KR20220000688A - Pharmaceutical composition for the treatment of hypoxic ischemic encephalopathy comprising mesenchymal stem cell line expressing brain-derived neurotrophic factor - Google Patents

Abstract

The present invention relates to the treatment use for hypoxic ischemic encephalopathy (HIE) of stem cells expressing brain-derived neurotrophic factor. A pharmaceutical composition for treatment of HIE of the present invention is capable of mass production and expresses BDNF as a stem cell capable of maintaining the same effect, in particular, the stem cell is an immortalized mesenchymal stem cell, which has a high cell proliferation rate, and has high stability due to low possibility of abnormal differentiation and proliferation, thereby being able to be usefully used as the pharmaceutical composition for treatment of HIE.

Description

Pharmaceutical composition for the treatment of hypoxic ischemic encephalopathy comprising immortalized stem cell line expressing brain-derived neurotrophic factor {Pharmaceutical composition for the treatment of hypoxic ischemic encephalopathy comprising mesenchymal stem cell line expressing brain-derived neurotrophic factor}

The present invention relates to the use of stem cells expressing brain-derived neurotrophic factor for the treatment of hypoxic ischemic encephalopathy.

Brain-Derived Neurotrophic Factor (BDNF) is expressed in the brain, retina, motor neurons, kidneys, saliva, prostate, etc. It is expressed in the lower (hypothalamus), basal forebrain, etc. BDNF occupies an important part in long-term memory, and in animals where learning has taken place, BDNF mRNA is expressed at a high level in the hippocampus of the brain. It has been reported that the increase in expression in the hippocampus is also associated with the level of learning.

In addition, BDNF is a substance showing the most excellent activity for neurotrophin, which promotes the generation of nerve cells. In fact, in the case of mice in which BDNF is genetically removed, severe mortality is observed after birth, and neuronal loss is observed in the cerebellum, olfactory bulb, visual cortex, and cochlear. On the other hand, when the BDNF gene is partially removed, neurotrophin expression is reduced, long-term potentiation (LTP) in the hippocampus and changes in sensory and nociceptive systems are observed. problem is also observed.

Therefore, BDNF is being used as a target material in the development of drugs for treating neurodegenerative disorders. Experimental evidence that BDNF is involved in neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and Amyotrophic lateral sclerosis (ALS) was established in 1990. It has been reported since the early 1990s. Various studies have reported that genetic abnormalities of BDNF are associated with diseases. In fact, in patients suffering from these neurodegenerative diseases, the amount of BDNF is decreased compared to normal people. In particular, with respect to Huntington's disease, it has been reported that a problem occurs in BDNF transport as the disease progresses through various cell and animal experiments. Attempts have been made to treat diseases through a method of externally supplying reduced BDNF, but there is a problem in that it is difficult to effectively deliver BDNF to the brain so far.

Meanwhile, treatment methods for diseases using cells are being developed around the world, and many studies are being conducted on cell therapy products using adult stem cells. Mesenchymal stem cells (MSCs), which are adult stem cells, are multipotent cells capable of differentiating into bone, cartilage, muscle, fat, fibroblast, and the like. The MSC can be obtained relatively easily from various adult tissues, such as bone marrow, umbilical cord blood, and fat. MSC has the specificity of moving to the site of inflammation or damage, and thus has a great advantage as a carrier for delivering therapeutic drugs. In addition, by inhibiting or activating the functions of immune cells such as T cells, B cells, dendritic cells and natural killer cells, it is possible to regulate the immune function of the human body. In addition, MSCs have the advantage that they can be cultured relatively easily in vitro. Due to these characteristics, studies for the use of MSC as a cell therapy are being actively conducted.

However, despite the advantages of MSCs, there are the following problems in producing MSCs that can be clinically used as cell therapeutics. First, there is a limit to the proliferation of MSCs, making it difficult to mass-produce them. Second, since the obtained MSCs are mixed with various types of cells, it is difficult to maintain the same effect during production. Third, when only MSC is used, the therapeutic effect is not high. Finally, there is a possibility that MSCs injected into the body can become cancer cells in the body.

Meanwhile, Korean Patent Registration No. 1585032 discloses a cell therapeutic agent containing mesenchymal stem cells cultured in a hydrogel. The literature provides a composition that can be administered immediately by shortening the pretreatment process in the process of isolating mesenchymal stem cells for use as a cell therapeutic agent. not mentioned Therefore, there is a need for research on safe and effective mesenchymal stem cells that can be used as cell therapeutics.

In particular, a study of transforming mesenchymal stem cells using adenovirus to express brain-derived neurotrophic factor (BDNF) has been conducted (Kari Pollock et al ., Molecular Therapy, vol. .24 no.5: 965-977, 2016). However, the mesenchymal stem cells used in the above study are general mesenchymal stem cells obtained from tissues, and there is a limitation in the proliferation of MSCs, so it is difficult to mass-produce them. In addition, the expression level of the BDNF gene is not uniform, so it is difficult to expect the same effect during production.

On the other hand, hypoxic ischemic encephalopathy (HIE) is a disease in which irreversible brain damage occurs due to insufficient supply or passage of oxygen and blood flow to the brain. In particular, it is known that it can cause death or permanent disability in newborns and children. In the case of newborns, it is caused by problems at birth, such as newborn housework, or in children, heart surgery, infectious upper airway obstruction, burns, excessive bleeding during surgery, improper It can also be caused by blood transfusions, sudden infant death syndrome, inadequate anesthesia, metabolic, infectious, post-traumatic, or other neurological causes. Hypoxic ischemic encephalopathy causes various complications such as changes in cognitive and arousal states, various types of epilepsy, autonomic nervous system failure, and movement disorders. Hypothermic therapy may be used as a treatment method for hypoxic ischemic encephalopathy, but complications may occur due to such treatment, so there is a need for research on a therapeutic agent or treatment method with a low incidence of complications.

Republic of Korea Patent No. 1585032

Kari Pollock et al., Molecular Therapy, vol.24 no.5: 965-977, 2016

Accordingly, the present inventors studied to develop a stem cell therapeutic agent capable of mass production and maintaining the same effect as a therapeutic agent having a therapeutic effect on hypoxic ischemic encephalopathy (HIE). As a result, the transfected mesenchymal stem of the present invention It was confirmed that cells (MSCs) exhibit a therapeutic effect on HIE. In addition, the present invention was completed by confirming that the MSC can be subcultured (proliferated) for more than 160 days and can stably express the brain-derived neurotrophic factor (BDNF) gene.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating HIE using a stem cell line expressing BDNF.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating HIE comprising stem cells expressing BDNF.

As an aspect of the present invention, the pharmaceutical composition of the present invention may contain an immortalized stem cell line comprising a gene encoding BDNF.

In another aspect of the present invention, the pharmaceutical composition of the present invention may contain a lentiviral vector for transducing cells to express the BDNF protein.

As another aspect of the present invention, the pharmaceutical composition of the present invention may contain the host cells transfected with the lentivirus as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition for preventing or treating HIE comprising stem cells expressing BDNF. The stem cells may be transduced with a recombinant vector expressing BDNF protein, or may include a virus into which the vector is introduced.

As used herein, the term "brain-derived neurotrophic factor (BDNF)" protein is the most abundantly distributed neurotrophin in the brain. It is known to promote the growth of neurons, and to regulate the synthesis, metabolism, release and activity of neurotransmitters. In addition, it is known that BDNF protein is decreased in the prefrontal cortex or hippocampus of depressed patients, and can treat depression by increasing its concentration.

The BDNF protein according to the present invention may be a human-derived protein. The protein may be a polypeptide having the amino acid sequence of SEQ ID NO: 1. The BDNF protein may have about 80%, 90%, 95%, or 99% or more homology to the amino acid sequence of SEQ ID NO: 1. Meanwhile, the gene encoding the BDNF protein may be the nucleotide sequence of SEQ ID NO: 2. In addition, the nucleotide sequence encoding the BDNF protein may have about 80%, 90%, 95%, or 99% or more homology with the nucleotide sequence of SEQ ID NO: 2.

The recombinant vector of the present invention may be a lentiviral vector. The "lentiviral vector" is a kind of retrovirus. The vector is also referred to as a lentiviral transfer vector interchangeably. The lentiviral vector is inserted into the genomic DNA of a cell to be infected to stably express the gene. In addition, the vector can deliver genes to dividing cells and non-dividing cells. Since the vector does not induce an immune response of the human body, its expression is continuous. In addition, there is an advantage in that a gene of a large size can be delivered compared to an adenoviral vector, which is a viral vector used in the prior art.

The recombinant lentiviral vector of the present invention can regulate the expression of a gene loaded therein by a promoter. The promoter may be a cytomegalovirus (CMV), respiratory syncytial virus (RSV), human elongation factor-1 alpha (EF-1α) or tetracycline response elements (TRE) promoter. According to an embodiment of the present invention, the recombinant lentiviral vector can regulate the expression of BDNF protein by one promoter. The promoter is operably linked to a gene encoding a protein to be expressed.

According to an embodiment of the present invention, the BDNF protein may be linked to a TRE promoter. The TRE promoter may activate transcription of a gene linked to the promoter by a tetracycline transactivator (tTA) protein. Specifically, the tTA protein binds to the TRE promoter and activates transcription in the absence of tetracycline or doxycycline. On the other hand, when they are present, the tTA protein cannot bind to the TRE promoter and thus cannot activate transcription. Therefore, the expression of BDNF protein can be regulated depending on the presence of tetracycline or doxycycline.

The term "operably linked" means that a particular polynucleotide is linked to another polynucleotide so that it can exert its function. That is, the fact that a gene encoding a specific protein is operably linked to a promoter means that the gene can be transcribed into mRNA and translated into a protein by the promoter.

The recombinant lentivirus of the present invention comprises the steps of transforming a host cell with a lentiviral vector, a packaging plasmid and an envelope plasmid; And it can be obtained through the step of isolating the lentivirus from the transformed host cell.

The terms "packaging plasmid" and "envelope plasmid" are plasmids loaded with genes encoding proteins. These plasmids may provide, in addition to the lentiviral vector, the helper constructs (eg, plasmids or isolated nucleic acids) necessary to produce the lentivirus. This construct contains elements useful for manufacturing and packaging the lentiviral vector in a host cell. Such elements include structural proteins such as GAG precursors; processing proteins such as pol precursors; proteases, envelope proteins, and expression and regulatory signals necessary for production of proteins and lentiviral particles in host cells.

For the production of recombinant lentivirus, Clonetech Laboratories' Lenti-X Lentiviral Expression System or Addgene's packaging plasmid (eg, pRSV-Rev, psPAX, pCl-VSVG, pNHP, etc.) or envelope plasmid (eg, pMD2.G, pLTR-G, pHEF-VSVG, etc.) can be used.

As one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating HIE containing the host cell transduced with the recombinant lentivirus.

The term “transduction” refers to transfer of a gene loaded into a recombinant lentiviral vector through viral infection.

Host cells according to the present invention are human embryonic stem cells (hES), bone marrow stem cells (BMSC), mesenchymal stem cells (MSC), human neural stem cells (human neural stem cells) stem cells, hNSCs), limbal stem cells, or oral mucosal epithelial cells. According to an embodiment of the present invention, the host cell may be a mesenchymal stem cell.

The term "mesenchymal stem cell (MSC)" refers to a multipotent stromal cell that can be differentiated into various cells including osteocytes, chondrocytes and adipocytes. Mesenchymal stem cells can be differentiated into cells of specific organs, such as bone, cartilage, fat, tendon, nervous tissue, fibroblasts, and muscle cells. These cells can be isolated or purified from adipose tissue, bone marrow, peripheral nerve blood, umbilical cord blood, periosteum, dermis, mesoderm-derived tissue, and the like.

The transfected host cell according to the present invention may be an immortalized one. Specifically, the host cell may be one into which hTERT and/or c-Myc genes are introduced.

In addition, the transfected host cell according to the present invention may further include a thymidine kinase (TK) gene.

The term "thymidine kinase" used in the present invention refers to an enzyme that catalyzes the reaction of producing thymidylic acid while thymidine is transformed into a triphosphate form by binding phosphate at the γ-position of ATP to thymidine. It is known that modified thymidine cannot be used for DNA replication and thus induces death of cells containing it. Any known sequence of the TK protein may be used.

As used herein, the term "hTERT" refers to telomerase reverse transcriptase. The telomerase reverse transcriptase synthesizes DNA complementary to the RNA template of telomerase, forms an RNA-DNA hybrid, and becomes double-ringed DNA and is inserted into the chromosome of the host cell. Then, in the host cell, instead of telomeres, telomerase attaches to chromosome telomeres and continues to produce telomeres, thereby making immortalized cells.

As used herein, the term “c-Myc” refers to a gene encoding a transcription factor located on chromosome 8 of a human. The protein encoded by c-Myc, which regulates the expression of about 15% of intracellular genes, is a transcription factor, and when the transcription factor is overexpressed, cell activity and proliferation are promoted.

The host cell can be prepared in the following way:

1) first infecting the host cell with a lentivirus containing the c-Myc gene;

2) secondary infection of the lentivirus containing the tTA gene into the primary infected host cell;

3) a third step of infecting the second-infected host cells with a lentivirus containing the BDNF gene.

In step 1), c-Myc is a gene that immortalizes host cells, and other genes known as immortalization genes other than c-Myc can be used. In one embodiment, the c-Myc protein may be a polypeptide having the amino acid sequence of SEQ ID NO:3. Meanwhile, the gene encoding the c-Myc protein may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 4, respectively.

In step 2), tTA is a gene capable of regulating the expression of a target protein, and refers to a tetracycline transactivator. The Tet-off system used in the present invention can regulate the expression of a target protein according to the presence or absence of tetracycline or doxycycline in the same manner as described above.

The BDNF-expressing mesenchymal stem cells of the present invention are characterized in that the expression of BDNF protein is suppressed by treatment with doxycycline ex vivo by the TRE promoter, and BDNF is expressed when doxycycline is removed.

That is, the present invention relates to a pharmaceutical composition for preventing or treating HIE containing the recombinant lentivirus or transfected host cell as described above. The recombinant lentivirus or host cell of the present invention can exhibit a neuroprotective effect through the expression of BDNF, and in particular, it was confirmed that it has an effective therapeutic effect on HIE through intracellular and animal experiments.

In the present invention, the hypoxic ischemic encephalopathy (HIE) refers to a state in which the function and structure of the brain are changed due to the lack of oxygen and blood flow delivered to the brain. It is a disease caused by various causes. In the present invention, "prophylaxis or treatment of HIE" includes alleviation, alleviation and improvement of symptoms of the disease, and includes reducing the likelihood of HIE induced.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier. The carrier is commonly used in the manufacture of pharmaceuticals, and is lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrol money, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In addition, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive selected from the group consisting of lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, preservatives, and combinations thereof.

The carrier may be included in an amount of from about 1% to about 99.99% by weight, preferably from about 90% to about 99.99% by weight, based on the total weight of the pharmaceutical composition of the present invention, and the pharmaceutically acceptable additive is about 0.1 It may be included in an amount of about 20% by weight to about 20% by weight.

The pharmaceutical composition may be prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier and excipient according to a conventional method, or may be prepared by internalizing it in a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer.

In addition, the present invention provides a method for preventing or treating hypoxic ischemic encephalopathy as described above, comprising administering the pharmaceutical composition of the present invention to a subject.

The subject may be a mammal, specifically a human. The administration route and dosage of the pharmaceutical composition may be administered to the subject in various ways and amounts depending on the patient's condition and presence or absence of side effects, and the optimal administration method and dosage may be selected within an appropriate range by a person skilled in the art. In addition, the pharmaceutical composition may be administered in combination with other drugs or physiologically active substances with known therapeutic effects on the disease to be treated, or formulated in the form of a combination preparation with other drugs.

When the pharmaceutical composition is administered parenterally, examples thereof include subcutaneous, ocular, intraperitoneal, intramuscular, oral, rectal, orbital, intracerebral, intracranial, intravertebral, intraventricular, intrathecal, There are intranasal and intravascular administration, preferably intraventricular or intravascular administration.

The administration may be administered more than once, 1 to 4 times, and specifically divided into two administrations. In the case of repeated administration, it may be administered at intervals of 12 to 48 hours, 24 to 36 hours, 1 week, 2 weeks to 4 weeks, and specifically, may be administered at intervals of 24 hours or 1 week or more. The administration may be administered in an amount of ^{1.0x10 6} to 1.0x10 ¹² TU, specifically 1.0x10 ⁸ to 1.0x10 ¹⁰ TU per day in the case of lentivirus. Meanwhile, cells may be administered in an amount of ^{1.0x10 5} to 1.0x10 ¹¹ cells, specifically 1.0x10 ⁷ to 1.0x10 ^{9 cells per day.} In the case of a large dose, it may be administered several times a day.

The pharmaceutical composition for treating HIE comprising the BDNF-expressing mesenchymal stem cells of the present invention as an active ingredient is capable of mass production and expresses BDNF as a cell therapeutic agent capable of maintaining the same effect, in particular, the cells are immortalized As mesenchymal stem cells, they have a high cell proliferation rate, but have a low possibility of abnormal differentiation and proliferation, and thus have high stability, so they can be usefully used as a therapeutic pharmaceutical composition for HIE.

1 is a graph comparing cell proliferation rates of immortalized MSCs (STEM24) and non-immortalized MSCs (BM-MSCs):
X-axis: incubation period; and
Y-axis: cumulative population doubling level (PDL).
FIG. 2 is a graph showing the BDNF protein expression level according to the presence or absence of doxycycline in clones in which BDNF protein expression was confirmed.
FIG. 3 shows a comparison of the expression of surface antigen proteins CD44 and CD105, which are unique characteristics of MSCs, and the expression of surface antigen proteins of bone marrow-derived MSCs after genetic manipulation of the BM102 monoclonal cell line.
4 is a graph confirming the proliferation rate while culturing immortalized MSC cells transduced with a lentivirus containing the BDNF gene for a long time:
X-axis: incubation period; and
Y-axis: cumulative population doubling level (PDL).
5 is a graph confirming the cell proliferation rate of BM102 according to the presence or absence of doxycycline treatment during long-term culture.
X-axis: incubation period; and
Y-axis: cumulative population doubling level (PDL).
6 is a view confirming that morphological characteristics of cells are maintained for each passage of the BM102 cell line with or without doxycycline treatment.
7 is a graph showing the expression level of BDNF protein in the BM102 cell line with or without doxycycline treatment.
8 is a view confirming that the gene was introduced into the BM102 cell line.
9 is a graph confirming that there is no tumorigenicity even after genetic manipulation of the BM102 cell line.
10 shows the concentration-dependent neuroprotective effect of the BM102 cell line in the OGD model (in virtro) through cell viability.
11 shows the neuroprotective effect of the BM102 cell line administration in the OGD model (in virtro) through cell viability, cytotoxicity, and the degree of oxidative stress.
12 shows the results of the TUNEL analysis confirming whether or not apoptosis according to the administration of the BM102 cell line in the OGD model (in virtro).
13 is a schematic diagram of an experiment confirming the therapeutic effect of HIE according to the administration of the BM102 cell line through a short-term in vivo evaluation model.
14 is a result showing the measurement value of the non-injured area of the brain that appears when the BM102 cell line is administered in a short-term in vivo evaluation model.
15 shows the results of measuring the secretion amount of pro-inflammatory cytokines expressed in the brain upon administration of the BM102 cell line in a short-term in vivo evaluation model.
16 is a comparison result of histological scores appearing for each brain region when the BM102 cell line is administered in a short-term in vivo evaluation model.
17 is a schematic diagram of an experiment confirming the therapeutic effect of HIE according to the administration of the BM102 cell line through a long-term in vivo evaluation model.
18 is a long-term in vivo evaluation model, a result showing the measurement value of the non-injured area of the brain that appears when the BM102 cell line is administered.
19 shows the results of measuring the secretion amount of pro-inflammatory cytokines expressed in the brain when the BM102 cell line is administered in a long-term in vivo evaluation model.
20 is an immunostaining result comparing the histological scores appearing for each brain region when the BM102 cell line is administered in an in vivo evaluation model.
21 shows the sensorimotor nerve analysis results of the animal model when the BM102 cell line is administered in the in vivo evaluation model.

Hereinafter, examples will be given to describe the present specification in detail. However, the embodiments according to the present specification may be modified in various other forms, and the scope of the present specification is not to be construed as being limited to the embodiments described below. The embodiments of the present specification are provided to more completely explain the present specification to those of ordinary skill in the art.

Example 1. Preparation of immortalized mesenchymal stem cells (MSCs)

Example 1.1 Preparation of Lentiviral Vector Containing Immortalization Gene

In order to immortalize MSCs, a lentiviral vector including the immortalization gene c-Myc was prepared. At this time, in order to use the Tet-off system, a gene construct expressing tTA protein was inserted together.

First, a pBD lentiviral vector was prepared by replacing the EF promoter with the CMV promoter in the expression cassette of the pWPT vector (Bioneer synthesis). The c-Myc gene (SEQ ID NO: 4) was inserted into the pBD lentiviral vector so that expression could be regulated by the CMV promoter. The constructed vector was named pBD-1.

Example 1.2 Production of Lentiviruses Containing Immortalization Genes

Using the lentiviral vector prepared in Example 1.1, a lentivirus containing an immortalized gene was produced in the following way.

First, lenti-X cells (Clontech Laboratories, USA) were cultured in a 150 mm dish using DMEM medium containing 10% fetal bovine serum. Meanwhile, lentiviral vectors were extracted and quantified from DH5α E. coli cells using the EndoFree Plasmin Maxi Kit (Qiagen, USA).

에서 약 5분 동안 방치한 뒤, 세포가 탈착된 것을 확인하였다. 탈착된 세포를 7 ㎖의 10% 우태아 혈청이 포함된 DMEM 배지를 첨가하여 중화시켰다. 중화된 세포는 50 ㎖ 튜브에 모아서 1,500rpm으로 5분 동안 원심분리하였다. 상층액을 제거하고 10 ㎖의 10% 우태아 혈청이 포함된 DMEM 배양배지를 첨가하여 세포를 재현탁하였다.After washing the cultured lenti X cells with PBS, 3 ml of TrypLE™ Select CTS™ (Gibco, USA) was added. 37 cells

After leaving it for about 5 minutes, it was confirmed that the cells were detached. The detached cells were neutralized by adding 7 ml of DMEM medium containing 10% fetal bovine serum. The neutralized cells were collected in a 50 ml tube and centrifuged at 1,500 rpm for 5 minutes. The supernatant was removed and the cells were resuspended by adding 10 ml of DMEM culture medium containing 10% fetal bovine serum.

Suspended cells were counted with a hematocrit, and then aliquoted to ^{1.2x10 7 cells in a 150 mm dish.} When the seeded cells were cultured to a cell confluency of about 90%, 12 μg of lentiviral vector, 12 μg of psPAX (Addgene; gag-pol expression, packaging plasmid) and 2.4 μg of pMD.G plasmid (Addgene; vesicular) stomatitis virus G protein, VSV-G expression, envelope plasmid) mixture was transduced into the cells. To aid in transduction, lipofectamine (Invitrogen, USA) and plus reagent (Invitrogen, USA) were used. After 6 hours of transduction, the medium was exchanged with DMEM containing 10% fetal bovine serum. After further culturing for 48 hours, the supernatant was collected.

The obtained supernatant was mixed with a lentivirus concentration kit (Lenti-X concentrator, Clontech Laboratories, USA), and then incubated at 4°C overnight. The virus was obtained by centrifugation at 4°C and 4,000 rpm for 2 hours, and it was resuspended in 0.5 ml of DMEM without FBS. As a result, the lentivirus produced from the pBD-1 lentivirus vector was prepared at a concentration of ^{8.3x10 6 copies/ml.}

Example 1.3 Preparation of immortalized mesenchymal stem cells

Immortalized MSCs were prepared using the lentivirus containing the immortalized gene produced in Example 1.2.

First, bone marrow-derived MSCs were prepared as follows. Specifically, bone marrow aspirate was obtained from the iliac crest of a healthy donor. This was mixed with 20 IU/ml heparin in a sterile container to inhibit coagulation. The bone marrow mixture was centrifuged for 7 minutes at 4°C and 739 G, the supernatant was removed, and mixed with 10 times the volume of sterilized water. This was centrifuged again under the same conditions to obtain a cell pellet. The obtained pellet was suspended in DMEM-low glucose (11885-084, Gibco, USA) medium containing 20% FBS and 5 ng/ml of b-FGF (100-18B, Peprotech, USA) and dispensed into a culture flask. did This was cultured at 37° C., 5% CO ₂ conditions for 24 to 48 hours, and then replaced with a fresh medium. It was subcultured while replacing it with a new medium every 3 to 4 days, and after 2 weeks of culture, MSC was checked using a fluorescence cytometer.

The prepared MSCs with the pBD-1 lentivirus produced in Example 1.2. were infected with retronectin (Retronectin, Clontech Laboratories, USA) at an MOI of 100. As a result, the cell proliferation rates of MSCs containing the immortalized gene and MSCs not including the immortalized gene are shown in FIG. 1 .

As shown in Figure 1, MSC cells infected with the lentivirus containing the immortalization gene c-Myc, as immortalized by the immortalization gene, the proliferation rate increased based on the culture 30 days after infection, and up to 50 days A high growth rate was maintained. On the other hand, the cell proliferation rate of normal MSC cells decreased sharply after 20 days of culture.

Example 2. Construction of a lentivirus containing the tTA gene

Example 2.1. Construction of Lentiviral Vector Containing tTA Gene

In the pBD lentiviral vector prepared in Example 1.1., a tetracycline trans activator (tTA) gene (SEQ ID NO: 6) was inserted so that expression could be controlled by the CMV promoter. The constructed vector was named pBD-2.

Example 2.2. Production of Lentiviruses Containing the tTA Gene

Using the lentiviral vector containing the tTA gene constructed in Example 2.1, lentiviruses were produced in the same manner as described in Example 1.2. The produced lentivirus was prepared at a concentration ^{of 7.2x10 6 copies/ml.}

The immortalized MSCs prepared in Example 1.3 were infected with the lentivirus containing the tTA gene at an MOI of 100 to prepare cells expressing the tTA gene.

Example 3. Construction of Lentivirus Containing BDNF Gene

Example 3.1. Construction of Lentiviral Vector Containing BDNF Gene

The BDNF gene (SEQ ID NO: 2) was inserted into the pBD lentiviral vector constructed in Example 1.1.

First, in order to confirm the effect of the promoter on BDNF expression, a lentiviral vector was prepared to express the BDNF gene using the CMV promoter and the TRE promoter, respectively, and MSC expressing the BDNF gene was prepared using this. As a result, in the case of a cell line to which the CMV promoter was applied, it was confirmed that the MSC expressing BDNF was morphologically changed during long-term subculture, and the expression rate of BNDF decreased as the subculture progressed.

Accordingly, the BNDF gene was inserted so that expression was regulated by the TRE promoter. The TRE promoter can control the expression of genes linked to the promoter depending on whether doxycycline is added or not.

Example 3.2. Production of Lentiviruses Containing the BDNF Gene

Using the lentiviral vector containing the BDNF gene constructed in Example 3.1., Example 1.2. Lentiviruses were produced in the same manner as described in The produced lentivirus was prepared at a concentration ^{of 4.7x10 6 copies/ml.}

Example 4. Preparation of MSCs transfected with lentiviruses containing BDNF gene

Immortalized MSCs transfected with the tTA gene prepared in Example 2.2. were infected with the lentivirus containing the BDNF gene produced in Example 3.2. at an MOI of 100 to prepare cells expressing the BDNF gene. The transduced cells were cultured in a medium supplemented with 2 μg/ml doxycycline (doxycycline, 631311, Clontech, USA) to suppress the expression of BDNF protein during culture.

The cells were cultured to form colonies using a clone select imager. After numbering each well inoculated with cells in a 96-well plate, the presence or absence of BDNF protein expression was confirmed with the human BDNF DuoSet ELISA kit (R&D systems, DY248, USA) among 200 clones that confirmed colony formation. Clones #10, #22, #28, #65, #110, #116, #126, #596, #669 and #741 expressing 1.5 ng/ml or more were selected. Thereafter, clones #28 and #110 expressing BDNF protein at a level of 0.5 ng/ml or less upon doxycycline treatment were selected ( FIG. 2 ).

As a result, clone #28 showing a stable proliferation pattern and the best BDNF protein expression level was named 'BM102 cell line' and deposited as a patent strain (Accession No. KCTC13876BP), and then the experiment was carried out.

Experimental Example 1. Confirmation of surface antigen protein expression in BM102 cell line

Surface antigen protein expression of bone marrow-derived MSC and BM102 cell line into which the BDNF gene was inserted before gene insertion was analyzed using a human MSC assay kit (Stemflow™, Cat No562245, BD). The experiment was performed according to the manual included in each kit, and the experimental results are shown in FIG. 3 .

As a result, as shown in Figure 3, the BM102 cell line demonstrated that the expression of surface antigen proteins CD44 and CD105, which are unique characteristics of MSCs, is similar to that of bone marrow-derived MSCs even after genetic manipulation to express BDNF. .

Experimental Example 2. Confirmation of proliferation rate of BM102 cell line

The proliferation rate of the BM102 cell line established in Example 4 was confirmed.

^{After inoculating 0.2x10 6} to 0.8x10 ⁶ BM102 cell lines in a T175 flask, culture was performed for 3 or 4 days with or without doxycycline, and PDL (Proliferation Doubling Level) and cell viability were measured. PDL was calculated by the formula "PDL=X+3.222 (logY-logI)", where X denotes the initial PDL, I denotes the initial number of cells inoculated into the flask, and Y denotes the final number of cells. The proliferation rates of the established cell lines are shown in FIGS. 4 and 5 .

As a result, as shown in FIG. 4 , the BM102 cell line proliferated stably for more than 100 days. In addition, as shown in FIG. 5 , it was confirmed that the cell proliferation rate of the BM102 cell line cultured up to 80 days without doxycycline was gradually decreased compared to the BM102 cell line cultured with doxycycline added.

Experimental Example 3. Morphological confirmation of the BM102 cell line according to subculture

In order to confirm the morphological change according to the passage culture of the BM102 cell line established in Example 4, the proliferation rate of the BM102 cell line was confirmed in Experimental Example 2, and pictures of the cells were taken under a microscope, and the morphology of the BM102 cells is shown in FIG. indicated.

As a result, as shown in FIG. 6 , it was confirmed that there was no morphological change in the BM102 cell line even after long-term culture up to 100 days. However, when cultured without the addition of doxycycline, it was confirmed that the BM102 cell line was morphologically changed due to the expression of BDNF. This means that the expression of BDNF protein affects BM102 cells, thereby changing cell properties.

Experimental Example 4. Confirmation of expression of BDNF protein in BM102 cell line

The expression of BDNF protein in the BM102 cells established in Example 4 was confirmed by ELISA analysis. Specifically, the expression level of BDNF protein was confirmed with the human BDNF DuoSet ELISA kit. Experiments were performed according to the manual included in each kit. 7 shows the expression level of BDNF protein induced by expression from ^{about 1×10 5} cells in a medium in which doxycycline was removed and in a medium in which doxycycline was not removed for 48 hours.

As a result, as shown in FIG. 7 , about 0.2 ng of BDNF was detected in the medium with doxycycline, whereas about 740 ng of BDNF was detected in the medium without doxycycline.

target protein expression level BDNF 740 ng/10 ⁵ cells

As shown in Table 1, it was confirmed that about 740 ng/10 ⁵ cells of the BDNF protein were expressed in the BM102 cell line of the present invention.

Experimental Example 5. Confirmation of BDNF transgene in BM102 cell line

PCR was performed to confirm whether the gene was introduced into the BM102 cell line prepared in Example 4. Specifically, the BM102 cell line was transferred to a 15 ml tube containing 9 ml PBS and then cell-downed at 1,500 rpm for 5 minutes. After the PBS was completely removed, the pellet was suspended in 200 μl PBS in a 1.5 ml tube and then transferred. Thereafter, gDNA was prepared using NucleoSpin® Tissue (MN, 740952.250) and a mixture was prepared as shown in Table 2 below, and then PCR was performed in the steps of Table 3 below. At this time, 100 ng of BM102 plasmid DNA was added as a positive control and 1 μl of purified water was added as a negative control. The BM102 plasmid DNA can be separated and purified according to the method described in Korean Patent Application Laid-Open No. 2017-0093748.

Forward primer (SEQ ID NO: 7) (10 pmol/μl, Cosmogenetec synthesis) 1 μl Reverse primer (SEQ ID NO: 8) (10 pmol/μl, Cosmogenetek synthesis) 1 μl sample (100 ng/μl) 1 μl Purified water 17 μl total volume 20 μl

step Temperature hour number One 95℃ 5 minutes One 2 95℃ 30 seconds 35 62℃ 30 seconds 72℃ 40 seconds 3 72℃ 7 minutes One 4 4℃ indefinitely One

A 1% (v/v) agarose gel was placed in the electrophoresis kit. 10 μl of DNA Size Marker was loaded into the first well, and 10 μl of each was loaded in the order of negative control, positive control, and three BM102 samples from the next well. Thereafter, electrophoresis was performed at 100 V for 20 minutes, and a photograph of the gel was taken and the results are shown in FIG. 8 . As a result, as shown in FIG. 8, PCR products of the same size (0.6 kb) as the positive control group were confirmed for all three BM102 cell lines.

Experimental Example 6. Confirmation of tumorigenicity of BM102 cell line in vitro

Bone marrow-derived MSC, BM102 cell line, positive control group (HeLa) and negative control group (NIH3T3) on soft agar gels using CytoSelectTM 96-well Cell Transformation Assay. Colony formation by anchorage-independent growth was confirmed, and by measuring absorbance by staining with MTS solution, tumor formation due to cell transformation was quantified.

As a result, colonies were formed by cell transformation in the positive control group as shown in FIG. 9, and the BM102 cell line had no transformation ability during cell culture, from which colonies were not formed in the negative control group, bone marrow-derived MSCs and BM102 cell lines. It was confirmed that there was no tumorigenicity in vitro.

Experimental Example 7. Confirmation of HIE prevention and treatment effect of BM102 cell line in vitro

Experimental Example 7.1. Cell preparation and transplantation

All animal experiments were performed using frozen stem cells provided by SL Weizen, and the cells were stored in liquid nitrogen until testing and then thawed in a 37°C water bath and maintained at a temperature of 4°C until use. In the control group, only the vehicle was treated.

Experimental Example 7.2. HIE in vitro model

7.2.1. In vitro model production

Sprague Dawley (SD) rats to release the nerve cells in the cerebral cortex area, and then 7 days after the culture was 1 hour exposure to _{95% N 2, 5% CO} 2 and 37 ℃ environment using glucose- free culture medium in vitro oxygen glucose deprivation ( OGD) model was fabricated. After making the OGD model, a sample (Primary MSC or BM102) was put into a 1 μm pore upper chamber where cell migration is difficult, and the change was measured after co-culture for 24 hours.

7.2.2. Cell staining evaluation (TUNEL assay)

After the production of the OGD model, the cells were fixed and washed with a fixative, followed by TUNEL (apoptosis indicator) staining. The stained slides were photographed at 400X magnification using a fluorescence microscope. The number of TUNEL positive cells was measured and analyzed with the photograph taken.

7.2.3. biochemical evaluation

Cell viability (CCK-8) and cytotoxicity (lactate dehydrogenase assay) were measured according to the reagent manufacturer's standard test method.

In addition, for the measurement of oxidative stress, it was measured according to the standard test method of the reagent manufacturer using a malondialdehyde assay kit.

7.2.4. Statistical processing

The SPSS (ver.18.0) program was used, and statistical differences were analyzed for each group through one way-ANOVA with Tukey's post-test. A case where the P value between groups was less than 0.05 was judged to be statistically significant.

Experimental Example 7.3. Neuronal survival improvement effect according to BM102 cell line treatment concentration

In the OGD model, 1x10 ³ , 1x10 ⁴ , 5x10 ⁴ , 1x10 ⁵ , 1x10 ⁶ BM102 cells were co-cultured and tested. As shown in FIG. 10 , it was confirmed that neuronal cell viability was statistically significantly increased in the BM102 group in the OGD model compared to the primary MSC group. Significantly increased the BM102 cells with a cell viability statistically from 1X10 ^4-treated groups was confirmed to be seen the ⁴ 5X10, 1X10 ⁵ treatment groups in the primary MSC and processed with BM102, statistically significant improvement neuron survival efficacy.

Experimental Example 7.4. Measurement of cell survival, cytotoxicity and oxidative stress following treatment with BM102 cell line

The experiment measured the cell viability, cytotoxicity and oxidative stress of the group treated with ^{1x10 5} BM102 cells in the OGD model. As shown in FIG. 11 , it was confirmed that the survival rate of nerve cells was statistically significantly decreased in the OGD model compared to the untreated control group (NC). The group treated with primary MSC showed a tendency to increase neuronal survival compared to the OGD control group, but the group treated with BM102 showed a statistically significant increase in neuronal survival compared to the group treated with OGD control and primary MSC.

In the LDH (cytotoxicity) and Malondialdehyde (oxidative stress) measurement results, it was confirmed that the measured values of LDH and Malondialdehyde were statistically significantly decreased in the group treated with BM102 than in the group treated with primary MSC.

Experimental Example 7.5. Cell death assay following treatment with BM102 cell line

^{In the case of the group treated with 1x10 5} BM102 cells in the OGD model, as shown in FIG. 12, it was confirmed that the number of TUNEL positive cells in the OGD model increased statistically significantly compared to the normal control (NC) group. In the group treated with primary MSC, there was a tendency to decrease the number of TUNEL positive cells compared to the OGD control group, but in the group treated with BM102, the number of TUNEL positive cells was statistically significantly decreased compared to the group treated with OGD control and primary MSC. Confirmed.

From the above results, it was confirmed that, when OGD-induced neurons and BM102 cells were co-cultured, the survival rate of neurons was increased, and cytotoxicity and oxidative stress were reduced. In addition, since apoptosis of neurons is reduced, it can be seen that the BM102 cell line has a protective effect on neurons.

Experimental Example 8. Confirmation of short-term therapeutic effect of BM102 cell line in HIE-induced animal model

Experimental Example 8.1. Cell Preparation and Transplantation Methods

All animal experiments were performed using frozen stem cells provided by SL Weizen, and the cells were stored in liquid nitrogen until transplantation, thawed in a 37°C water bath and maintained at a temperature of 4°C until administration. Primary MSC or BM102 was prepared according to the dose, and the designated coordinates (x=+0.5, y=+1.2, z= -2.7 mm). In the control group, only 10 μL of the vehicle was injected.

Experimental Example 8.2. animal testing

8.2.1. HIE model production and BM102 cell administration group selection

After tying the right carotid artery of 7-day-old male Sprague dawley (SD) rats, they _{were exposed to 8% O 2} in a special chamber for 2 hours and 30 minutes to prepare an in vivo hypoxic ischemic encephalopathy model. The experimental group was a total of five groups including the control group administered with the excipient (CryoStor® CS10), and the rats that had finished hypoxic ischemic encephalopathy induction were treated with hypothermia at 32°C for 24 hours in all experimental groups. It was performed at the age of 10 days after birth, 2 days after administration.

Group 1: HIE model + CS10 administration (n=11)

Group 2: HIE model + Primary MSC 1X10 ⁵ /10ul administration (n=7)

Group 3: HIE model + BM102 1X10 ⁴ /10ul administration (n=9)

Group 4: HIE model + BM102 5X10 ⁴ /10ul administration (n=14)

Group 5: HIE model + BM102 1X10 ⁵ /10ul administration (n=14)

A schematic diagram of the above experimental procedure is shown in FIG. 13 .

8.2.2. Tissue storage and sample preparation for analysis

For pathological analysis, brain tissues of 10-day-old rats were stored in formalin solution at room temperature. The tissue was fixed on a paraffin block, sliced to a thickness of 4 μm (-3.60mm to -3.80mm/bregma) to make an empty slide, and then used for analysis.

In addition, the brains of 10-day-old rats were stored at -80°C for biochemical analysis. After grinding the brain samples stored before analysis, the supernatant was separated by centrifugation at 8,000 g for 20 minutes, and the separated supernatant was used for analysis.

8.2.3. Heamatoxylin & Eosin staining

H&E staining was performed by exposing an empty slide on which the rat brain was fixed to H&E (hematoxylin & eosin) solution for 10 minutes each. The remaining area of the right hemisphere from which the hypoxic ischemic encephalopathy model was derived was calculated as a ratio to the area of the left normal hemisphere.

8.2.4. Enzyme Immunoassay (ELISA)

The levels of pro-inflammatory cytokines IL-1α, IL-1β, IL-6 and TNF-α were measured using the enzyme immunoassay (ELISA) in the brain tissue homogenate of 10-day-old rats.

8.2.5. MRI analysis

MRI was performed immediately after modeling (7 days old) and at the 5th week (42 days old) at the end of the long-term experiment, respectively. Through MRI taken 7 days after birth, it was confirmed that the right brain tissue was damaged by about 50% compared to the left, and group randomization was performed by measuring the damaged area of each individual.

8.2.6. statistical analysis

The SPSS (ver.18.0) program was used, and statistical differences by group were analyzed through non-parametric tests with Mann-Whitney U test. A case where the P value between groups was less than 0.05 was judged to be statistically significant.

Experimental Example 8.3. Measurement of brain intact area

In 10-day-old rats, the ratio of the intact volume in the right hemisphere increased significantly from the group administered with ^{BM102 5X10 4} /10ul compared to the excipient-administered group (CS10, vehicle), ^{and in the group administered with BM102 1X10 5} /10ul compared to the primary MSC It was confirmed that the ratio of the intact volume in the right hemisphere was significantly increased. (Fig. 14)

Experimental Example 8.4. Measurement of proinflammatory cytokine secretion

Significant reduction of IL-1α, IL-1β, IL-6 and TNF-α, known as pro-inflammatory cytokines, from the group administered with ^{BM102 5X10 4} /10ul compared to the excipient-administered group (CS10, vehicle) in the brain tissue of 10-day-old rats appeared, and a decrease in pro-inflammatory cytokines was observed in the group administered with ^{BM102 1X10 5} /10ul compared to primary MSC. (Fig. 15)

Experimental Example 8.5. Histology score

When the degree of brain damage in 10-day-old rats was observed and quantified by regions such as cortex, hippocampus, striatum, and thalamus, the histological score was higher in the excipient-administered group (CS10, vehicle) and the group administered with ^{BM102 1X10 5/10ul compared to the primary MSC.} There was a significant decrease, and when the values were summed up and viewed as a whole, the degree of histological damage was significantly reduced from the ^{excipient administration group (CS10, vehicle) and the group administered with BM102 5X10 4/10ul compared to the primary MSC (Fig. 16).}

From the above results, it was confirmed that when BM102 was administered after 24 hours for the HIE-induced animal model, after 2 days of administration, there was an effect of restoring HIE as a result of histological and biochemical evaluations compared to the control group, and through this It can be seen that there is a short-term therapeutic effect.

Experimental Example 9. Confirmation of long-term therapeutic effect of BM102 cell line in HIE-induced animal model

Experimental Example 9.1. Cell preparation and transplantation

Stem cells and transplantation methods used in this experiment were prepared in the same manner as in Experimental Example 8.1.

Experimental Example 9.2. animal testing

9.2.1. HIE model production and BM102 cell administration group selection

The animal model used in the experiment was prepared in the same manner as in Experimental Example 8.2.1, and was classified into the following experimental groups. The experiment was conducted for 5 weeks for long-term evaluation.

Group 1: HIE model + CS10 administration (n=18)

Group 2: HIE model + Primary MSC 1X10 ⁵ /10ul administration (n=12)

Group 3: HIE model + BM102 1X10 ⁴ /10ul administration (n=17)

Group 4: HIE model + BM102 5X10 ⁴ /10ul administration (n=17)

Group 5: HIE model + BM102 1X10 ⁵ /10ul administration (n=15)

A schematic diagram of the above experimental procedure is shown in FIG. 17 .

9.2.2. Tissue storage and sample preparation for analysis

Pathological and biochemical analysis were performed using the brain tissue of a 42-day-old rat according to the same method as the short-term in vivo experiment (Experimental Example 8.2.2.).

9.2.3. Enzyme Immunoassay (ELISA)

The levels of pro-inflammatory cytokines IL-1α, IL-1β, IL-6 and TNF-α were measured using enzyme immunoassay (ELISA) in the brain tissue homogenate of 42-day-old rats.

9.2.4. MRI analysis

MRI was performed immediately after modeling (7 days old) and at the 5th week (42 days old) at the end of the long-term experiment, respectively. Through MRI taken 7 days after birth, it was confirmed that the right brain tissue was damaged by about 50% compared to the left, and group-random analysis was performed by measuring the damaged area of each individual. The prognosis for HIE was confirmed by re-imaging MRI at 42 days of age. For MRI analysis, volumetry was performed on a total of 8 consecutive pictures. MRI was analyzed by calculating the ratio of the area of the left normal hemisphere to the remaining area of the right hemisphere inducing HIE.

9.2.5. Immunostaining

GFAP (indicator of reactive gliosis), ED-1 (indicator of activated microglia), and TUNEL (indicator of cell death) staining were performed on an empty slide on which the brains of 42-day-old rats were fixed. The penumbra area for brain injury was photographed in the HIE model at a magnification of 400X using a confocal microscope on the stained slides. The photographed pictures were analyzed by measuring the intensity of GFAP and the number of ED-1 and TUNEL-positive cells.

In the case of GFAP intensity value, ED-1 or TUNEL-positive cell count, subtract the intact volume ratio of the right hemisphere from 1 measured by MRI, and then calculate the value of the damaged volume ratio of the right hemisphere did Statistical analysis was performed after correcting the value of the damaged volume ratio by multiplying the GFAP intensity value and the number of ED-1 or TUNEL positive cells.

※ Correction formula: volume ratio of damaged area ⅹ histological analysis value

9.2.6. behavioral evaluation

To measure the sensorimotor function of the HIE model, negative geotaxis and rotarod test were performed.

Negative intelligence was evaluated once a week from 14 days old to 42 days old, a total of 5 times. After placing the rat's head down on the inclined surface, the body was rotated 180 degrees to measure the duration of the head turning upwards on the inclined surface.

Rotarod evaluation was performed for 3 consecutive days from 40 days to 42 days after birth. The maximum measurement time was 5 minutes, and the latency on the rotating cylindrical rod was measured.

9.2.7. statistical processing

The SPSS (ver. 18.0) program was used, and statistical differences were analyzed for each group through one way-ANOVA with Tukey's post-test and non-parametric tests with Wald-Wolfowitz test. A case where the P value between groups was less than 0.05 was judged to be statistically significant.

Experimental Example 9.3. MRI imaging analysis

Through MRI of 7-day-old rats, it was confirmed that severe ischemic brain damage occurred evenly in all HIE induction groups. In MRI of 42-day-old rats, which is the end point of the experiment, it was confirmed that the ratio of the non-injured area of the right hemisphere was significantly increased in the group administered with ^{BM102 1X10 5/10ul compared to the excipient-administered group (CS10, vehicle).} (Fig. 18)

Experimental Example 9.4. Measurement of proinflammatory cytokine secretion

Significant reduction of IL-1α, IL-1β, IL-6 and TNF-α, known as pro-inflammatory cytokines, in the group administered with ^{BM102 1X10 5} /10ul compared to the vehicle administered group (CS10, vehicle) in the brain tissue of 42-day-old rats has appeared (Fig. 19)

Experimental Example 9.5. Analysis result by immunostaining method

In the case of activated microglia, the number of ED-1 positive cells increased by HIE was significantly reduced in the group administered with ^{BM102 1X10 5} /10ul compared to the vehicle-administered group (CS10, vehicle).

Reactive astrocytes were significantly reduced in the number of GFAP-positive cells increased by hypoxic ischemic encephalopathy in the group administered with ^{BM102 1X10 5} / 10ul compared to the excipient-administered group (CS10, vehicle).

Cell death was significantly reduced in the number of TUNEL positive cells increased by hypoxic ischemic encephalopathy in the group administered with ^{BM102 1X10 5} /10ul compared to the excipient administration group (CS10, vehicle).

The results are shown in FIG. 20 .

Experimental Example 9.6. behavioral function analysis

As shown in FIG. 20 , as a result of the negative intelligence evaluation, the duration was significantly reduced in the group administered with ^{BM102 1X10 5} /10ul compared to the excipient administration group (CS10, vehicle) in the behavioral evaluation performed on the 35th and 42nd days of life.

In addition, in the rotarod evaluation results, no significant results were observed between groups until 40 to 41 days of age, but in the behavioral evaluation conducted at 42 days of age, the persistence was significant in the group administered with ^{BM102 1X10 5/10ul compared to the excipient-administered group (CS10, vehicle).} increased significantly. (Fig. 21)

From the above results, it was confirmed that when BM102 was administered after 24 hours for HIE-induced animal models, there was an effect of restoring HIE as a result of histological and biochemical evaluations compared to the control group in long-term evaluation 5 weeks after administration, Also, it was confirmed that the sensorimotor function damaged by HIE was restored in the behavior evaluation result. Through these results, it can be seen that when the BM102 cells of the present invention are administered, there is a long-term therapeutic effect of HIE.

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

<110> SLBIGEN Inc SAMSUNG LIFE PUBLIC WELFARE FOUNDATION <120> Pharmaceutical composition for the treatment of hypoxic ischemic encephalopathy comprising mesenchymal stem cell line expressing brain-derived neurotrophic factor <130> 1067431 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of BDNF <400> 1 Met Phe His Gln Val Arg Arg Val Met Thr Ile Leu Phe Leu Thr Met 1 5 10 15 Val Ile Ser Tyr Phe Gly Cys Met Lys Ala Ala Pro Met Lys Glu Ala 20 25 30 Asn Ile Arg Gly Gln Gly Gly Leu Ala Tyr Pro Gly Val Arg Thr His 35 40 45 Gly Thr Leu Glu Ser Val Asn Gly Pro Lys Ala Gly Ser Arg Gly Leu 50 55 60 Thr Ser Leu Ala Asp Thr Phe Glu His Val Ile Glu Glu Leu Leu Asp 65 70 75 80 Glu Asp Gln Lys Val Arg Pro Asn Glu Glu Asn Asn Lys Asp Ala Asp 85 90 95 Leu Tyr Thr Ser Arg Val Met Leu Ser Ser Gln Val Pro Leu Glu Pro 100 105 110 Pro Leu Leu Phe Leu Leu Glu Glu Tyr Lys Asn Tyr Leu Asp Ala Ala 115 120 125 Asn Met Ser Met Arg Val Arg Arg His Ser Asp Pro Ala Arg Arg Gly 130 135 140 Glu Leu Ser Val Cys Asp Ser Ile Ser Glu Trp Val Thr Ala Ala Asp 145 150 155 160 Lys Lys Thr Ala Val Asp Met Ser Gly Gly Thr Val Thr Val Leu Glu 165 170 175 Lys Val Pro Val Ser Lys Gly Gln Leu Lys Gln Tyr Phe Tyr Glu Thr 180 185 190 Lys Cys Asn Pro Met Gly Tyr Thr Lys Glu Gly Cys Arg Gly Ile Asp 195 200 205 Lys Arg His Trp Asn Ser Gln Cys Arg Thr Thr Gln Ser Tyr Val Arg 210 215 220 Ala Leu Thr Met Asp Ser Lys Lys Arg Ile Gly Trp Arg Phe Ile Arg 225 230 235 240 Ile Asp Thr Ser Cys Val Cys Thr Leu Thr Ile Lys Arg Gly Arg 245 250 255 <210> 2 <211> 768 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of BDNF <400> 2 atgttccacc aggtgagaag agtgatgacc atcctgttcc tgaccatggt gatcagctac 60 ttcggctgca tgaaggctgc ccctatgaag gaggccaaca tcagaggaca gggcggcctg 120 gcctaccccg gcgtgagaac ccacggcacc ctggagagcg tgaacggccc caaggccggc 180 agcagaggcc tgaccagcct ggccgacacc ttcgagcacg tgatcgagga gctgctggac 240 gaggaccaga aggtgagacc caacgaggag aacaacaagg acgccgacct gtacaccagc 300 agagtgatgc tgagcagcca ggtgcccctg gagcctcccc tgctgttcct gctggaggag 360 tacaagaact acctggacgc cgccaacatg agcatgagag tgagaagaca cagcgacccc 420 gccagaagag gcgagctgag cgtgtgcgac agcatcagcg agtgggtgac cgccgccgac 480 aagaagaccg ccgtggacat gagcggcggc accgtgaccg tgctggagaa ggtgcccgtg 540 agcaagggcc agctgaagca gtacttctac gagaccaagt gcaaccccat gggctacacc 600 aaggagggct gcagaggcat cgacaagaga cactggaaca gccagtgcag aaccacacag 660 agctacgtga gagccctgac catggacagc aagaagagaa tcggctggag attcatcaga 720 atcgacacca gctgcgtgtg caccctgacc atcaagagag gcagataa 768 <210> 3 <211> 454 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of c-Myc <400> 3 Met Asp Phe Phe Arg Val Val Glu Asn Gln Gln Pro Pro Ala Thr Met 1 5 10 15 Pro Leu Asn Val Ser Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr Asp 20 25 30 Ser Val Gln Pro Tyr Phe Tyr Cys Asp Glu Glu Glu Asn Phe Tyr Gln 35 40 45 Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser Glu Asp Ile 50 55 60 Trp Lys Lys Phe Glu Leu Leu Pro Thr Pro Pro Leu Ser Pro Ser Arg 65 70 75 80 Arg Ser Gly Leu Cys Ser Pro Ser Tyr Val Ala Val Thr Pro Phe Ser 85 90 95 Leu Arg Gly Asp Asn Asp Gly Gly Gly Gly Ser Phe Ser Thr Ala Asp 100 105 110 Gln Leu Glu Met Val Thr Glu Leu Leu Gly Gly Asp Met Val Asn Gln 115 120 125 Ser Phe Ile Cys Asp Pro Asp Asp Glu Thr Phe Ile Lys Asn Ile Ile 130 135 140 Ile Gln Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys Leu Val 145 150 155 160 Ser Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser Gly Ser 165 170 175 Pro Asn Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser Leu Tyr 180 185 190 Leu Gln Asp Leu Ser Ala Ala Ala Ser Glu Cys Ile Asp Pro Ser Val 195 200 205 Val Phe Pro Tyr Pro Leu Asn Asp Ser Ser Ser Pro Lys Ser Cys Ala 210 215 220 Ser Gln Asp Ser Ser Ala Phe Ser Pro Ser Ser Asp Ser Leu Leu Ser 225 230 235 240 Ser Thr Glu Ser Ser Pro Gln Gly Ser Pro Glu Pro Leu Val Leu His 245 250 255 Glu Glu Thr Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln Glu 260 265 270 Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Ala Pro 275 280 285 Gly Lys Arg Ser Glu Ser Gly Ser Pro Ser Ala Gly Gly His Ser Lys 290 295 300 Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser Thr His 305 310 315 320 Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp Tyr Pro Ala 325 330 335 Ala Lys Arg Val Lys Leu Asp Ser Val Arg Val Leu Arg Gln Ile Ser 340 345 350 Asn Asn Arg Lys Cys Thr Ser Pro Arg Ser Ser Asp Thr Glu Glu Asn 355 360 365 Val Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg Asn Glu 370 375 380 Leu Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu Glu 385 390 395 400 Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr Ala 405 410 415 Tyr Ile Leu Ser Val Gln Ala Glu Glu Gln Lys Leu Ile Ser Glu Glu 420 425 430 Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys Leu Glu Gln 435 440 445 Leu Arg Asn Ser Cys Ala 450 <210> 4 <211> 1365 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of c-Myc <400> 4 atggatttct ttcgcgtcgt cgagaaccag cagccacccg ccactatgcc tctgaacgtg 60 tcttttacta acaggaacta tgatctggat tacgacagcg tgcagcccta cttctattgc 120 gatgaggaag agaactttta tcagcagcag cagcagagcg agctgcagcc acctgcacct 180 tccgaagaca tttggaagaa attcgagctg ctgcctacac cacccctgtc tccaagtcgg 240 agaagcggcc tgtgttcacc cagctacgtg gccgtcactc ctttcagcct gaggggggac 300 aatgatggcg ggggaggctc cttttctaca gccgatcagc tggaaatggt gactgagctg 360 ctggggggag acatggtcaa ccagagcttc atttgcgatc ctgacgatga aacttttatc 420 aagaacatca tcatccagga ctgtatgtgg tcaggcttta gcgccgctgc aaagctggtg 480 tctgagaaac tggcaagtta tcaggccgct cggaaagata gtgggtcacc taacccagct 540 agaggacact ccgtgtgctc tacaagctcc ctgtacctgc aggacctgag cgcagccgct 600 tccgagtgta ttgatccctc cgtggtcttc ccctatcctc tgaatgactc tagttcaccc 660 aagagttgcg catcacagga cagctccgcc ttttcacctt ctagtgatag cctgctgtca 720 agcactgaat cctctccaca gggcagccca gagccactgg tgctgcatga agagacccct 780 ccaaccacaa gttcagattc cgaagaggaa caggaggacg aggaagagat cgatgtggtc 840 tctgtggaaa agcgccaggc tccaggaaaa cgaagcgagt ccggctctcc aagtgcagga 900 ggacactcca agccacctca ttctcccctg gtgctgaaaa ggtgccacgt ctccacccac 960 cagcataact acgcagcccc accctctaca cgaaaggact atccagctgc aaagcgcgtg 1020 aaactggata gcgtgagagt cctgaggcag atcagtaaca atcggaagtg tacttcaccc 1080 agaagctccg acaccgaaga gaacgtgaaa aggcgcaccc ataatgtcct ggaacgccag 1140 cgacggaatg agctgaagag gtccttcttt gccctgcgcg atcagattcc tgaactggag 1200 aacaatgaga aggctccaaa agtggtcatt ctgaagaaag ccacagctta catcctgtct 1260 gtgcaggccg aagagcagaa actgatcagt gaagaggacc tgctgagaaa acgcagggaa 1320 cagctgaaac ataaactgga acagctgaga aactcttgtg cttaa 1365 <210> 5 <211> 248 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of tTA <400> 5 Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu 1 5 10 15 Leu Asn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln 20 25 30 Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys 35 40 45 Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His 50 55 60 Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg 65 70 75 80 Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly 85 90 95 Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr 100 105 110 Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu 115 120 125 Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys 130 135 140 Val Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr 145 150 155 160 Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu 165 170 175 Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190 Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro 195 200 205 Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala 210 215 220 Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp 225 230 235 240 Phe Asp Leu Asp Met Leu Pro Gly 245 <210> 6 <211> 747 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of tTA <400> 6 atgtcaaggc tggataaaag caaagtgatt aactccgctc tggaactgct gaacgaagtc 60 ggcattgagg ggctgaccac acgcaagctg gcacagaagc tgggagtgga gcagcccacc 120 ctgtactggc acgtgaagaa caagcgcgcc ctgctggacg ccctggccat cgagatgctg 180 gatcggcacc acacacactt ctgccctctg gagggcgaga gctggcagga cttcctgcgg 240 aacaatgcca agagctttag atgtgccctg ctgtcccaca gggatggagc aaaggtgcac 300 ctgggcacca gaccaacaga gaagcagtac gagaccctgg agaaccagct ggccttcctg 360 tgccagcagg gcttttctct ggagaatgcc ctgtatgccc tgagcgccgt gggacacttc 420 accctgggat gcgtgctgga ggaccaggag caccaggtgg ccaaggagga gagagagaca 480 cctaccacag actccatgcc ccctctgctg aggcaggcca tcgagctgtt tgatcaccag 540 ggcgccgagc cagccttcct gtttggcctg gagctgatca tctgcggcct ggagaagcag 600 ctgaagtgtg agtctggagg accagcagat gccctggacg atttcgacct ggatatgctg 660 cccgccgacg ccctggacga ttttgatctg gacatgctgc ctgctgatgc cctggatgat 720 tttgacctgg atatgctgcc tggataa 747 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for BDNF <400> 7 gcatcgacaa gagacactgg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for BDNF <400> 8 caacaccacg gaattgtcag 20

Claims (7)

Brain-derived neurotrophic factor (brain-derived neurotrophic factor, BDNF) containing stem cells expressing the protein hypoxic ischemic encephalopathy (hypoxic ischemic encephalopathy) prevention or treatment pharmaceutical composition. According to claim 1,
The stem cells are human embryonic stem cells, bone marrow stem cells, mesenchymal stem cells, human neural stem cells, limbal stem cells, or a pharmaceutical composition for the prevention or treatment of hypoxic ischemic encephalopathy, characterized in that selected from oral mucosal epithelial cells. The method of claim 1,
The stem cell is a pharmaceutical composition for preventing or treating hypoxic ischemic encephalopathy, characterized in that the immortalized stem cell line. The method of claim 1,
The stem cell is, the BDNF protein-derived amino acid sequence of SEQ ID NO: 1 will be infected with a recombinant lentivirus comprising a protein, hypoxic ischemic encephalopathy prevention or treatment pharmaceutical composition. 5. The method of claim 4,
The gene encoding the BDNF protein-derived amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 2, a pharmaceutical composition for preventing or treating hypoxic ischemic encephalopathy. The method of claim 1,
The stem cell is a pharmaceutical composition for preventing or treating hypoxic ischemic encephalopathy, characterized in that the cell line of accession number KCTC13876BP. The method of claim 1,
The pharmaceutical composition is a pharmaceutical composition for preventing or treating hypoxic ischemic encephalopathy, characterized in that it is administered intraventricularly or intravascularly.

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