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (lie, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids Asp, Glu), basic amino acids (His, Lys, Arg, Gin, Asn) and sulfur containing amino acids (Cys, Met). Such functional equivalents also include variants in which a portion of the amino acid is deleted in the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14. The deletion or substitution of the amino acid is preferably located in a region that is not directly related to the physiological activity of the polypeptide of the present invention. In addition, deletion of the amino acid is preferably located in a portion not directly involved in the physiological activity of the protein. Also included are variants in which some amino acids are added at both ends or sequences of the amino acid sequence of the protein. Also included within the scope of the functional equivalents of the present invention are polypeptide derivatives in which some of the chemical structures of the polypeptides are modified while maintaining the basic skeleton of the polypeptide according to the present invention and its physiological activity. This includes, for example, structural modifications to alter the stability, shelf stability, volatility, or solubility of the protein.

The nucleic acid encoding the IGF-1 or IL-10 may be prepared by a recombinant method known in the art (Sambrook, Fritsch and Maniatis, Molecular Cloning, A laboratory Manual, Cold Spring Harbor laboratory press, 1989; Short Protocols in Molecular Biology, John Wiley and Sons, 1992). For example, there are techniques for PCR amplification, chemical synthesis, or cDNA sequence production to amplify nucleic acid from a genome.

The IGF-1 protein having the amino acid sequence represented by SEQ ID NO: 13 may be encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 15 or SEQ ID NO: 17, but not limited thereto.

The IL-10 protein having the amino acid sequence represented by SEQ ID NO: 14 may be encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 18, but not limited thereto.

The term "transformed" in the present invention means all the activities in which a specific trait occurs, disappears or is controlled through mutation such as insertion, deletion or substitution on the DNA base sequence. In particular, in the present invention, Which means that the genetic properties of

In the present invention, the transformation may be mediated by a vector comprising at least one selected from the group consisting of a nucleic acid encoding IGF-1 and a nucleic acid encoding IL-10. That is, the transformation can be carried out by 1) introducing a vector into which a gene multiplex expression system capable of simultaneously expressing two genes, IGF-1 and IL-10, or 2) expressing IGF-1 And a method of introducing IL-10-expressing vector and IL-10-expressing vector into cells simultaneously or sequentially.

Most preferably, the transformation is carried out by a single vector to which a gene-multiple expression system is applied. The present inventors have produced a novel vector system for co-expression of IGF-1 and IL-10. This is well illustrated in the examples herein.

In an embodiment of the present invention, two different promoters of the pCHO vector (EF2 / CMV hybrid promoter and CMV / EF1 hybrid promoter) were cut and then introduced into a shuttle vector and a unique vector system And then transformed into adenovirus. This is because we tried to overcome the disadvantages of 2A or IRES system which was commonly used to simultaneously express two genes. Since the two systems commonly use one promoter, the efficiency of protein expression is low and the amount of expression of two proteins is different. In addition, the 2A system also has the disadvantage that separation of the two proteins does not occur during translation. In order to overcome the above disadvantages, the present inventors produced a unique expression vector system employing the EF2 / CMV hybrid promoter and the CMV / EF1 hybrid promoter, and utilized the two proteins of IGF-1 and IL-10 with high efficiency Gt;

The nucleic acid encoding said IGF-1 or IL-10 is operably linked to an expression control sequence and inserted into an expression vector. The term 'operably linked' as used herein means that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by other nucleic acid fragments. Also, an " expression control sequence " means a DNA sequence that regulates the expression of a nucleic acid sequence operably linked to a particular host cell. Such regulatory sequences include promoters for initiating transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation. Examples of the promoter include a CMV promoter, a CAG promoter (Hitoshi Niwa et al., Gene, 108: 193-199, 1998), and a promoter that induces the expression of a target gene at all times in all time periods. 1991), the CaMV 35S promoter (Odell et al., Nature 313: 810-812, 1985), the Rsyn7 promoter (US patent application Ser. No. 08 / 991,601) , Rice Actin promoter (McElroy et al., Plant Cell 2: 163-171, 1990), ubiquitin promoter (Christensen et al., Plant Mol. Biol. 12: 619-632, 1989), ALS promoter U.S. Patent Application No. 08 / 409,297), EF2 / CMV hybrid promoter, CMV / EF1 hybrid promoter, and the like.

Preferably, the vector of the present invention is characterized by using the EF2 / CMV hybrid promoter and the CMV / EF1 hybrid promoter simultaneously. The combination of genes operatively linked to each promoter is not particularly limited. For example, a polynucleotide encoding IL-10 is contained in the EF2 / CMV hybrid promoter and a polynucleotide encoding IGF-1 is encapsulated in the CMV / EF1 hybrid promoter Or the EF2 / CMV hybrid promoter may be operably linked to a polynucleotide encoding IGF-1, and the CMV / EF1 hybrid promoter may be operably linked to a polynucleotide encoding IL-10.

In the present invention, the vector is most preferably

A polynucleotide encoding an EF2 / CMV hybrid promoter and IL-10 operably linked thereto; And

A polynucleotide encoding a CMV / EF1 hybrid promoter and IGF-1 operably linked thereto.

In one embodiment of the present invention, a unique expression vector system to which the EF2 / CMV hybrid promoter and the CMV / EF1 hybrid promoter are applied is produced, and the protein alone or in co-expression according to the combination of genes linked to each promoter, . As a result, when the IL-10 expression gene is linked to the EF2 / CMV hybrid promoter and the IGF-1 expression gene is linked to the CMV / EF1 hybrid promoter, co-expression and co-expression (co -secretion).

The vector may further comprise a sequence encoding a poly A tail (polyadenylation sequence). The sequence encoding the poly A tail is not particularly limited in its kind and includes SV40-derived polyadenylation sequence (poly A), beta globin polyA, HSV TK polyA, CMV polyA sequence, and the like. The poly A tail (poly A tail, polyadenylation sequence) is preferably, but not limited to, located downstream of the gene insertion site. In one embodiment of the present invention, CMV pA is constructed downstream of the gene insertion site connected to the EF2 / CMV hybrid promoter and SV40 pA is constructed downstream of the gene insertion site connected to the CMV / EF1 hybrid promoter (see FIG. 1) .

The vector may further comprise a secretion signal sequence. The secretory signal sequence may be preferably linked to a nucleic acid encoding IGF-1 or / and IL-10. The secretory signal sequence includes Igκ chain leader, secretory signal sequence of seminal RNase, SEC2 sequence (N-terminal 28 amino acids of human fibrillin-1), a FIB sequence (nucleotides 208-303 derived from rat fibronectin mRNA sequence), or a signal peptide of FGF-4.

Also, in the present invention, 'expression vector' refers to a plasmid, a virus vector, or the like which can insert a nucleic acid encoding IGF-1 or / and IL-10 and can express the nucleic acid in a host cell. Or other media. In the present invention, it may preferably be a viral vector.

The type of the viral vector is not particularly limited as long as it is known in the art. For example, the viral vector may be an adenoviral vector, an adeno-associated virus vector, a retroviral vector, A herpes simplex virus vector and a lentiviral vector. The term " herpes simplex virus vector "

In the present invention, a viral vector containing a nucleic acid encoding IGF-1 and IL-10 is preferably a gene expression system (a gene expression system unique to the present invention to which the EF2 / CMV hybrid promoter and CMV / EF1 hybrid promoter is applied) May be constructed on a shuttle vector and the shuttle vector may be constructed through homologous recombination with a rescue vector, and a schematic diagram thereof is provided in FIG.

The present invention relates to a use of a cell which secretes IGF-1 and IL-10, which is transformed with a nucleic acid encoding the insulin like growth factor-1 (IGF-1) and IL-10 (interleukin-10) Lt; / RTI > That is, the present invention relates to a method for preventing or treating fibrosis, liver disease, cell or tissue transplant rejection inhibition, graft-versus-host disease, inflammatory disease, autoimmune disease, A pharmaceutical composition for the prevention or treatment of a disease to be selected.

The liver disease is known to be a liver disease in the art, and the kind thereof is not particularly limited as long as the effect of preventing or ameliorating or treating a symptom or a disease by IGF-1 is known. For example, hepatic fibrosis, liver cirrhosis, Fatty liver. ≪ / RTI >

The fibrosis is known as a disease caused by tissue fibrosis in the art, and the kind thereof is not particularly limited as long as the effect of preventing or ameliorating or treating a symptom or a disease is known by IGF-1. For example, , Pulmonary fibrosis, dermal fibrosis, cardiac fibrosis, joint fibrosis, nerve fibrosis, muscular fibrosis and peritoneal fibrosis.

The inflammatory disease is known as a disease mediated by an inflammatory reaction in the art, and the kind thereof is not particularly limited as long as the effect of preventing or ameliorating or treating a symptom or a disease by IL-10 is known. However, for example, Inflammatory bowel disease, inflammatory vascular disease, inflammatory rheumatoid arthritis, recurrent stenosis, psoriasis, multiple sclerosis and inflammatory lung disease, in a mammal selected from the group consisting of gastritis, rhinitis, conjunctivitis, asthma, bronchitis, dermatitis, atopic dermatitis, .

The autoimmune disease is known in the art as an autoimmune disease, and the type thereof is not particularly limited as long as the effect of preventing or ameliorating a disease or a symptom is known by IL-10. However, examples thereof include rheumatoid arthritis, Atherosclerosis, aphthous stomatitis, chronic thyroiditis, ulcerative colitis, multiple myositis, multiple sclerosis, autoimmune hemolytic anemia, autoimmune encephalomyelitis, fibromyalgia and temporal lobe Arteriosclerosis or arteriosclerosis.

The term "cell therapeutic agent" used in the present invention is a drug used for the purpose of treatment, diagnosis and prevention with cells and tissues prepared by isolation, culture and special manipulation from animals (especially human) Refers to a drug used for therapeutic, diagnostic and prophylactic purposes through a series of actions such as homologous or xenogeneic cell propagation, selection, or otherwise altering the biological characteristics of a cell to restore it. The cell therapy agent is classified into a somatic cell therapy agent and a stem cell treatment agent depending on the degree of cell differentiation, and the present invention can be particularly related to a stem cell treatment agent.

The term " prophylactic " as used herein refers to any action that inhibits the onset or delayed progression of the desired disease upon administration of the composition of the present invention.

As used herein, the term " treatment " means any action in which the symptoms of the desired disease are alleviated or beneficially altered upon administration of the composition of the present invention.

The cells that secrete IGF-1 and IL-10 according to the present invention may be supplied in the form of a pharmaceutical composition for administration into humans. The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. The term " pharmaceutically acceptable " as used herein refers to a substance which is not toxic to cells or humans exposed to the composition. The carrier can be used without limitation as long as it is known in the art such as a buffer, a preservative, an anhydrous agent, a solubilizer, an isotonic agent, a stabilizer, a base, an excipient, a lubricant and a preservative. The pharmaceutical compositions of the present invention may be prepared according to techniques commonly used in the form of various formulations. For example, in the case of an injectable preparation, it may be prepared in the form of a unit-containing ampoule or a composition containing a plurality of doses. For general principles of pharmaceutical formulations of the pharmaceutical compositions according to the present invention, reference can be made to the following references: Cell Therapy; Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, G. Morstyn & W. Sheridan, Cambridge University Press, 1996; And Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

The pharmaceutical composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration Lt; / RTI > The pharmaceutical composition of the present invention can be formulated into a parenteral administration preparation according to the administration route as described above. These formulations are described in Remington's Pharmaceutical Science, 15th edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a commonly known formulary for all pharmaceutical chemistries.

The total effective amount of the cell treatment agent of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol administered for a long period in multiple doses. In the pharmaceutical composition of the present invention, the content of the active ingredient may be varied depending on the degree of the disease. The dose of the cell treatment agent of the present invention may be appropriately adjusted depending on various factors such as the route of administration and the number of treatments of the pharmaceutical composition as well as the patient's age, body weight, health condition, sex, severity of disease, A person skilled in the art will be able to determine an appropriate effective dose depending on the specific disease use for the cell treatment agent of the present invention. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, administration route and administration method as long as the effect of the present invention is exhibited.

The present invention relates to IGF-1 and IL-10 secretory cells produced by a unique gene introduction system, and the cells produced in the present invention have excellent coexpression and coexistence of IGF-1 and IL-10. Based on this, the cells of the present invention inhibit the activation and proliferation of hepatic stellate cells, thereby being excellent in the treatment and prevention of liver diseases such as liver fibrosis and cirrhosis, and also in suppressing inflammation, immunoregulation and regeneration And can be used for a composition for treating and preventing other diseases.

Figure 1 shows the unique adenoviral vector system produced in the present invention to co-express and co-secrete IGF-1 and IL-10. Specifically, the present invention shows a vector system in which a specific shuttle vector and an adenovirus plasmid constructed to coexpress IGF-1 and IL-10 are recombined.
FIG. 2 shows the result of confirming that PacI was treated and electrophoresis after the recombination reaction in order to first verify whether the recombination of the shuttle vector and the adenovirus plasmid was normally performed.
Figure 3 shows the primer design sites on which these sequence constructs were based, with respect to the primers used for secondary verification of whether the recombinant adenovirus vectors obtained by recombination of shuttle vectors and adenovirus plasmids were constructed according to the intended configuration. Specifically, the primers were constructed based on the regions (P1 and P2) where the gene was actually introduced, the adenovirus gene itself (P3), and the P4 region in which the adenovirus should be eliminated due to homologous recombination with pShuttle, Primer sequence information is shown in Table 1.
FIG. 4 shows the result of inserting IGF-1 or IL-10 gene into mesenchymal stem cells and then examining the expression level at the gene level. No treat is a control group with no treatment, and Empty is a group treated with adenovirus that does not introduce the gene into mesenchymal stem cells. In addition, IGF-1 or IL-10 is a group treated with adenovirus expressing each gene to mesenchymal stem cells. At this time, PCR was performed to confirm the expression of the gene, and it was verified that the expression amount of IGF-1 or IL-10 optimized for the codon inserted into the adenovirus vector was higher than that of the endogenous gene (endogenous IGF-1 or IL-10) Respectively.
FIG. 5 shows the results of analysis of protein expression of IGF-1 or / and IL-10 gene introduced into mesenchymal stem cells by measurement of secretory proteins present in cell culture medium. The expression of the two proteins secreted from the cells was measured by an ELISA method. No treat is a control group with no treatment, and Empty (labeled E) / E is a group of adenovirus treated with mesenchymal stem cells without any genes. The preceding part based on the / (slash) symbol means a gene inserted into the multiple cloning site according to the first promoter (EF2 / CMV hybrid promoter), and the following part means a gene inserted into the second promoter (CMV / EF1 hybrid promoter) Means a gene inserted at the following multiple cloning site. For example, in a recombinant adenovirus system capable of expressing two genes at the same time, E / IGF-1 does not insert anything in the multiple cloning site according to the first promoter (EF2 / CMV hybrid promoter) and the second promoter (CMV / EF1 Hybrid promoter) is a group of recombinant adenovirus system containing IGF-1 and introducing adenovirus into mesenchymal stem cells.
Figure 6 shows the results of verifying the inhibitory effect of IGF-1 on collagen synthesis in liver disease. LX2 (hepatic stellate cells), a representative cell line of liver disease, was treated with TGFβ1 and then treated with IGF-1 at different concentrations to evaluate collagen synthesis inhibitory ability.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Genetically engineered recombinant vector system

<1-1> Construction of Recombinant Adenovirus Vector

A special recombinant adenovirus vector system expressing IGF-1 (NCBI Reference Sequence: NP_001104754.1, SEQ ID NO: 13) and IL-10 (NCBI Reference Sequence: NP_000563.1, SEQ ID NO: 14) was constructed. Adenovirus as a basic construct was purchased from Agilent Technologies, Inc. AdEasy Adenoviral Vector System pAdEasy-1 vector (Catalog # 240005) and pShuttle vector (Catalog # 240006). The pShuttle vector lacks its own promoter, and the restriction enzyme recognition sequence at the site where the gene can be cloned is limited, so that five restriction enzyme recognition sites of Scal, Nhel, Mrel, Fsel and Ascl are replaced between XhoI and HindIII (See Fig. 1). Then, as shown in FIG. 1, a system for expressing the two genes IGF-1 and IL-10 was introduced at the site from Scal to Ascl, and the system was constructed using the pCHO 1.0 plasmid (Freedom® pCHO 1.0 Vector (CMV / EF1 hybrid promoter), multiple cloning site (MCS), and poly A tail (CMV pA and SV40 pA, respectively) required for the expression of the two proteins (EF2 / CMV hybrid promoter, CMV / EF1 hybrid promoter, Quot;) portion was extracted. The shape of the final shuttle vector of the new system after reconstruction with the pAdEasy-1 vector is shown in FIG. Using the shuttle vector newly generated in the present invention, six vectors (pAd_E / IGF-1, pAd_IGF-1 / E, pAd_E / IL-10, pAd_IL-10 / E, pAd_IGF-1 / IL-10, pAd_IL-10 / IGF-1). Using these vectors, cell lines expressing the IGF-1 or / and IL-10 gene described below were prepared.

The IGF-1 gene sequence (SEQ ID NO: 17) and the IL-10 gene sequence (SEQ ID NO: 18) inserted into the vector are represented by GenBank ID: NM_001111284.1 (IGF-1, SEQ ID NO: On the basis of the sequence information described in NM_000572.2 (IL-10, SEQ ID NO: 16), a plasmid in which the amino acid sequence to be translated was identical but the base sequence was replaced with a codon having a high preference for human cells was applied.

<1-2> Confirmation of normal production of recombinant vector system

The recombinant adenovirus vector prepared in <1-1> was firstly verified whether or not the recombinant adenovirus vector was normally recombined. The recombinant adenovirus vectors were digested with restriction enzyme PacI and then electrophoresed on agarose gel. PacI restriction enzymes exist in two adenoviral vector sequences and are homologous recombination with pShuttle, so that they are used as one of the basic recombinant adenovirus vector production assays.

As a result, as shown in Fig. 2, it was confirmed that normal recombination occurred between the two vectors.

It was also verified in a second way that the recombinant adenovirus vector was constructed in the intended configuration. In order to verify the sequence of the prepared recombinant adenovirus vector, primers for four regions (P1, P2, P3, P4) were designed as shown in FIG. Sequence information of the primers used is shown in Table 1 below. As a result of sequencing using the above primers, the desired sequence was sequenced without any mutations such as mutations in the P1, P2 and P3 regions in all of the six recombinant vectors produced, whereas sequencing data for the P4 region And confirmed that they did not appear. Thus, it was verified that a desired recombinant adenovirus vector was produced.

Marker order Source ^* P1 (primer region # 1) SEQ ID NO: 1 sense 5'-CCT CTC TTC CCC AGA CCT-3 ' pCHO vector sequence information P2 (primer region # 2) SEQ ID NO: 2 sense 5'-ACA ACA AAA CCG AAC AAA AG-3 ' pCHO vector sequence information P3 (primer region # 3) SEQ ID NO: 3 sense 5'-AAA ACC ATC CTG CAT ACC AA- 3 ' Adeno vector sequence information P4 (primer region # 4) SEQ ID NO: 4 sense 5'-ATC CTA CGT CTC GAC CGA TG-3 ' Adeno vector sequence information

Origin: Designed in sequence in the backbone of pCHO and pAdeno vector.

&Lt; Example 2 >

Generation of co-expression and co-secretion cells

<2-1> Extraction, culture and stem cell function of human mesenchymal stem cells

Derived human mesenchymatous stem cell (MSC) was obtained by the following method. Specifically, adipose tissue was cut into surgical scalpels and washed three times with phosphate buffered saline (PBS) (Sigma, St. Louis, Mo.). Subsequently, the finely cut fat tissue was transferred into a 50 ml conical tube, filled with phosphate buffered saline, shaken thoroughly, and centrifuged. Then, the cells were filled up to 50 ml with Dulbecco's modified Eagle medium (DMEM) containing 0.2% collagenase, and reacted at 37 ° C for 90 minutes. After centrifugation at 2000 rpm for 10 minutes, undissolved fat tissue was removed from the upper layer, followed by washing, centrifugation and removal with general DMEM. The isolated adipose-derived stem cells were proliferated by culturing in a 5% CO ₂ incubator at 37 ° C using serum-free, chemically defined media. It can also be grown using DMEM supplemented with 10% fetal bovine serum (FBS). The proliferation of mesenchymal stem cells is mediated by stem cell-positive antigens CD44, CD105, CD29 and stem cell negative antigens CD34 and CD45 using fluorescence-activated cell sorting (FACS) Respectively.

The mesenchymal stem cells thus prepared were used for the production of cell therapeutic agents and evaluation of efficacy in the following experiments.

<2-2> Verification of gene expression by intracellular gene transfer and RT-PCR

The pAd_E / IGF-1 and pAd_E / IL-10 recombinant vectors prepared in Example 1 were introduced into mesenchymal stem cells obtained in <2-1> through adenoviruses. Methods for infecting adenovirus were performed according to a conventional method with reference to the manufacturer's manual. In the present invention, viruses were treated within a MOI range of 10 to 500. In order to verify whether the mesenchymal stem cells transfected with IGF-1 and IL-10, respectively, actually express the gene of interest, they were confirmed by polymerase chain reaction (RT-PCR). Expression of the inserted gene was optimized by codon optimization in the present invention, and GAPDH was used as a housekeeping gene.

The RT-PCR was performed as follows. Cells obtained from each group were washed three times with PBS, and the cells were recovered with trypsin-EDTA and extracted with TRIzol (Life Technologies, Inc. Grand Island, NY) Respectively. CDNA was prepared with 1 ㎍ of the extracted RNA and amplified at 27 cycles under RT-PCR (denaturation at 95 캜 for 30 seconds, annealing at 55 캜 for 30 seconds, synthesis at 72 캜 for 30 seconds) And the change in gene expression was measured. The primer sets used in the RT-PCR are shown in Table 2 below.

Marker order Source ^* IGF-1 order
Number 5 sense 5'-GAG ACA GGG GCT TTT ATT TC-3 ' NM_001111284.1 order
Number 6 Anti
sense 5'-AGT TCT TGT TTC CTG CAC TC-3 ' Codon optimized IGF-1 order
Number 7 sense 5'-TGA AGA TGC ACA CCA TGA-3 ' NM_001111284.1
(Codon optimized for human) order
Number 8 Anti
sense 5'-GTG GGC TTG TTG AAG TAG AA- 3 ' IL-10 order
Number 9 sense 5'-CTT TCA AAT GAA GGA TCA GC-3 ' NM_000572.2 order
Number 10 Anti
sense 5'-CTC TTG TTT TCA CAG GGA AG-3 ' Codon optimized IL-10 order
Number 11 sense 5'-AGC GAG ATG ATC CAG TTC TA-3 ' NM_000572.2
(Codon optimized for human) order
Number 12 Anti
sense 5'-GAA ATC TGT GGC ATC TTC TC-3 '

The PCR results are shown in FIG. In the mesenchymal stem cells transformed to express each gene, it was verified that the amount of IGF-1 or IL-10 expressed by the codon-optimized IGF-1 or IL-10 expressed in the present invention was larger than that of endogenous IGF-1 or IL-10.

<2-3> Protein secretion of mesenchymal stem cells into which IGF-1 or / and IL-10 is introduced

When a gene encoding IGF-1 and IL-10 is introduced into a mesenchymal stem cell through the recombinant adenoviral vector system of the present invention, the transformed mesenchymal stem cell line secretes the two proteins smoothly ELISA was performed to confirm the presence or absence of the disease. IL-10, pAd_IL-10 / E, pAd_IGF-1 / IL-10 and pAd_IL-10 / IGF- 1). Adenovirus infection was performed according to a conventional method in the art. In this experiment, viruses were treated within a MOI range of 10 to 500.

After the day of virus treatment, the amount of protein secreted from each cell was measured. The amount of secreted protein was examined by the following method. IGF-1 human elisa kit (Abcam, ab100545) was used for IGF-1 and IL-10 human elisa kit (Invitrogen, KHC0101) for IL-10 The amount of protein was measured.

The amount of protein secretion in each experimental group is shown in FIG. It was confirmed that IGF-1 is expressed within the range of about 1 to 40 ng / mL, and IL-10 is expressed within the range of about 5 to 2000 ng / mL. In particular, when the pAd_IGF-1 / IL-10 and pAd_IL-10 / IGF-1 vectors were designed to express both genes simultaneously, it was confirmed that the amount of secreted protein was significantly different depending on the gene insertion position. From this, it was confirmed that IL-10 is secreted most efficiently by the EF2 / CMV hybrid promoter such as pAd_IL-10 / IGF-1 and when IGF-1 is co-expressed and secreted by the CMV / EF1 hybrid promoter.

&Lt; Example 3 >

Verification of usefulness as a cell therapy agent

The efficacy of IGF-1 and IL-10 gene co-expression and co-secretion cells (hereinafter referred to as IGF-IL-MSC) prepared in Example 2 above was evaluated.

<3-1> Confirmation of efficacy by single secretion factor treatment

In order to test whether IGF-1 inhibits collagen synthesis, which is the cause of fibrosis, IGF-1 inhibited collagen synthesis in LX2 cells, a typical cell line of liver disease. first, LX2 cells were activated with TGFβ1 (PeproTech), and then the ability of collagen synthesis inhibition was evaluated by treating IGF-1 at various concentrations as shown in FIG.

As a result, as shown in FIG. 6, it was confirmed that collagen type 1 was increased in activated LX2 cells when TGFβ1 was treated at 2 ng / ml, and collagen type 1 expression was decreased when IGF-1 was treated at 1 ng / Respectively.

<3-2> Immunophenotypic analysis

Flow cytometry analysis was performed to analyze surface expression marker expression of IGF-IL-MSC. In subculture 5, we obtained a rapidly dividing homogeneous population of cells with fibroblastoid morphology. Cells were fixed with 70% ethanol at 4 ° C and stained for 30 minutes on ice with primary antibodies recognizing various surface molecules. Phycoerythrin-conjugated (PE) mouse anti-human CD29 (BD Biosciences, USA), FITC (fluorescein isothiocyanate-conjugated) mouse anti-human CD31 (BD Biosciences), PE mouse anti- Human CD34 (BD Biosciences Pharmingen, San Diego, CA, USA), PE mouse anti-human CD45 (BD Biosciences), FITC mouse anti- Mouse anti-human CD90 (BD Biosciences), and PE anti-human CD105 / endoglin (R & D system, Minneapolis, MN, USA). The immunophenotype of MSC was analyzed using a FACS Caliber flow cytometer (BD Biosciences, Bedford, MA, USA) with CELLQuest software (BD Biosciences).

CD29, CD44, CD73, CD90, CD105, CD31, CD34 and CD45 were used to analyze the surface markers of the IGF-IL-MSCs prepared in the present invention. Analysis of flow cytometry showed that CD29, CD44, CD73, CD90, and CD105 surface markers were highly expressed in IGF-IL-MSC, and CD31, CD34 and CD45 surface markers were absent. This indicates that the MSC surface markers obtained in Example <2-1> were not changed by the introduction of IGF-1 and IL-10 gene.

<3-3> Identification of stemness

IGF-IL-MSC was evaluated for its ability to proliferate and its stemness was confirmed. Cell proliferation and viability were evaluated by WST-1 cell proliferation reagent (WST-1 Cell Proliferation Reagent, Roche, USA) and trippin blue bezel assay. WST-1 cell proliferation assay for 5x10 cells in 96-well plates ³ cells / well were seeded (seeding) to each well. Cells were cultured for 4 days (0 days), 24 days (1 day), 48 days (2 days) and 72 hours (3 days) and then 10 μl WST-1 reagent was added to each well. The reaction proceeded at 37 [deg.] C for 2 hours with 5% CO2. The absorbance of each sample was measured at 450 nm using a microplate reader. For trypan blue exclusion analysis, cells were seeded at 6 x 10 ⁵ cells / well in 6-well culture plates and incubated at 37 [deg.] C with 5% CO2. After 1, 2, 3, 5, 7 and 9 days, the cells were collected and stained with 0.4% trypan blue solution. The counts of viable cells were performed using a phase contrast microscope with a hemocytometer.

As a result of the experiments, it was confirmed that IGF-IL-MSC cells had originally the same or up-growing capacity as MSC, and thus it was found to be useful as a stem cell treatment agent. These proliferative activities were also confirmed by the IGF-IL-MSC injection experiments for the following liver disease mouse models.

<3-4> Analysis of differentiability of hepatocytes

IGF-IL-MSC is cultured in the hepatocyte differentiation medium for 2 weeks. The culture medium was supplemented with 2% fetal bovine serum (FBS, HyClone, USA), 4.9 mM nicotinamide (Sigma), 20 ng / mL hepatocyte growth factor (HGF) DMEM (Dulbecco modified Eagles medium) (Hyclone) containing fibroblast growth factor 4 (FGF) (Sigma) was used. The cells were then induced for 2 weeks in a second step in DMEM (Hyclone) supplemented with 2% FBS, 30 ng / mL oncostatin M (Gibco), 10 nM dexamethasone (Sigma) and 10 μL / mL ITS. The expression of hepatocyte-specific genes (alpha fetoprotein, transferrin, albumin) was examined by RT-PCR and Western blotting.

As a result, it was confirmed that IGF-IL-MSC enhanced the ability to differentiate into hepatocytes.

<3-5> in vivo  Evaluation of liver disease treatment and inflammation inhibition

Animals were sacrificed three times a week (monthly, weekly, monthly) with 1% DMN (Dimethylnitrosamine) at 1 mL / kg body weight for 8 h at the end of the refinement period. (IP) injection for 4 weeks (28 days) to establish a chronic cirrhosis model.

The test group consisted of 1) adenovirus-treated group in which pAd_E / IGF-1 or pAd_E / IGF-1 vector was introduced, 2) MSC combination treatment in which vectors of pAd_E / IGF-1 or pAd_E / IGF- 3) IGF-IL-mouse MSC, and 3) IGF-IL-human MSC.

Body weight, liver weight, etc. were measured while observing clinical symptoms. The formation of fibrosis by MT staining was confirmed. To measure the amount of collagen in tissue, hydroxyproline was measured as a component of collagen. Immunohistochemistry and serum biochemical tests were performed to examine the lesion characteristics of the test group.

As a result of the experiment, it was confirmed that the IGF-IL-MSC of the present invention showed the best therapeutic effect. The IGF-IL-MSC of the present invention shows a synergistic effect through mechanisms such as the co-secretion ratio of the proteins, when compared with the case where the single expression system of IGF-1 or IL-10 protein is used in combination. The ability is excellent. In addition, it showed excellent effect in inhibiting inflammation.

In the present invention, in order to maximize the effect of improving mesenchymal stem cells using mesenchymal stem cells, IGF-1 which helps regeneration of liver tissue, IL-10 which suppresses inflammation and has immunoregulatory action is introduced into mesenchymal stem cells simultaneously, Mesenchymal stem cells expressing two genes were constructed and their effects were verified. The mesenchymal stem cells transformed to express specific genes together with the paracrine effect of the mesenchymal stem cells themselves were found to be maximized in function.

As described above, the present invention relates to IGF-1 and IL-10 secretory cells produced by a unique gene introduction system, and the cells produced by the present invention are capable of expressing IGF-1 and IL- This is excellent. Based on this, the cells of the present invention inhibit the activation and proliferation of hepatic stellate cells and thus can be used for the treatment and prevention of liver diseases such as liver fibrosis or cirrhosis. Further, the cells of the present invention can inhibit inflammation, It can be applied to a composition for treatment and prevention of other diseases that are required, and thus the use of the composition is highly likely to be industrially applicable.

<110> Xcell Therapeutics Inc. <120> Cell secreting IGF-1 and IL-10, and use thereof <130> NP16-0151 <160> 18 <170> KoPatentin 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> P1 primer <400> 1 cctctcttcc ccagacct 18 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P2 primer <400> 2 acaacaaaac cgaacaaaag 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P3 primer <400> 3 aaaaccatcc tgcataccaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P4 primer <400> 4 atcctacgtc tcgaccgatg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IGF-1 forward primer <400> 5 gagacagggg cttttatttc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IGF-1 reverse primer <400> 6 agttcttgtt tcctgcactc 20 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized IGF-1 forward primer <400> 7 tgaagatgca caccatga 18 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized IGF-1 reverse primer <400> 8 gtgggcttgt tgaagtagaa 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> IL-10 forward primer <400> 9 ctttcaaatg aaggatcagc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> IL-10 reverse primer <400> 10 ctcttgtttt cacagggaag 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized IL-10 forward primer <400> 11 agcgagatga tccagttcta 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized IL-10 reverse primer <400> 12 gaaatctgtg gcatcttctc 20 <210> 13 <211> 137 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > Homo sapiens insulin like growth factor 1 (IGF-1) <400> 13 Met Ile Thr Pro Thr Val Met Met His Thr Met Ser Ser His Leu   1 5 10 15 Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala              20 25 30 Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe          35 40 45 Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly      50 55 60 Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys  65 70 75 80 Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu                  85 90 95 Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp             100 105 110 Met Pro Lys Thr Gln Lys Glu Val His Leu Lys Asn Ala Ser Arg Gly         115 120 125 Ser Ala Gly Asn Lys Asn Tyr Arg Met     130 135 <210> 14 <211> 178 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens interleukin 10 (IL10) <400> 14 Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val   1 5 10 15 Arg Ala Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His              20 25 30 Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe          35 40 45 Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu      50 55 60 Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys  65 70 75 80 Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro                  85 90 95 Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu             100 105 110 Gly Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg         115 120 125 Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn     130 135 140 Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu 145 150 155 160 Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile                 165 170 175 Arg Asn         <210> 15 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> Wild type Human IGF-1 Class II / Ea GeneBank: NM_001111284.1 <400> 15 atgattacac ctacagtgaa gatgcacacc atgtcctcct cgcatctctt ctacctggcg 60 ctgtgcctgc tcaccttcac cagctctgcc acggctggac cggagacgct ctgcggggct 120 gagctggtgg atgctcttca gttcgtgtgt ggagacaggg gcttttattt caacaagccc 180 acagggtatg gctccagcag tcggagggcg cctcagacag gcatcgtgga tgagtgctgc 240 ttccggagct gtgatctaag gaggctggag atgtattgcg cacccctcaa gcctgccaag 300 tcagctcgct ctgtccgtgc ccagcgccac accgacatgc ccaagaccca gaaggaagta 360 catttgaaga acgcaagtag agggagtgca ggaaacaaga actacaggat gtag 414 <210> 16 <211> 537 <212> DNA <213> Artificial Sequence <220> <223> Wild type Human IL-10 GenBank ID: NM_000572.2 <400> 16 atgcacagct cagcactgct ctgttgcctg gtcctcctga ctggggtgag ggccagccca 60 ggccagggca cccagtctga gaacagctgc acccacttcc caggcaacct gcctaacatg 120 cttcgagatc tccgagatgc cttcagcaga gtgaagactt tctttcaaat gaaggatcag 180 ctggacaact tgttgttaaa ggagtccttg ctggaggact ttaagggtta cctgggttgc 240 caagccttgt ctgagatgat ccagttttac ctggaggagg tgatgcccca agctgagaac 300 caagacccag acatcaaggc gcatgtgaac tccctggggg agaacctgaa gaccctcagg 360 ctgaggctac ggcgctgtca tcgatttctt ccctgtgaaa acaagagcaa ggccgtggag 420 caggtgaaga atgcctttaa taagctccaa gagaaaggca tctacaaagc catgagtgag 480 tttgacatct tcatcaacta catagaagcc tacatgacaa tgaagatacg aaactga 537 <210> 17 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized hIGF-1 DNA sequence <400> 17 atgatcaccc ccaccgtgaa gatgcacacc atgagcagca gccacctgtt ctacctggcc 60 ctgtgcctgc tgaccttcac cagctctgcc acagccggcc ctgagacact gtgtggcgct 120 gaactggtgg acgccctgca gttcgtgtgc ggcgacagag gcttctactt caacaagccc 180 accggctacg gcagcagctc cagaagggct cctcagaccg gcatcgtgga cgagtgctgc 240 ttcagaagct gcgacctgcg gcggctggaa atgtactgcg cccctctgaa gcccgccaag 300 agcgctagat ctgtgcgggc ccagagacac accgacatgc ccaagaccca gaaagaagtg 360 cacctgaaga acgccagcag aggcagcgcc ggcaacaaga actaccggat gtga 414 <210> 18 <211> 537 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized hIL-10 DNA sequence <400> 18 atgcatagca gcgccctgct gtgctgtctg gtgctgctga caggcgtgcg ggcttctcct 60 ggacagggca cccagagcga gaacagctgc acccacttcc ccggcaacct gcccaacatg 120 ctgcgggacc tgagagatgc cttcagcaga gtgaaaacat tcttccagat gaaggaccag 180 ctggacaacc tgctgctgaa agagagcctg ctggaagatt tcaagggcta cctgggctgc 240 caggccctga gcgagatgat ccagttctac ctggaagaag tgatgcccca ggccgagaac 300 caggaccccg acattaaggc ccacgtgaac agcctgggcg agaacctgaa aaccctgcgg 360 ctgcggctga gaagatgcca cagatttctg ccctgcgaga acaagagcaa ggccgtggaa 420 caagtgaaga acgccttcaa caagctgcag gaaaagggca tctacaaggc catgtccgag 480 ttcgacatct tcatcaacta catcgaggcc tacatgacca tgaagatccg gaactga 537

Claims (17)

Cells that secrete IGF-1 and IL-10, transformed with nucleic acids encoding insulin like growth factor-1 (IL-10) and IL-10 (interleukin-10).
The cell according to claim 1, wherein the cell is selected from the group consisting of embryonic stem cells, adult stem cells, induced pluripotent stem cells, mesenchymal stem cells and undifferentiated progenitor cells.
The cell according to claim 2, wherein the mesenchymal stem cell is derived from at least one tissue selected from the group consisting of fat, bone marrow, umbilical cord, umbilical cord blood, placenta, synovial membrane, periosteum and chondrocyte.
2. The cell of claim 1, wherein said IGF-1 comprises the amino acid sequence of SEQ ID NO: 13.
The cell according to claim 1, wherein the nucleic acid encoding the IGF-1 comprises the nucleotide sequence of SEQ ID NO: 15 or SEQ ID NO: 17.
The cell of claim 1, wherein said IL-10 comprises the amino acid sequence of SEQ ID NO: 14.
The cell according to claim 1, wherein the nucleic acid encoding the IL-10 comprises the nucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 18.
2. The cell of claim 1, wherein said transformation is mediated by a vector comprising at least one selected from the group consisting of a nucleic acid encoding IGF-1 and a nucleic acid encoding IL-10.
9. The method of claim 8,
A polynucleotide encoding an EF2 / CMV hybrid promoter and IL-10 operably linked thereto; And
Wherein the vector is a recombinant vector comprising a polynucleotide encoding a CMV / EF1 hybrid promoter and IGF-1 operably linked thereto.
9. The method of claim 8,
A polynucleotide encoding an EF2 / CMV hybrid promoter and IGF-1 operably linked thereto; And
Wherein the vector is a recombinant vector comprising a CMV / EF1 hybrid promoter and a polynucleotide encoding IL-10 operably linked thereto.
9. The cell of claim 8, wherein said vector further comprises a secretory signal sequence.
9. The method of claim 8, wherein the vector is selected from the group consisting of an adenoviral vector, an adeno-associated virus vector, a retroviral vector, a herpes simplex virus vector, Wherein the cell is one or more selected from the group consisting of a lentiviral vector.
A pharmaceutical composition for preventing or treating diseases selected from the group consisting of fibrosis, liver disease, inhibition of rejection of cell or tissue transplant rejection, graft-versus-host disease, inflammatory disease and autoimmune disease, comprising the cell of claim 1 as an active ingredient.
14. The composition of claim 13, wherein the liver disease is selected from the group consisting of liver fibrosis, cirrhosis, hepatitis, and fatty liver.
The composition according to claim 13, wherein the fibrosis is selected from the group consisting of renal fibrosis, liver fibrosis, pulmonary fibrosis, dermal fibrosis, cardiac fibrosis, joint fibrosis, nerve fibrosis, muscle fibrosis and peritoneal fibrosis.
14. The method of claim 13, wherein the inflammatory disease is selected from the group consisting of venous sinusitis, gastritis, rhinitis, conjunctivitis, asthma, bronchitis, dermatitis, atopic dermatitis, inflammatory growth disease, inflammatory liver disease, inflammatory vascular disease, inflammatory rheumatoid arthritis, , Multiple sclerosis, and inflammatory lung disease.
14. The method of claim 13, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, diabetes, atopic dermatitis, alopecia areata, psoriasis, pemphigus, asthma, aphthas stomatitis, chronic thyroiditis, ulcerative colitis, Wherein the composition is selected from the group consisting of multiple sclerosis, autoimmune hemolytic anemia, autoimmune encephalomyelitis, fibrosingitis, and temporal arteritis.

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