KR20220061920A - Gene carrier comprising exosome derived from human peripheral blood and uses thereof - Google Patents

Abstract

The present invention relates to a gene carrier comprising exosomes derived from human peripheral blood, and uses thereof. More specifically, the present invention can provide: a carrier for regulating gene expression in heart cells containing exosomes derived from human peripheral blood; and a pharmaceutical composition for preventing or treating heart disease.

Description

Gene carrier comprising exosome derived from human peripheral blood and uses thereof

It relates to a gene carrier comprising exosomes derived from human peripheral blood and uses thereof.

Cardiovascular disease (CVD) is the disease with the highest mortality rate in the world, and according to the World Health Organization (WHO) report, it is expected that 23.6 million people will die from heart disease by 2030. In addition, myocardial infarction (myocardial infarction) is a fatal disease that occupies the number one cause of sudden death in adults, and continues to increase. Causes of heart disease include smoking, eating, overweight, obesity, and problems with blood sugar metabolism, and research to prevent and treat heart disease through lowering blood pressure, lowering serum cholesterol, and psychological treatment is continuing.

Meanwhile, recent studies report that some miRNAs including miR-21 regulate the pathological progression of myocardial infarction, for example, the level of cardiac fibrosis. However, the miRNA-based therapy has technical limitations in that miRNAs must be safely, efficiently, and specifically delivered to target cells in vivo. Under this technical background, research on a drug or gene delivery system having excellent delivery efficiency to cardiac cells is being actively conducted (Korean Patent Registration No. 10-1896082), but the situation is still insufficient.

One aspect is an exosome derived from human peripheral blood; And it is to provide a pharmaceutical composition for preventing or treating heart disease, comprising a genetic material selected from mRNA, tRNA, rRNA, siRNA, miRNA, gDNA, pDNA, or cDNA encapsulated in the exosome.

Another aspect is exosomes derived from human peripheral blood; And to provide a delivery system for regulating the gene expression of cardiac cells comprising a genetic material selected from mRNA, tRNA, rRNA, siRNA, miRNA, gDNA, pDNA, or cDNA encapsulated in the exosome.

Other objects and advantages of the present application will become more apparent from the following detailed description in conjunction with the appended claims and drawings. Content not described in this specification will be omitted because it can be sufficiently recognized and inferred by those skilled in the technical field or similar technical field of the present application.

Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.

One aspect is exosomes derived from human peripheral blood; And it provides a pharmaceutical composition for preventing or treating heart disease, comprising a genetic material selected from mRNA, tRNA, rRNA, siRNA, miRNA, gDNA, pDNA, or cDNA encapsulated in the exosome.

As used herein, the term “prevention” refers to any action that suppresses or delays the onset of heart disease by administering the pharmaceutical composition according to the present invention.

As used herein, the term “treatment” refers to any action in which symptoms for heart disease are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

The "heart disease" which is a disease to be prevented or treated by the pharmaceutical composition may be an ischemic heart disease. The ischemic heart disease is a disease that occurs due to insufficient blood supply to cardiac cells or heart muscle, and refers to a disease in which cell death such as myocardial cells is induced and tissue is permanently damaged. The heart disease may be, for example, angina pectoris, myocardial infarction, atherosclerosis, cardiac arrhythmias, heart attack, or heart attack.

As used herein, the term "exosome" refers to a small vesicle with a membrane structure secreted from various cells, and refers to a vesicle that is released into the extracellular environment after fusion of the polycystic body and the plasma membrane. According to one embodiment, the exosome may be derived or isolated from human peripheral blood.

In one embodiment, the exosome may further include a genetic material selected from mRNA, tRNA, rRNA, siRNA, miRNA, gDNA, pDNA, or cDNA having a therapeutic effect on heart disease, for example, The exosome may have the genetic material encapsulated therein.

In addition, as the genetic material, various materials known in the art to have a therapeutic effect on heart disease may be applied. For example, the genetic material may be a genetic material related to FGF family, VEGF, PlGF, PDGF, EGF, GM-CSF, PGE2, TNF-α, TGF-β, and IL-1 (Korean Circulation J 2000; 30(6):772-779). In addition, the genetic material may be a genetic material related to miRNA having a therapeutic effect on heart disease, for example, increasing the expression of STRN gene in cardiac cells or increasing the expression of STRN and NOS genes in cardiac cells. It may be a substance. The genetic material may be, for example, miR-21 inhibitor, for example, the genetic material represented by SEQ ID NO: 1.

In one embodiment, the exosome may have a diameter of 50 to 150 nm. The diameter of the exosomes may be, for example, 50 to 130 nm, 50 to 110 nm, 50 to 90 nm, 50 to 70 nm, 70 to 130 nm, 70 to 110 nm, 70 to 90 nm, 90 to 130 nm, 90 to 110 nm.

In one embodiment, the exosome may express a protein marker selected from Alix, LAMP2, CD9, CD63, CD81, and combinations thereof.

According to one embodiment, it was confirmed that exosomes derived from human peripheral blood were effectively uptaked into H9C2 and HL-1 cardiac cell lines (Example 1), and after encapsulating a specific genetic material in the exosomes, As a result of applying this to cardiac cell lines and myocardial infarction animal model, significant therapeutic efficacy was confirmed (Examples 2 and 3). In addition, exosomes derived from human peripheral blood showed safety in vivo, and it was confirmed that the exosomes derived from human peripheral blood according to an embodiment exhibited excellent absorption into cardiac cells and ability to transfer genetic material (implementation). Examples 4 and 5). In addition, exosomes derived from human peripheral blood loaded with miR-21 inhibitor according to an embodiment showed significant therapeutic efficacy in myocardial infarction animals, and their detailed treatment mechanisms could also be experimentally confirmed (Example 6 and 7).

Therefore, the pharmaceutical composition according to an embodiment may be utilized as an active ingredient for the treatment of heart disease.

The pharmaceutical composition may include a pharmaceutically effective amount of exosomes derived from human peripheral blood alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents. In the above, the pharmaceutically effective amount means an amount sufficient to prevent, improve, and treat symptoms of heart disease. The pharmaceutically effective amount of the exosome may be 5×10 ³ to 5×10 ¹⁰ particles/day/weight (kg), and may be 5×10 ⁹ particles/day/kg body weight. However, the pharmaceutically effective amount may be appropriately changed depending on the severity of heart disease symptoms, the age, weight, health status, sex, administration route, and treatment period of the patient. In addition, the "pharmaceutically acceptable" means a composition that is physiologically acceptable and does not normally cause gastrointestinal disorders, allergic reactions such as dizziness or similar reactions when administered to humans. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-agglomeration agents, lubricants, wetting agents, flavoring agents, emulsifiers and preservatives and the like may be further included.

The pharmaceutical composition may be formulated and administered in a unit dosage form suitable for administration in a patient's body according to a method conventional in the pharmaceutical field, and the preparation may be administered to heart cells or cardiomyocytes by one or several administrations. effective dosages to develop As a formulation suitable for this purpose, an injection such as an ampoule for injection is preferable as a formulation for parenteral administration. The injection ampoule may be prepared by mixing with an injection solution immediately before use, and physiological saline, glucose, mannitol, Ringer's solution, etc. may be used as the injection solution.

In the pharmaceutical composition, in addition to the active ingredient, one or more pharmaceutically acceptable conventional inert carriers, for example, preservatives, analgesics, solubilizers or stabilizers in the case of injections, and the like, in the case of formulations for topical administration It may further include a base (base), excipients, lubricants or preservatives and the like.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, and humans by various routes such as parenteral and oral administration, and the administration method may be administered by any method commonly used in the art. Although not limited thereto, it may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

Another aspect may provide a method for treating heart disease, comprising administering the pharmaceutical composition to a subject.

As used herein, the term "subject" refers to a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, dogs, cats, horses and cattle. means mammals.

Another aspect is exosomes derived from human peripheral blood; And it is possible to provide a vehicle for regulating gene expression in cardiac cells comprising a genetic material selected from mRNA, tRNA, rRNA, siRNA, miRNA, gDNA, pDNA, or cDNA encapsulated in the exosome.

Among the terms or elements mentioned in the following gene expression control delivery system, those mentioned in the description of the pharmaceutical composition are as described above.

According to one embodiment, it was confirmed that exosomes derived from human peripheral blood were effectively uptaked into H9C2 and HL-1 cardiac cell lines (Example 1), and after encapsulating a specific genetic material in the exosomes, As a result of applying this to cardiac cell lines and myocardial infarction animal model, it was confirmed that the gene expression level of cardiac cells could be effectively regulated (Examples 2 and 3). Accordingly, the delivery system according to an embodiment may be utilized for regulating gene expression in cardiac cells.

In one embodiment, when the genetic material is a material having a therapeutic effect on heart disease, the carrier may be applied to gene therapy for heart disease.

According to the composition of one aspect, exosomes derived from human peripheral blood encapsulated with genetic material can significantly improve the efficiency of delivery of the genetic material to cardiac cells, and thereby, the genetic properties of cardiac cells can be easily adjusted. Accordingly, the composition according to an aspect may be applied as a core technology in the field of heart disease treatment, particularly gene therapy.

1 is a graph showing the characteristics of exosomes derived from human peripheral blood according to an embodiment, and FIG. 1A is a result of observing the exosomes with a transmission electron microscope, and FIG. 1B is a nanoparticle tracking analysis. It is the result of confirming the size of the exosome, and FIG. 1C is the result of confirming the expression pattern in the exosome through western blotting.
2 is a view showing the uptake of exosomes derived from human peripheral blood according to an embodiment into cardiac cell lines was confirmed by confocal microscopy. B is the result of confirming the uptake into the HL-1 cardiac cell line.
3 is a view illustrating the effect of transferring human peripheral blood-derived exosomes to the H9C2 cardiac cell line according to an embodiment, and quantitatively comparing the levels of miR-21 in each H9C2 cardiac cell line.
4 is a view illustrating the effect of transferring human peripheral blood-derived exosomes to the H9C2 cardiac cell line according to an embodiment. FIG. 4A is a quantitative comparison of Smad7 mRNA levels, and FIG. 4B is This is the result of quantitatively comparing the protein level of Smad7.
5 is a graph illustrating the effect of transferring human peripheral blood-derived exosomes to the H9C2 cardiac cell line according to an embodiment. FIG. 5A is a quantitative comparison of PTEN mRNA levels, and FIG. 5B is This is the result of quantitatively comparing the protein level of PTEN.
6 is a view illustrating the effect of transferring human peripheral blood-derived exosomes to HL-1 cardiac cell lines according to an embodiment, and quantitatively comparing the levels of miR-21 in each HL-1 cardiac cell line.
7 is a view illustrating the effect of transferring human peripheral blood-derived exosomes to the HL-1 cardiac cell line according to an embodiment. FIG. 7A is a quantitative comparison of Smad7 mRNA levels, and FIG. B is the result of quantitatively comparing the protein level of Smad7.
FIG. 8 is a view illustrating the effect of transferring human peripheral blood-derived exosomes to the HL-1 cardiac cell line according to an embodiment, and FIG. 8A is a quantitative comparison of PTEN mRNA levels, and FIG. B is the result of quantitative comparison of the protein level of PTEN.
9 is an animal model of myocardial infarction, confirming the genetic material transfer effect of exosomes derived from human peripheral blood according to an embodiment. FIG. 9A is a quantitative comparison of Smad7 mRNA levels, and FIG. 9B is a result of quantitatively comparing the mRNA level of PTEN, and FIG. 9C is a result of quantitatively comparing the protein level of PTEN.
10 is a result of confirming the pathological change according to an embodiment after processing the exosomes derived from human peripheral blood loaded with the genetic material to the lesion site of the myocardial infarction animal model.
11 is as confirming the biosafety of exosomes derived from human peripheral blood according to an embodiment, and FIG. 11A is the result of confirming the change in cell viability according to the treatment dose of exosomes derived from human peripheral blood, FIG. 11B is the result of confirming the change in cell viability according to the treatment time of exosomes derived from human peripheral blood.
12 is a result confirming the change in the expression level of inflammatory cytokines when human peripheral blood-derived exosomes according to an embodiment are added or treated to the H9C2 cardiac cell line.
13 is a result of histological confirmation of whether an inflammatory response is induced when the exosomes derived from human peripheral blood according to an embodiment are added or processed to tissues of various organs.
14 is a view showing the characteristics of exosomes derived from human peripheral blood loaded with a genetic material according to an embodiment, and FIG. 14A is a result of observing the exosomes with a transmission electron microscope, and FIG. 14B is a Western Blot. It is the result of confirming the expression pattern in the exosome through ting.
15 is a view illustrating the effect of transferring genetic material from human peripheral blood-derived exosomes loaded with genetic material according to an embodiment to the H9C2 cardiac cell line, and confirming the uptake into the cardiac cell line by confocal microscopy.
16 is a view illustrating the effect of transferring genetic material to the H9C2 cardiac cell line of human peripheral blood-derived exosomes loaded with the genetic material according to Example under normal and hypoxic conditions. FIG. 16A shows miR- in each H9C2 cardiac cell line. It is a result of quantitatively comparing the level of 21, and FIG. 16B is a result of quantitatively comparing the level of miR-21 according to the concentration of exosomes in the H9C2 cardiac cell line.
17 is a result of evaluating Rnase resistance of exosomes derived from human peripheral blood loaded with a genetic material according to an embodiment.
18 is a result of confirming the change in left ventricular contracture rate when adding or processing exosomes derived from human peripheral blood loaded with genetic material according to an embodiment in an animal model of myocardial infarction.
19 is a result confirming the change in the expression level of collagen I and collagen III in the case of adding or processing exosomes derived from human peripheral blood loaded with genetic material according to an embodiment in an animal model of myocardial infarction.
20 is a result of confirming the binding potential of the 3'-UTR region of STRN and miR-21 through the miRNA target prediction program (www.targetscan.org).
21 is a result confirming the change in luciferase activity when cardiac cell lines are treated with the synthesized luciferase vector system and scrambled miRNA as miR-21 mimic or as a control.
22 is a result confirming the change in the STRN protein expression level according to the expression level of miR-21 in the H9C2 cardiac cell line.
23 is a result confirming the change in the expression level of NOS3 protein when STRN-specific siRNA is added or treated in H9C2 cardiac cell line.
24 is a result confirming the change in the expression level of STRN protein and NOS protein when miR-21 mimic or miR-21 inhibitor is treated in H9C2 cardiac cell line under hypoxic conditions.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Isolation of exosomes derived from human peripheral blood and identification of properties thereof

In this example, using the Exoquick exosome precipitation kit, exosomes were isolated from human peripheral blood (250 μl) according to the manufacturer's instructions. Thereafter, through transmission electron microscopy, nanoparticle tracking analysis, and Western blotting analysis, detailed characteristics of the isolated exosome were investigated. In addition, through PKH26 fluorescence staining (red) and confocal microscopy, it was attempted to determine whether exosomes derived from human peripheral blood were uptaken into H9C2 and HL-1 cardiac cell lines.

As a result, as shown in FIG. 1 , the exosomes derived from human peripheral blood were identified as circular structures having an average diameter of about 104 nm, and proteins such as Alix, LAMP2, CD9, CD63, and CD81 from the exosomes. Markers were detected. In addition, as shown in FIG. 2 , it was confirmed that the exosomes were effectively absorbed into both H9C2 and HL-1 cardiac cell lines.

Example 2. Delivery of genetic material using exosomes derived from human peripheral blood

In this example, it was attempted to confirm the effect of transferring the genetic material of the exosome of Example 1, specifically, the effect of transferring the genetic material to the cardiac cell line. To this end, the negative control miRNA, miR-21 mimic, and miR-21 inhibitor were loaded into exosomes isolated from human peripheral blood using the Exo-Fect ^TM Exosome Transfection Kit (SBI System Biosciences, USA), respectively. The miRNA (SMC-2003), miR-21 mimic (5'-UAGCUUAUCAGACUGAUGUUGA-3', SMM-002, SEQ ID NO: 2), miR-21 inhibitor (5'-UAGCUUAUCAGACUGAUGUUGA-3'; SMI-002, SEQ ID NO: 1) ) was obtained from Bioneer (Daejeon, Korea).

2-1. miRNA delivery into the H9C2 cardiac cell line

The H9C2 cardiac cell line was cultured in a 100 mm culture dish, and after one day, 150 μg of control, mimic or inhibitor-loaded exosomes of miRNA were added to the cell line, and the cell line was re-incubated under hypoxic conditions after 24 hours. cultured. Then, by using the reverse transcriptase polymerase chain reaction and western blotting analysis, the miRNA delivery efficiency of exosomes derived from human peripheral blood to the H9C2 cardiac cell line was confirmed. In addition, mRNA and protein levels of Smad7 or PTEN, which are direct targets of miR-21, were analyzed.

As a result of analyzing the miR-21 level of the H9C2 cardiac cell line, as shown in FIG. 3 , it was confirmed that the miR-21 level in the H9C2 cell line was regulated by the mimic or inhibitor-loaded exosomes. That is, in the H9C2 cell line under hypoxic conditions, the addition of miR-21 mimic-loaded exosomes increased the expression of miR-21 compared to cells under normoxic and hypoxic conditions (Exo+21-M), whereas miR Addition of the -21 inhibitor-loaded exosomes decreased the expression of miR-21 (Exo+21-I).

In addition, as shown in FIGS. 4 and 5, the addition of miR-21 mimic-loaded exosomes down-regulated both mRNA and protein levels of Smad7 and PTEN (Exo+21-M), whereas miR-21 inhibitor The addition of the loaded exosomes upregulated both mRNA and protein levels of Smad7 and PTEN (Exo+21-I). Considering that miRNA exerts its function by binding to the 3'-untranslated region (3'-UTR) of the target mRNA and inhibiting the expression of the target gene, the above experimental results were analyzed for the expression level of miR-21. It can be seen that the result is consistent with

2-2. miRNA delivery into HL-1 cardiac cell line

In the same manner as in Example 2-1, the miRNA delivery efficiency of exosomes derived from human peripheral blood to the HL-1 cardiac cell line was confirmed, and the mRNA and protein levels of Smad7 or PTEN, a direct target of miR-21, were analyzed. did

As a result of analyzing the level of miR-21 in the HL-1 cardiac cell line, as shown in FIG. 6 , in the HL-1 cell line under hypoxic conditions, the addition of miR-21 mimic-loaded exosomes showed that the levels of miR-21 in the hypoxic and normoxic conditions were reduced. While the expression of miR-21 was increased compared to the cells (Exo+21-M), the addition of the miR-21 inhibitor-loaded exosomes decreased the expression of miR-21 (Exo+21-I). On the other hand, when miRNA negative control-loaded exosomes (Exo+NC) were added, no change in expression level was observed compared to cells under hypoxic conditions. In addition, as shown in FIGS. 7 and 8, the addition of miR-21 mimic-loaded exosomes down-regulated both mRNA and protein levels of Smad7 and PTEN (Exo+21-M), as shown in FIGS. ), the addition of miR-21 inhibitor-loaded exosomes upregulated both mRNA and protein levels of Smad7 and PTEN (Exo+21-I).

Combining these experimental results, it can be seen that the exosomes derived from human peripheral blood according to an embodiment can be applied to the transfer of genetic material to cardiac cells.

Example 3. Confirmation of treatment efficacy using an animal model of myocardial infarction

In this example, after inducing myocardial infarction in C57BL/6 mice, it was evaluated by reverse transcriptase polymerase chain reaction and Western It was evaluated using a blotting technique. In addition, it was attempted to confirm the therapeutic efficacy of myocardial infarction using the exosomes derived from human peripheral blood. Specifically, after myocardial infarction was induced in C57BL/6 mice, miR-21 mimic or inhibitor-loaded human peripheral blood-derived exosomes were delivered to the lesion site in the heart, and then, Hematoxylin & By performing eosin (H&E) and Masson's Trichrome (MT) staining, pathological changes according to the delivery of each of the exosomes were observed.

As a result, as shown in FIG. 9, the addition of miR-21 mimic-loaded exosomes down-regulated Smad7 mRNA, PTEN mRNA and protein levels, similar to the results of Example 2 (Exo+21) -M), the addition of miR-21 inhibitor-loaded exosomes upregulated Smad7 mRNA, PTEN mRNA and protein levels (Exo+21-I). On the other hand, when miRNA negative control-loaded exosomes (Exo+NC) were added, no change in expression level was observed compared to cells under hypoxic conditions.

In addition, as shown in FIG. 10 , with respect to myocardial disorders and fibrosis in mice, the miR-21 mimic-loaded exosome-treated group significantly increased the area of the fibrotic lesion site compared to the control group (3.758). ± 0.203 vs. 6.010 ± 0.571, p = 0.020), the miR-21 inhibitor-loaded exosome-treated group significantly reduced the area of the fibrotic lesion (3.758 ± 0.203 vs. 2.594 ± 0.288, p = 0.030).

These experimental results show that exosomes derived from human peripheral blood according to an embodiment can be usefully used for transferring genetic material to heart cells, and that these properties can be extended to genetic treatment of heart diseases including myocardial infarction. it will indicate

Example 4. Toxicity evaluation of exosomes derived from human peripheral blood

In this example, it was attempted to confirm the biosafety of the exosome of Example 1. To this end, exosomes derived from human peripheral blood were added to the H9C2 cardiac cell line, and changes in cell viability according to dose (5, 10, 20, 40 μg/ml) or treatment time were confirmed, and in addition, TNF-α, By comparing the expression levels of inflammatory cytokines such as HMGB1 and IL-10, it was evaluated whether the inflammatory response was induced.

As a result, as shown in FIG. 11 , no dose or time-dependent cytotoxicity was observed for exosomes derived from human peripheral blood, and as shown in FIG. 12 , when 40 μg/ml of exosomes were treated However, it was found that the induction of the inflammatory response was at a negligible level. In addition, as a result of histological evaluation of whether an inflammatory response was induced by the administration of the exosomes derived from human peripheral blood (200ug) using experimental animals, as shown in FIG. 13 , not only the heart but also the liver, spleen, kidney, It did not cause an inflammatory response in other organs such as the lungs.

Example 5. Genetic material transfer to cardiac cells using exosomes derived from human peripheral blood

In this example, it was attempted to confirm the effect of transferring the genetic material of the exosome of Example 1, specifically, the effect of transferring the genetic material to the cardiac cell line. The genetic material mentioned in Example 2, miRNA, was loaded into exosomes derived from human peripheral blood. Thereafter, the exosomes loaded with the genetic material were observed under a transmission microscope, and expression changes of Alix, CD9, CD63, and CD81 were confirmed through western blotting. In addition, the exosome loaded with the genetic material was added to the H9C2 cardiac cell line in the same manner as in Example 2 and cultured. Then, the H9C2 cardiac cell line treated with the exosomes fluorescently labeled with PKH26 was observed with a confocal microscope.

As a result, as shown in FIG. 14 , after miRNA loading, significant changes in the shape and marker protein of the exosome were not observed, and as shown in FIG. absorption was confirmed.

In addition, under normal and hypoxic conditions, in order to check the effect of regulating the gene expression level in cardiac cells through the exosome loaded with the genetic material, miR-21 mimic (5'-UAGCUUAUCAGACUGAUGUUGA-3', SMM-002), miR The exosome loaded with -21 inhibitor (5'-UAGCUUAUCAGACUGAUGUUGA-3'; SMI-002) was added to the cardiac cell line in the same manner as in Example 2 and cultured, and then the miR-21 expression change was confirmed.

As a result, as shown in FIG. 16 , miR-21 expression in the H9C2 cardiac cell line was regulated by miR-21 mimic or inhibitor-loaded exosomes. Specifically, the addition of miR-21 mimic-loaded exosomes showed higher miR-21 expression than the control under normoxic and hypoxic conditions, whereas the addition of miR-21 inhibitor-loaded exosomes decreased miR-21 expression. became In addition, uptake of miRNA-loaded exosomes induced the regulation of miR-21 levels in H9C2 cells in a dose-dependent manner with respect to exosomes, and treatment with exosomes alone did not affect miR-21 expression.

In addition, in order to evaluate the RNase resistance of the miRNA-loaded exosomes, RNase A, an enzyme capable of degrading RNA, was treated to the exosomes, and then the H9C2 cardiac cell line was added. As a result, as shown in Figure 17, RNase treatment did not affect the total amount of miR-21 loaded in the exosomes, which shows the resistance ability of the exosomes to Rnase.

Example 6. Re-verification of treatment efficacy using an animal model of myocardial infarction

In this example, after myocardial infarction was induced in C57BL/6 mice in the same manner as in Example 3, exosomes derived from peripheral blood loaded with miR-21 inhibitor were delivered to the heart lesion site. Then, one week after the induction of myocardial infarction, the cardiac function of the mouse was evaluated by echocardiography. In addition, the expression levels of collagen I and collagen III in cardiac cells of the animal model were evaluated through Western blotting.

As a result, as shown in FIG. 18 , compared to the group treated with the miRNA negative control-loaded exosomes (Exo+NC) and miR-21 mimic-loaded exosomes, the miR-21 inhibitor-loaded exosomes In the treated group, the left ventricular ejection fraction (LVEF), which is a cardiac function evaluation index, was significantly increased. In addition, as shown in FIG. 19 , the miR-21 inhibitor-loaded exosome-treated group significantly reduced the expression of collagen related to cardiac fibrosis.

These experimental results show that exosomes derived from human peripheral blood according to an embodiment can be usefully used for the delivery of genetic material, specifically miRNA-21 inhibitor, to cardiac cells, and based on this effect, the This indicates that exosomes can be broadly applied to the genetic treatment of heart diseases including myocardial infarction.

Example 7. Confirmation of the treatment mechanism of heart disease

In this example, it was attempted to confirm the mechanism of the effective efficacy verified in Example 6. First, as shown in FIG. 20, as a result of searching for a gene having a nucleotide sequence capable of binding to miR-21 using the miRNA target prediction program (www.targetscan.org), miR in the 3'-UTR region of STRN It was confirmed that -21 has the possibility of binding.

Based on these results, in order to confirm whether miR-21 and STRN actually bind, the nucleotide sequence of the STRN 3'-UTR region to which miR-21 binds was cloned into pmirGLO Dual-luciferase Vector (Promega, USA). Thereafter, when cardiac cell lines were treated with the synthesized luciferase vector system and scrambled miRNA as miR-21 mimic or as a control, as shown in FIG. 21 , the luciferase activity was significantly increased in the miR-21 mimic-treated group. On the other hand, no significant change was observed when the vector system in which the nucleotide sequence of the STRN 3'-UTR region to which miR-21 binds was mutated and the cell line were treated with miR-21 mimic or scrambled miRNA as a control. These results indicate that miR-21 affects the expression level of STRN by binding to the STRN 3'-UTR region.

In addition, by treating miR-21 with mimic or inhibitor, STRN mRNA and protein expression levels in the H9C2 cardiac cell line were confirmed by overexpression and inhibition of miR-21, respectively. In order to confirm the role of STRN on NOS3, STRN-specific After inhibiting the expression level of STRN using siRNA, the change in the expression level of NO3 was confirmed accordingly.

As a result, as shown in FIG. 22 , when miR-21 was overexpressed, the expression levels of STRN mRNA and protein were greatly reduced, and on the contrary, when the expression level of miR-21 was suppressed, STRN mRNA and protein expression levels were decreased. increased. In addition, as shown in FIG. 23 , the STRN-specific siRNA-transfected H9C2 cardiac cell line reduced the protein level of STRN by about ~50% or more, and the NOS3 protein expression was also significantly reduced along with the decrease of the STRN expression level.

Next, the effect of miR-21 expression changes on STRN and NOS protein expression levels in the H9C2 cardiac cell line exposed to hypoxia was confirmed through western blotting. As a result, as shown in FIG. 24 , after exposure to the hypoxic condition for 24 hours, the protein expression levels of STRN and NOS3 were reduced compared to the control (normoxia) not exposed to the hypoxic condition. In addition, this expression level was further down-regulated when miR-21 mimic was treated, and on the contrary, the change was reversed when miR-21 inhibitor was treated.

These experimental results indicate that the miR-21 inhibitor according to an embodiment exhibits effective therapeutic efficacy through an increase in STRN expression followed by an increase in NO3 expression.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Gene carrier comprising exosome derived from human peripheral blood and uses it <130> PN143880KR <150> KR 10-2019-0011309 <151> 2019-01-29 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-21 inhibitor <400> 1 uagcuuauca gacugauguu ga 22 <210> 2 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-21 mimic <400> 2 uagcuuauca gacugauguu ga 22

Claims (7)

As a pharmaceutical composition for the prevention or treatment of myocardial infarction comprising an exosome encapsulated with a miRNA 21 inhibitor,
The exosome is for regulating the gene expression of cardiac cells, and is an exosome derived from human peripheral blood,
The pharmaceutical composition will increase the expression of the STRN gene in cardiac cells, the pharmaceutical composition. The pharmaceutical composition of claim 1, wherein the exosome expresses a protein marker selected from Alix, LAMP2, CD9, CD63, CD81, and combinations thereof. The pharmaceutical composition of claim 1, wherein the exosome has a diameter of 50 to 150 nm. The pharmaceutical composition of claim 1, wherein the miRNA 21 inhibitor consists of the sequence of SEQ ID NO: 1. exosomes derived from human peripheral blood; and
As a vehicle for regulating gene expression in cardiac cells, which is applied to gene therapy of myocardial infarction comprising a miRNA 21 inhibitor encapsulated in the exosome,
The vehicle for regulating gene expression is a vehicle for regulating gene expression in cardiac cells, which increases the expression of STRN gene in cardiac cells. The method according to claim 5, wherein the exosome has a diameter of 50 to 150 nm, the delivery system for regulating the gene expression of cardiac cells. The vehicle according to claim 5, wherein the exosome expresses a protein marker selected from Alix, LAMP2, CD9, CD63, CD81, and a combination thereof.

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