ANTI-INFLAMMAATORY AND ANTI-VIRAL COMPOSITION BASED ON PLACENTA-DERIVED EXTRACELLULAR VESICLE

Abstract

The present invention relates to kinds and effects of various materials derived from the placenta, in particular, kinds and effects of diverse biomolecules including: extracellular vesicle (EV) derived from the placenta, umbilical cord and/or cord blood, placental mesenchymal stem cell (pMSC) derived from the placenta, umbilical cord and/or cord blood, EVs derived from the above bio-materials, as well as miRNA and cargo contained in the above EVs. In this regard, miRNA contained in the EV efficiently degrades RNA genome of corona virus, thereby exhibiting direct anti-viral effects. Further, indirect effects of preventing and treating different virus infections owing to antiinflammatory effects of various biomolecules inherently included in EV, can be identified, thereby completing the present invention.

Claims

1. A cell-free composition, comprising biomolecules extracted from placenta, umbilical cord and/or cord blood along with at least one carrier, excipient or diluents.

2. The cell-free composition according to claim 1, wherein the biomolecules are extracellular vesicles (EV) derived from the placenta and placental mesenchymal stem cell (pMSC).

3. The cell-free composition according to claim 1, wherein the biomolecules are miRNA contained in the cellular vesicles (EV) of the placental mesenchymal stem cell (pMSC).

4. The cell-free composition according to claim 3, wherein the miRNA is one or more selected from the group consisting of hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-miR-181a-5p, hsa-miR-26a-5p, hsa-miR-34a-5p, hsa-miR-23a-3p, hsa-miR-125b-5p, hsa-miR-125a-5p, hsa-miR-103a-3p, hsa-miR-223-3p, hsa-miR-25-3p, hsa-miR-26b-5p, hsa-miR-193a-5p, hsa-miR-1307-3p, hsa-miR-155-5p, hsa-miR-185-5p, hsa-miR-424-3p and hsa-miR-23b-3p.

5. The cell-free composition according to claim 3, wherein the biomolecules included in the cell-free composition are one or more selected from the EV and the miRNA.

6. An anti-viral composition comprising the cell-free composition according to claim 1.

7. The anti-viral composition according to claim 6, wherein the virus is one or more selected from the group consisting of corona virus, HIV, influenza, enterovirus, porcine reproductive and respiratory syndrome virus, C type hepatitis virus and bovine. viral diarrhea virus.

8. The anti-viral composition according to claim 7, wherein the corona virus is corona-19 virus (NMC-nCoVO2) or SARS-CoV-2 virus.

9. A pharmaceutical composition for prevention or treatment of viral infection, comprising the anti-viral composition according to claim 6.

10. The pharmaceutical composition according to claim 9, wherein the viral infection is caused by corona viral infection.

11. A food composition for prevention or improvement of viral infection, comprising the anti-viral composition according to claim 6.

12. An anti-inflammatory composition, comprising the cell-free composition according to claim 1.

13. An anti-viral composition comprising the cell-free composition according to claim 2.

14. An anti-viral composition comprising the cell-free composition according to claim 3.

15. An anti-viral composition comprising the cell-free composition according to claim 4.

16. An anti-viral composition comprising the cell-free composition according to claim 5.

17. An anti-inflammatory composition, comprising the cell-free composition according to claim 2.

18. An anti-inflammatory composition, comprising the cell-free composition according to claim 3.

19. The anti-viral composition according to claim 16, wherein the virus is corona-19 virus (NMC-nCoVO2) or SARS-CoV-2 virus.

20. A pharmaceutical composition for prevention or treatment of viral infection, comprising the anti-viral composition according to claim 19.

Description

DESCRIPTION OF DRAWINGS

[0052] FIG. 1 illustrates a functional mechanism for anti-viral effects of tissue-EV, tissue extract EV, MSC-EV and miRNA included therein with regard to SARS-CoV-2 virus, and it could be seen that direct anti-viral effects include down control of SARS-CoV-2RNA induced by direct binding of miRNA in sites such as 3'UTR, 5'URT or a coding sequence of SARS-CoV-2, while indirect anti-viral effects include adjustment of inflammatory mRNA expression through miRNA and protein inherent in EV so as to regenerate damaged tissues, while controlling anti-inflammatory circumstances through miRNA and protein of the regenerated tissues so as to inhibit cytokine storm.

[0053] FIG. 2 illustrates observed results of pMSC-EV characteristics (A: pMSC-EV is positive for CD63 measured by fluorescence microscopy. B: an average size of pMSC-EV is 121.8 mm in diameter for 1 ml of extract. C: Representative TEM image. D: Western blott’s results of pMSC-EV with regard to CD81, CD9, annexin A2 and HS970).

[0054] FIG. 3 illustrates a potential binding site at which miRNA of the present invention can be bound to 3'UTR of SARS-CoV-2.

[0055] FIG. 4 illustrates results of analyzing qPCR in order to confirm whether miRNA of the present invention exists in EV.

[0056] FIG. 5 illustrates results of luciferase analysis in order to identify direct anti-viral effects of EV according to the present invention (A: a recombinant plasmid prepared by cloning a PCR fragment to a luciferase reporter plasmid between luciferase ORF and the synthetic poly(A) sequence. B: activity of opposite luciferase in SK-N-BE(2)C cells transfected with the recombinant plasmid).

[0057] FIG. 6 illustrates results of confirming whether miRNA of the present invention is effective for various corona viruses (A: a binding site of miRNA as well as 3'UTR of each of five corona viruses. B: interaction between miRNA of the present invention and other viruses).

[0058] FIG. 7 schematically illustrates an experimental method for identification of indirect anti-virus effects of EV and miRNA, respectively.

[0059] FIG. 8 illustrates results of confirming effects after treatment of BEAS-2B (gastrointestinal ephithelia) with EV and miRNA (A: EV, B: miRNA).

[0060] FIG. 9 illustrates results of confirming protection and recovery effects when treating LL-24 with EV and miRNA (A: EV, B: miRNA).

[0061] FIG. 10 illustrates results of confirming protection and recovery effects of MEF (fibroblast) and BV2 (microglial cell line), in particular, in FIGS. 10a and 10b, respectively.

[0062] FIG. 11 illustrates results of confirming expression levels of inflammation relevant factors after treatment of neuron cells, M2, with EV.

[0063] FIG. 12 shows photographed images before and after treatment of each of cell lines with PKH26-labeled EV.

[0064] FIG. 13 is a conceptual view illustrating viruses and processes of treating or improving viral infection symptoms with EV and miRNA of the present invention through direct and indirection routes.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF INVENTION

Experimental Method and Material

Preparation of Placenta Extract and EV

[0065] Human placenta not involving symptoms of internal, obstetrical or surgical complication has been obtained. Each placenta was carefully incised, washed with PBS several times, mechanically pulverized, and digested with 0.5% collagenase IV (Sigma, St. Louis MO, USA) at 37° C. for 30 minutes to obtain the placenta extract and EV.

Cell Line and Virus

[0066] A cell line of renal epithelial cells of African green monkey, that is, vero cells were used. The cells were incubated in DMEM medium (Dulbecco’s Modified Eagle’s medium, Gibco, USA).

[0067] In early February in 2020, corona-19 virus (NMC-nCoVO2) and SARS-CoV-2 virus separated from the respiratory system specimens of domestic infected patients were used for infection of vero E6 cells, and a virus titer was determined by 50% tissue culture infection dose (TCID.sub.50) with regard to cytopathic effects (CPE). Further, all experiments for viruses were implemented in biological safety level-3 (BLS-3) laboratories.

Assessment of Cytotoxicity

[0068] Cytotoxic effects of EV to vero cells were evaluated by MTT-assay.

[0069] More particularly, a vero cell layer in 96-well plate was washed with phosphate buffer saline (PBS) and treated with a continuous amount of EV. 1 x 10.sup.4 vero cells were incubated in 96-well plate at 37° C. under 5% CO.sub.2 for 24 hours. Absorbance was measured by a plate reader at a wavelength of 500 to 600 nm.

Assessment of Inhibitory Effects of Cytopathic Effects (CPE)

[0070] After introducing 1 x 10.sup.4 vero cells in 96 wells, using 50 .Math.L of culture solution containing EV and 50 .Math.L of solution containing SARS-CoV-2 virus were treated at 37° C., respectively. The cells inoculated with virus was used as a control. The infected cells were observed with 100% CPE under a microscope. The cells were stained with 1% crystal violet. The percentage of CPE in EV-treated cells was calculated (positive well/total well), and a result thereof was photographed.

Nano-Particle Tracking and Analysis

[0071] EV was diluted into 1 mL with PBS and inspected by a ZetaView nanoparticle tracking video microscope (Particle Metrix, Innig, Germany). A basic setting for EV or nanoparticles by a software manufacturer was chosen and, for each measurement, scanning was conducted three times.

Fluorescence Imaging of EV

[0072] EV was biotinylated using EZ-Link Sulfo-NHS-LC-LC-Biotin (Thermo Fisher Scientific, Waltham, MA, USA).

[0073] The biotinylated EV was loaded to Zeba spin desalting columns 7 K MWCO (Thermo Scientific), followed by removing the remaining free biotin.

[0074] For staining, 20 .Math.L of biotinylated EV was added to a circle drawn on a streptavidin coated glass slide (Arrayit Corporation) and, after 30 minutes, EV was fixed with BD Fix Perm (BD bioscience), followed by blocking the same with 0.2% BSA-PBS.

[0075] EV was subjected to immune fluorescent staining using anti-human CD 63 (1:100, Santa Cruz Bio-Technology in Dallas, Texas, USA, (SC-5275)) at room temperature for 2 hours, followed by incubation along with a secondary antibody conjugated with Alex Fluor 488 at room temperature for 1 hour.

Transmission Electron Microscopic (TEM) Imaging

[0076] 5 ml of aliquot of the diluted sample containing 0.5 .Math.g of protein was dropped on a hydrophilic grid. After several minutes, the grid was washed with distilled water and compared with 2% uranyl acetate for 20 seconds. Under JEM-1010 microscope (JEOL) operating at 80 kV, images were acquired.

Western Blotting

[0077] EV was dissolved in 10 x RIPA buffer containing protease cocktail tablets (Roche) and phosphatase inhibitors II and III (Sigma). According to BCA assay (Thermo Fisher Scientific), a protein concentration was determined. After adding 10% SDS-PAGE to 30 .Math.l aliquot of each sample, western blotting was conducted.

[0078] The blotting was blocked in 10% skim milk of TBS-T. The following antibodies to protein were obtained from the following suppliers: CD81 (1:1,000, Santa Cruz Biotechnology), Alix (1:1,000, Santa Cruz Biotechnology), CD9 (1:500, Santa Cruz), and GAPDH (1:10,000, sc-32233, Santa Cruz Biotechnology).

[0079] A primary antibody was diluted in TBS-T and incubated along with blotting at 4° C., followed by incubation using a secondary antibody for 1 hour. Using an enhanced chemiluminescent HRP substrate (Millipore), immune response was detected.

Cell Culture

[0080] A human liver cancer cell line HepG2 and a mouse microglial cell line BV2 were purchased from American Type Culture Collection (ATCC, Manassas, CA, USA). Each of these cell lines was cultured in a growth medium DMEM (Gibco, Carlsbad, CA, USA) supplied with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (P/S) at 37° C. under CO.sub.2. Both cell lines were sub-cultured by 2 to 4 days.

[0081] A normal lung cell line LL24 was also obtained from ATCC. LL24 cells were grown in a RPMI-1640 medium supplemented with 10% FBS and 1% P/S (Gibco/Life Technologies, USA, NY, USA) at 37° C. under 5% CO.sub.2 humidified atmosphere. Sub-culture was executed at an interval of 3 days.

[0082] MEF cells were isolated from mouse fetus on 13.5 days. The MEF cells were cultured in a DMEM medium supplemented with 10% FBS, 1% antibiotics and antifungal agent (250 ng/mL amphoterisin, 100 U/mL penicillin and 100 ug/mL streptomycin), 8 ug/ml tyrosin (Sigma) and 15 ug/mL gentamicin (Gibco) at 37° C. under 5% CO.sub.2. The MEF cells were sub-cultured at an interval of 3 days and used in 2.sup.nd to 5.sup.th subcultures.

[0083] Transformed human bronchial epithelial cell line BEAS-2B was cultured in 5 ug/mL of gentamicin (Gibco) and non-serum 1 X defined keratinocyte SFM (Gobco) at 37° C. under 5% CO.sub.2. The medium was changed at an interval of 2 to 3 days and the cells were sub-cultured at an interval of 4 to 5 days.

[0084] Neurons were isolated from human fetal tissues at 10 weeks, 12 weeks and 14 weeks after pregnancy with the consent of the mothers. The neurons were cultured in a DMEM/F12 medium (Gibco) supplemented with 5 ug/mL of a supplement, that is, B27 (Gibco), gentamicin (Gibco), human bFGF, 20 ng/mL of human EGF (Peprotech), 1 ug/mL of pocopherol and tocopherol acetate (Sigma Aldrich) at 37° C. under 5% CO.sub.2 and 3% O.sub.2.

EV Labeling with PKH26

[0085] EV was labeled by a PKH26 red fluorescent cell linker kit for general cell membranes (Sigma-Aldrich).

[0086] Specifically, after re-suspending 200 .Math.l/mL of EV pellets in diluents C, the solution was mixed with a dye solution (2 .Math.L of PKH26 ethanol dye and 500 .Math.L of diluents C) in 1:1 ratio for 5 minutes. Next, the same volume of 1% bovine serum albumin (BSA) was added thereto in order to combine EV with an excess of dye, followed by treating the produced solution in 7 K MWCO Zeba spin desalting column (Thermo Fisher Scientific) in order to remove the excess of dye. Thereafter, the sample was stored at -80° C.

LPS and EV Treatment

[0087] For experiments, the cells were seeded with the same density in each well of 96-well or 6-well plate (Nunc, Roskilde, Denmark). In order to determine recovery effects, the cells were stimulated with LPS (MEFs and BV2— 0.5 to 2 ug/mL, HepG2 and LL24: 2 to 4 .Math.g/mL), followed by EV treatment for 24 hours.

[0088] In order to determine protection effects, the cells were pre-treated with EV for 24 hours, and then, stimulated with LPS for 24 hours. After incubation for 24 hours, the cells were subjected to MTT assay or RNA extraction.

miRNA Transfection

[0089] in order to transfect miRNA, 6 x 10.sup.4 to 2 x 10.sup.5 cells were seeded in a 24-well plate, followed by infection with 20 nM miRNA (hsa-miR-92a-3p, hsa-miR-26a-5p, hsa-miR-23a-3p, hsa-miR-103-3p, and hsa-miR-181-5p) (Bioneer). As a Pre-miR miRNA negative control, lipofectamin 3000 was used.

MTT Assay

[0090] EV cytotoxicity was assessed by MTT assay. Briefly, the cells were seeded in a 96-well plate, treated with EV at a predetermined concentration, and stimulated with LPS for 24 hours. After removing the culture supernatant, the produced dark blue crystals were dissolved in DMSO. Absorbance was measured at 570 nm.

RNA Extraction and Quantitative PCR (qPCR)

[0091] Total RNAs for qPCR was extracted from BV2, MEF, HepG2, LL24, BEAS-2B and neuron cells, while miRNA was extracted from EV. Extraction of total RNA was implemented using TRIzol reagent (Invitrogen).

[0092] Using cDNA kit (iNtRON), cDNA was synthesized from 1 .Math.g of total RNA. Total exosome RNA and a protein separation kit (Thermo Fisher Scientific) were used to extract miNRA, followed by reverse transcription using miScript II RT (Qigen).

[0093] For PCR and qPCR, a primer of gene associated with inflammation was designed. A reaction mixture (total volume 20 .Math.L) contained 10 .Math.M primer mixture, SYBR-green (Enzynomics), water without nuclease (dark, nonionic) and 2 .Math.L of cDNA. Conditions are as follows: denaturalization/activation stage was conducted at 95° C. for 10 minutes, followed by repeating 40 cycles at 95° C. for 15 seconds, at 56° C. for 30 seconds and at 72° C. for 20 seconds. The reaction was conducted by StepOne Real Time PCR instrument (Applied Biosystems). Quantification of gene expression was based on C.sub.T value of each sample.

Transfection and Reporter Assay

[0094] 208 bp fragment of 3'UTR of SARS-CoV-2 genome was synthesized by Bionics. 3'UTR fragment was degraded with Xba I, inserted at the downstream of firefly luciferase gene in pGL3-control (Promega), thereby obtaining pGL3covi-3UTR-Luc.

[0095] All constructions were confirmed by sequencing. Human neuroblastoma SK-N-BE(2)C cells were maintained in a DMEM supplemented with 10% FBS. All culture media included 100 U/mL penicillin and 100 mg/mL streptomycin. For transfection, the cells in no-antibiotics DMEM were plated in 24-well plate with 1.2 x 10.sup.5 cells/well 1 day before transfection. The transfection was implemented using lipofectamine 2000 (Invitrogen). A total amount of DNA was 0.5 .Math.g/well, which comprised 0.2 .Math.g of pGL3covi-3UTR-Luc and 0.3 .Math.g of pRSV βgal as internal controls. Each transfection also contained labeled miRNA. 24 hours after transfection, cells of each well were dissolved in 100 .Math.L dissolution buffer (25 mM tris-phosphate [pH 7.8], 2 mM DTT, 2 mM CDTA [1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid], 10% glycerol and 1% triton X 100). The same volume of firefly luciferase substrate was added thereto, lucifease activity was measured by means of a luminoometer plate reader, followed by normalization with regard to beta-galactosidase.

RNA Sequencing and Data Process

[0096] A small RNA sequence analysis (“sequencing”) was performed using extracellular vesicles (EV) obtained from two placental tissue extracts as well as a hpMSC medium under eight (8) different conditions. The sequencing was implemented by BGISeq-500 in Bajing Gene Institute (BGI, Shenzhen, China). Reads were aligned on human reference genome (GRCh38) using a subread aligner, and a featureCounts tool was used to obtain miRNA read counts. The number of reading miRNA was standardized, and low expressive miRNA was filtrated by means of edger R package. Each miRNA expression was modified into CPM (counts per million). Reading quality was managed using qrqcR package, and a distribution of small RNA frequency was acquired using sRNA toolbox.

miRNA Target eStimation and Analysis of Function

[0097] In order to investigate a miRNA binding site in 3'UTR, PITA tool was used (Kertesz et al., 2007).

[0098] SARS-CoV-2 complete genome sequence (NC_045512.2) was obtained from NCBI standard sequence database (O’Leary O’Leary et al. 2016), and 3'UTR sequence was extracted from SARS-CoV-2 complete genome.

[0099] In order to expect a binding site of miRNA, a PITA tool with basic setting values was used.

[0100] A new miRNA binding site was estimated by miRDB user designated estimation tool (Chen and Xiaowei, 2020).

[0101] In order to determine biological functions of miRNAs, experimentally verified target of miRNAs was obtained from miRTarBase (Huang et al., 2020), miRNA to reach a rate of less than 1% was excluded from the analysis. [123] In order to investigate biological functions of miRNA, DAVID Bioinformatics Resource 6.8 (Huang et al., 2009) was used to perform GO term and KEGG pathway analysis.

[0102] Analyzed results of the function were visualized by Cytoscape 3.8 and Pathview R package.

ACE2 Expression Assay

[0103] Human tissue and cell line expression data was acquired from Human Protein Atlas database (Uhlen et al., 2015). Using the expression data, ACE2 expression of each cell or tissue type was investigated.

Analysis of Preserved miRNA Binding Site

[0104] In order to investigate the preserved region of miRNA binding site in SARS-CoV-2, 3'UTR sequence was obtained from NCBI reference sequence database, and a virus 3'UTR sequence relevant to SARS-CoV-2 was used.

[0105] Specifically, SARS corona virus (NC_004718.3), SARS-CoV-2 (NC 045512.2), SARS corona virus BJ01 (AY278488.2), bat SARS corona virus HKU3-1 (DQ022305.2) , bat SARS-like corona virus SL-CoVZC45 (MG772933.1) and bat SARS-like corona virus SL-CoVZXC21 (MG772934.1) were used.

[0106] The 3'UTR sequence of the corona virus was aligned by MUSCLE tool (Edgar et al., 2004), and MUSCLE assay adopted a basic value.

Statistical Assay

[0107] The statistical assay was performed according to ANOVA and HSD post-test of Tukey in R. Hit-map was created by expressing log12-CPM of miRNA in hpMSC-EV and using gplots R package (Warnes et al., 2015). P value in GO term assay and KEGG route assay was amended for multiple comparison using Benjamini-Hochberg method. Data was proposed by mean ± standard error, and p value or adjusted p value < 0.05 was considered to be significant.

[0108] Hereinafter, the present invention confirmed by the above-mentioned experimental method will be described in detail along with the accompanying drawings in terms of effects of the preset invention. However, such a description is proposed for helping understanding of the present invention, and the present invention is not restricted by these embodiments.

Example 1. Virus Inhibition Mechanism of Placenta-EV miRNA to SARS-CoV-2 Virus

[0109] FIG. 1 illustrates a functional mechanism for anti-viral effects of placenta-EV miRNA with regard to SARS-CoV-2 virus. Direct anti-viral mechanism is formed by directly binding miRNA in a region such as 3'UTR, 5'URT or a coding area of the virus so as to induce down control of SARS-CoV-2 RNA. Further, indirect anti-viral effects include adjustment of inflammatory mRNA expression through miRNA so as to inhibit cytokine storm.

[0110] In other words, according to the present invention, it was confirmed that a mesenchymal stem cell (MSC)-derived EV and miRNA and protein molecules included therein may regenerate damaged tissues, while controlling miRNA and anti-inflammatory circumstances.

[0111] EV separated from pMSC was positive to a typical EV marker, that is, CD63 (FIG. 2a). A size of EV was analyzed nanoparticle tracking assay (NTA) and transmission electron microscope inspection (TEM).

[0112] A hydraulic diameter of exosome measured by the nanoparticle tracking assay (NTA) is 121.8 nm (FIG. 2b), and representative TEM image thereof is shown in FIG. 2c. Additional features of EV separated by western blotting of EV marker are those of typical EV markers such as CD81, CD9, annexin A2 and HSP70 (FIG. 2d).

Example 2. Selection of miRNA Bound to SARS-CoV-2 Virus

[0113] On the ground that virus genome could become a target by human miRNA, the present inventors confirmed whether miRNA in EV may have interaction with 3'UTR of SARS-CoV-2 or not.

[0114] In SARS-CoV2 3'UTR, binding sites to miR-92a-3p, miR-26a-5p, miR-23a-3p, miR-103a-3p and miR-181a-5p are included.

[0115] Specifically, 3'UTR sequence of SARS-CoV-2 was aligned and the sequence was analyzed using MicroInspector software. As a result, total 18 miRNAs targeted 3'UTR of SARS-CoV-2 according to PITA software (Table 1), and it was expected that 27 miRNAs were bound to the entire genome site of SARS-CoV-2 virus (Table 2).

TABLE-US-00001 microRNA Target Energy Rank Start End hsa-miR-92a-3p 72 80 -13.8 1 hsa-miR-92a-3p 153 161 -9.01 1 hsa-miR-92b-3p 72 80 -18.8 3 hsa-miR-92b-3p 153 161 -11.16 3 hsa-miR-181a-5p 79 87 -18.7 5 hsa-miR-26a-5p 33 39 -14.9 12 hsa-miR-34a-5p 94 102 -13.25 20 hsa-miR-23a-3p 163 171 -8.1 23 hsa-miR-125b-5p 182 190 -11.9 36 hsa-miR-125a-5p 182 190 -10.4 37 hsa-miR-103a-3p 106 114 -12.6 39 hsa-miR-223-3p 189 197 -5.6 45 hsa-miR-25-3p 72 80 -15.5 51 hsa-miR-25-3p 153 161 -8.45 51 hsa-miR-26b-5p 33 39 -11.3 52 hsa-miR-193a-5p 38 46 -12.1 57 hsa-miR-1307-3p 66 74 -30.9 58 hsa-miR-155-5p 19 27 -9.2 63 hsa-miR-185-5p 52 60 -11.31 69 hsa-miR-424-3p 48 56 -9.4 72 hsa-miR-23b-3p 163 171 -11.3 74

[0116] Table 1 shows a list of 18 miRNAs targeting 3'UTR of SARS-CoV-2 virus.

TABLE-US-00002 miRNA Expression (%) Score hsa-miR-181a-5p 4.93 70 hsa-miR-26a-5p 1.67 68 hsa-miR-199a-3p 1.41 69 hsa-miR-320a-3p 1.22 73 hsa-miR-16-5p 0.83 99 hsa-miR-23a-3p 0.79 79 hsa-miR-181b-5p 0.77 70 hsa-miR-199b-3p 0.70 69 hsa-miR-122-5p 0.68 67 hsa-miR-378a-3p 0.52 55 hsa-miR-27a-3p 0.30 67 hsa-miR-103a-3p 0.27 85 hsa-miR-197-3p 0.27 82 hsa-miR-30d-5p 0.22 95 hsa-miR-26b-5p 0.15 68 hsa-miR-139-5p 0.13 83 hsa-miR-27b-3p 0.12 67 hsa-miR-15b-5p 0.11 99 hsa-miR-532-5p 0.11 61 hsa-miR-155-5p 0.10 51 hsa-miR-320c 0.08 73 hsa-miR-196b-5p 0.06 52 hsa-miR-424-3p 0.04 52 hsa-miR-29a-3p 0.04 91 hsa-miR-23b-3p 0.03 79 hsa-miR-15a-5p 0.03 99 hsa-miR-324-3p 0.01 70

[0117] Table 2 shows a list of 27 miRNAs expected to be bound to the entire genome site in SARS-CoV-2 virus. In Table 2, expression (%) was calculated by the corresponding miRNA CPM/total miRNA CPM, wherein with binding possibility will increase with higher score. According to referential literature, when the score is 80 or more, the binding is possible.

[0118] Table 3 below shows information on the binding site in SARS-CoV-2 genome and results of binding prediction. 5 miRNAs (miR-92a-3p, miR-26a-5p, miR-23a-3p, miR-103a-3p, miR-181a-5p) were selected based on thermodynamic energy score and high scores of two prediction tools, that is, PITA and miRDB.

TABLE-US-00003 PITA & UTR binding prediction minDB unconventional target sites prediction microRNA Expression (X) Seed location Seed match length match GU wooble microRNA-target hybridization energy Score Seed location hsa-miR-92a-3p 22.23476 29745 8 1 1 -13.8 - - hsa-miR-92b-3p 3.9711815 29748 8 1 1 -18.8 - - hsa-miR-181a-5p 4.9776 29758 8 1 1 -18.7 78 7430, 7528, 8221, 5916, 114512 12216, 18512, 29783, 27548 hsa-miR-26a-5p 1.67263 29707 6 6 9 - 14.8 68 454, 9596, 20513, 27848, 29707 hsa-miR-340-5p 0.973787 29768 8 1 1 -13.25 - - hsa-miR-23r-3p 0.798315 29817 1 0 -8.1 78 6456, 79011, 15302, 21244 hsa-miR-125b-5p 0.369575 23856 6 1 1 - 11.9 - - hsa-miR-125a-5p 0.324968 29856 6 1 1 -10.4 - - hsa-miR-103a-3p 0.289252 29789 8 1 1 -12.8 85 8827, 13083, 14561, 14780, 22343, 24235, 25313, 28371, 27101 20734, 20920, 29261 hsa-miR-223-3p 0.233218 29863 8 1 1 -536 - - hsa-miR-25-3p 0.153562 26746 6 1 1 -15.5 - - hsa-miR-25b-5p 0.149568 26707 6 0 0 -11.3 68 454, 9596, 20513, 273.48, 29707 hsa-miR-193a-3p 0.110145 28712 1 0 -12.1 - - hsa-miR-1907-3p 0.128069 26740 1 0 -30.9 - - hsa-miR-185-5p 048358 29693 1 0 -8.3 51 863, 5209, 17197, 28074 hsa-miR-185-5p 0.050111 29726 6 1 1 -11.31 - - hsa-miR-424-3p 0.03693 29722 6 1 0 -9.4 52 3054 18934, 20181, 29571 hsa-miR-23b-3p 0.07439 29837 6 1 0 -11.9 79 6450, 70011, 15302, 21747

[0119] Using PIA, it was predicted that potential binding sites at which finally selected five (5) miRNAs, that is, miR-92a-3p, miR-26a-5p, miR-23a-3p, miR-103a-3p and miR-181a-5p may be bound to 3'UTR of SARS-CoV-2 (FIGS. 3a, 3b). Using PITA, total free energy was obtained for each of the miRNAs. For example, the binding energy of miR-181a-5p to 3'UTR is microRNA target hybridization energy of -18.7 kcal/mol. This suggests that miRNA 3'UTR binding would be voluntarily performed. Low binding thermodynamic energy (kcal/mol) indicates stronger binding.

[0120] Next, in order to confirm whether the above 5 miRNAs exist in EV, qPCR analysis was implemented (FIG. 4). As a result, it was found that miRNAs in EV were highly expressed in the order of miR-92a-3p, miR-181a-5p, miR-26a-5p, miR-23a-3p and miR-103a-3p. That is, the expression of miR-92a-3p and miR-181a-5p was significantly higher in the same order as the predicted values. These results suggest that EV includes 5 miRNA, and therefore, can be used by itself as a therapeutic agent for SARC-CoV-2.

Example 3. Confirmation of Direct Anti-Viral Effects of EV and miRNA

[0121] In order to confirm whether the specific miRNAs selected in Example 2 alone or 5 miRNAs can directly interact with SARS-CoV-2 genome, luciferase assay was implemented.

[0122] According to luciferase-reporter assay, whether the specific miRNAs or 5 miRNAs separately could direct interact with SARS-CoV-2 genome was confirmed.

[0123] PCR fragments were cloned to the luciferase reporter plasmid between luciferase ORF and synthetic poly(A) sequence. The recombinant plasmid was named pGL3 SARS-CoV-3'-UTR Luc (FIG. 5a). Then, the recombinant plasmid was transfected to human neuroblastoma SK-N-BE(2)C cells and, 48 hours after transfection, luciferase activity was measured.

[0124] As shown in FIG. 5b, relative luciferase activity in SK-N-BE(2)C cells transfected with the recombinant plasmid was considerably reduced as compared to cells transfected with empty psi-control vector (ps < 0.0001).

[0125] Specifically, the relative luciferase activity was down controlled when 3'UTR of the corona virus was co-transfected to DF-1 cells by 5 miRNAs or expression vectors thereof. From the results, it was confirmed that luciperase activity of psi-59UTR was considerably reduced.

[0126] The above experimental results indicate that miR-92a-3p, miR-26a-5p, miR-23a-3p, miR-103a-3p and miR-181a-5, respectively, are bound to 3'UTR of SARS-CoV-2 so as to induce significant silence.

[0127] Based on the above results, it was confirmed that all of the above 5 miRNAs may be directly bound to 3'UTR of SARS-CoV-2, and at least one miRNA selected from the group consisting of miR-92a-3p, miR-26a-5p, miR-23a-3p, miR-103a-3p and miR-181a-5p may degrade SARS-CoV-2 virus genome, thereby inhibiting SARS-CoV-2 infection.

Example 4. Confirmation of Direct Anti-viral Ability to Different Corona Viruses and Other Viruses

[0128] In order to identify an assumption (or a hypothesis) that 3'UTR sequence would be preserved throughout corona virus family and a miRNA target site would be preserved over all of corona viruses, 5 corona viruses were randomly chosen and sequences in the 3'UTR region were compared by MUSCLE tool.

[0129] As shown in FIGS. 6a, 3'UTRs of five (5) corona viruses were mostly preserved to show only small variation. In particular, binding sites predicted to be combined with 5 miRNAs (indicated by red box) were also confirmed to be preserved very well.

[0130] In other words, as a result of confirming 3'UTRs of various corona viruses, very little mutation occurred in 3'UTR. Further, in consideration of high miRNA binding rate, it is understood that 5 selected miRNAs may have general virus inhibitory ability applicable to even newly generated viruses by mutation.

[0131] Further, it is already known that different miRNAs in EV may interact with other viruses (FIG. 6b).

[0132] Specifically, it was demonstrated that miR-150-5p, miR-223-3p and miR-29-3p may interact with HIV, thereby inhibiting virus translation and final latency T cells.

[0133] It is known that miR-23b-3p may block translation of entero-virus 71 while Let-7c-5p may reduce a significant matrix protein for influenza virus.

[0134] miR-181a-5p, miR-181b-5p, miR-23a-3p, miR-23b-3p and miR-378a-3p may degrade porcine reproductive and respiratory syndrome virus.

[0135] miR-122-5p and let-7c-5p may inhibit C type hepatitis virus and bovine viral diarrhea virus, respectively.

[0136] In other words, EV and different miRNAs included in EV may be bound to various viruses and 3'UTR with low mutation frequency, thereby exhibiting inhibitory effects. Such results suggest that EV can be used as a general therapeutic agent and/or preventive therapeutic agent with regard to diverse viral infections.

Example 5. Confirmation of Indirect Anti-Viral Effects of EV and miRNA

[0137] Viruses infecting the respiratory system, including corona virus, may penetrate cells expressing ACE2 receptor and cause cell damage.

[0138] ACE2 receptor is mainly present in the liver, kidneys, male genital tissues, muscle tissues and gastrointestinal, therefore, these organs may be damaged by viruses. In recent clinical studies, a number of patients showed liver damage or organ damage, thus supporting the above opinion.

[0139] In the children’s hospital in Uhan of China, 6 of 8 COVID-19 patients under treatment in the intensive care unit showed increase in C reactive protein, procalcitonin and dehydrogenated enzymes. Further, 4 of 8 patients had abnormal liver function. Further, cytokine storm was found from 3 of these patients and this symptom was more serious in patients of severe cases.

[0140] In another study, 74 of 651 patients (11.4%, average age: 46.14) showed at least one gastrointestinal (GI) symptom such as nausea, vomit or diarrhea, while 10.8% of the patients was confirmed to suffer from liver diseases.

[0141] Further, 29 (39.19%) of 74 COVID-19 patients had a high fever. Likewise, 23 patients (31.08%), 8 patients (10.81%) and 16 patients (21.62%) appealed fatigue, dyspnea and headache, respectively.

[0142] As expected from the above results, ACE2 receptor was expressed in LL24 (lung fibroblast), BEAS-2B cell (GI epithelia) and liver cell. Further, as demonstrated by Brain Atlas database, ACE2 expression level in the brain was not sufficiently studied. Although the expression level is low, when considering on the basis of the above clinical results, it was predicted that the brain may have a high possibility of becoming a target organ of SARS-CoV-2. Similarly, ACE2 is highly expressed in brain tumor cells and this may suggest that brain cells can become a target of viruses.

[0143] Accordingly, in order to confirm indirect anti-viral effects of miRNA derived from different EVs, cells expected to undergo over-immune response by SARS-COV2 such as liver cells, GI cells and brain cells were subjected to experiments.

[0144] Specific experimental procedures and cell culture methods are as described above. Briefly, the cell line was treated with EV and miRNA, after 24 hours, followed by LPS treatment so as to determine protection effects of EV and miRNA. On the contrary, the cell-line was firstly treated with LPS and, after 24 hours, followed by EV and miRNA treatment so as to determine recovery effects (FIG. 7).

Example 6. Anti-Inflammatory and Cytokine Storm Inhibitory Effects of EV and miRNA

[0145] With regard to BEAS-2B (gastrointestinal epithelia), LL24 (lung cell-line), HeoG2 (liver cancer cell-line), MEF (fibroblast), BV2 (microglial cell-line) and M2 (neuron cells), experiments for anti-inflammatory and cytokine storm inhibitory effects of EV and miRNA were implemented with and without LPS and EV treatment. Results of the experiments are shown in FIGS. 8 to 12.

[0146] BEAS-2B (gastrointestinal epithelia) was treated with EV, followed by LPS treatment to determine protection effects, and results thereof are shown in FIG. 8a. As a result, it was confirmed that some inflammatory factors, that is, all of IL-1, IL-6, IL-9 and TNF-a showed considerably decreased relative expression, as compared to a case where EV treatment is not involved.

[0147] FIG. 9a illustrates results of confirming protection and recovery effects when LL-24 was treated with EV. As a result, it was confirmed that, if treating with EV, all of the inflammatory factors such as IL-1, IL-6 and IL-8 have considerably reduced expression so as to exhibit excellent protection and recovery effects, in particular, very excellent recovery effects.

[0148] Further, as shown in FIG. 9b, after treating LL-24 with 5 miRNAs, respectively, protection effects were determined by LPS treatment. As a result, it was confirmed that treatment using miRNA-181a considerably reduced IL-6 expression to the same level as the control.

[0149] FIGS. 10a and 10b illustrate results of MEF (fibroblast) and BV2 (microglial cell-line) protection and recovery effects, respectively. It was confirmed that both of MEF and BV2 showed significantly reduced IL-1, IL-6 and TNF-a as inflammatory factors by EV treatment.

[0150] FIG. 11 illustrates results of confirming expression levels of inflammation related factors after EV treatment of M2 as a neuron cell. As a result, it was confirmed that remarkable effects were obtained for only IL-1.

[0151] FIG. 12 shows photographed images before and after treatment of each of cell-lines with PKH26-labeled EV. From the images, it could be seen that EV was successfully fused with each cell.

[0152] In other words, considering the above results together, it could be seen that EV and miRNA contained EV of the present invention exhibit effects of inhibiting inflammatory factors separately or in combination thereof so as to achieve anti-inflammatory effects, in addition, effects of inhibiting cytokine storm as an over-immune response.

Example 7. Viral Infection Treatment Effects of EV and miRNA

[0153] EV and miRNA of the present invention may treat or improve virus and viral infection symptoms through both of direct and indirect routes (FIG. 13).

[0154] From the above diverse experiments, the present inventors have found that the placenta, umbilical cord and/or cord blood-derived EVs, placental mesenchymal stem cells (pMSC) obtained from these tissues, and EVs derived therefrom may greatly improve pathological damage of the lungs and, in addition, other type inflammation of the lungs (pneumonia) and viral infection.

[0155] In particular, since EV contains regeneration effective factors of the stem cell, this may have a treatment mechanism similar to that of a mesenchymal stem cell (MSC) in treatment of pneumonia, while being considered to be much safer.

[0156] Specifically, intravenous administration of MSC may cause embolism and coagulation of blood, whereas EV having a size of 200 to 10.sup.8 nm is safe much more than MSC, and EV itself is also safer than MSC. Further, EV has advantages such as easy storage, no transformation into malignant or harmful cells, and less possibility of immune response.

[0157] Further, results of experiments of the present invention demonstrated that miRNAs contained in tissue-EV, tissue extract EV and pMSC-EV are directly bound to virus genome to inhibit transcription of RNA virus, thereby inhibiting proliferation of the virus genome.

[0158] As another important function of EV miRNA, 77 miRNAs existing in diverse EVs target mRNA stimulating immune response so as to exhibit effects of removing RNA transcribed by viruses (“viral RNAs”), thereby achieving strong indirect effects as well as direct anti-viral effects.

[0159] Further, the miRNA of the present invention may be combined with the viral RNA and may also mutually react with pre-inflammatory gene so as to greatly inhibit expression of an inflammatory factor, thereby solving a cytokine storm problem.

[0160] Further, miRNA of the present invention may exhibit effects of up controlling IFN-α/β level so as to prevent an IFN-α/β route, thereby inhibiting viral infection avoiding host immune response.

[0161] Further, it could be proved that 18 miRNAs of different EVs may directly and mutually react with 3'UTR of SARS-CoV-2, while 5 major miRNAs derived from EV may successfully inhibit SARS-CoV-2.

[0162] Some patients infected with corona virus showed liver damage and stomach damage other than main conditions related to the respiratory organ. Among 99 patients in Jinyintan hospital in Uhan, 43 persons had damaged liver function and, in particular, the liver of one patient was seriously damaged. Patients in mild cases are unclear whether liver function was damaged or not, however, patients in serious cases showed a decrease in main liver values, that is, alanine aminotransferase (ALT), aspartate aminotrasferase (AST) and lactic acid dehydrogenase (LDH).

[0163] Accordingly, in addition to direct virus inhibitory ability, anti-inflammatory effects of EV derived from the placenta, umbilical cord and/or cord blood may induce synergistic effects. Therefore, when using placenta-derived EV as a therapeutic agent of corona virus, remarkably excellent treatment effects may be achieved.

[0164] The above description of the present invention is provided for illustrative purpose, and those skilled in the art, to which the present invention pertains, will understand that the present invention is easily modified into other concrete forms without changing technical spirit or essential features of the present invention. Therefore, the examples described above should be construed as illustrative embodiments without limitation thereof. For example, individual components described in singular form may be separately embodied and some separate components described herein may also be embodied in a combination form.

[0165] The scope of the present invention may be defined by the following appended claims, and all alterations or modifications deduced from the meanings and ranges of the claims and equivalent concepts should be construed as being included in the scope of the present invention.