A method for non-invasively obtaining microRNAs from cells

Abstract

Provided is a method for noninvasively acquiring miRNA from cells, the method comprising: (1) a step which is for introducing, into cells, a nucleic acid encoding a miRNA-binding protein and a nucleic acid encoding a vesicle-forming protein, and in which the miRNA-binding protein includes a first portion formed of MID and PIWI domains of an argonaute protein and a second portion formed of a viral protein R, and the vesicle-forming protein includes a palmitoylation / myristoylation signal or a PH domain, a self-assembling domain, an ESCRT or ESCRT related factor-binding domain, and a Gag p6 domain, said step resulting in production of exosome-like vesicles including miRNA; (2) a step for collecting an extracellular liquid of the cells; and (3) a step for extracting miRNA from the extracellular liquid.

Description

[Technical Field]

[0001] The present invention relates to a method for non-invasively obtaining microRNA from cells. [Background technology]

[0002] MicroRNAs (miRNAs) are small, single-stranded, non-coding RNAs approximately 22 bases long that interact with mRNAs to regulate their expression. MiRNAs have been shown to be involved in various biological processes, including cell proliferation, differentiation, apoptosis, and the onset and pathology of various diseases, including cancer. It has been reported that they may serve as biomarkers reflecting cellular status (Non-Patent Document 1). Because miRNAs are present in body fluids, such as blood and urine, encapsulated in exosomes secreted by cells, liquid biopsies, which detect and quantify miRNAs in body fluids for non-invasive disease diagnosis, have attracted attention. Furthermore, because miRNAs are closely related to the onset and pathology of various diseases, exosomes containing miRNAs are also expected to serve as nucleic acid medicines.

[0003] However, because exosomes are formed by incorporating cytoplasm, miRNAs are only contained in exosomes at the same concentration as in the cytoplasm. Therefore, the amount of miRNA contained in exosomes is extremely small, making it difficult to obtain sufficient amounts of miRNA for high-precision diagnosis and drug development.

[0004] Recently, a method for inducing the production of exosome-like extracellular vesicles using artificially designed self-assembling protein nanocages has been proposed (Patent Document 1, Non-Patent Document 2). This method allows for the production of exosome-like vesicles containing a recombinant protein of interest expressed intracellularly. However, like exosomes, these extracellular vesicles are formed by incorporating cytoplasm, and therefore, it remains difficult to obtain miRNAs, which are naturally present in low amounts in the cytoplasm. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2016 / 138525 [Non-patent literature]

[0006] [Non-Patent Document 1] Liang, Y. et al.,Journal of Extracellular Vesicles,(2019),9(1):1697583,DOI:10.1080 / 20013078.2019.1697583 [Non-patent document 2] Votteler, J. et al.,Nature,(2016),540(7632):292-295,DOI:10.1038 / nature20607 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made with the aim of providing a method for non-invasively obtaining a larger amount of miRNA from cells. [Means for solving the problem]

[0008] The inventors succeeded in increasing the yield of miRNA by newly designing a miRNA-binding protein that is large enough to be encapsulated in exosome-like vesicles.

[0009] That is, according to one embodiment, the present invention provides a method for non-invasively obtaining microRNA from cells, the method comprising: (1) introducing into a cell a nucleic acid encoding a microRNA-binding protein and a nucleic acid encoding a vesicle-forming protein, wherein the microRNA-binding protein comprises a first portion consisting of the MID domain and PIWI domain of an Argonaute protein and a second portion consisting of viral protein R, and the vesicle-forming protein comprises a palmitoylation or myristoylation signal or a pleckstrin homology domain, a self-assembly domain, an ESCRT or ESCRT-associated factor-binding domain, and a Gag p6 domain, thereby producing exosome-like vesicles containing the microRNA; (2) recovering extracellular fluid from the cells; and (3) extracting the microRNA from the extracellular fluid.

[0010] Preferably, the microRNA-binding protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1.

[0011] The vesicle-forming protein preferably comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-6.

[0012] The cells may be in vitro cells and the extracellular fluid may be culture supernatant.

[0013] The cell may be an in vivo cell and the extracellular fluid may be a biological fluid.

[0014] Furthermore, according to one embodiment, the present invention provides an exosome-like vesicle comprising a nanocage composed of (a) a microRNA, (b) a microRNA-binding protein comprising a first portion consisting of the MID domain and PIWI domain of an Argonaute protein and a second portion consisting of viral protein R, and (c) a vesicle-forming protein comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 6.

[0015] Preferably, the exosome-like vesicles further contain (d) a membrane fusion protein. [Effects of the Invention]

[0016] The method of the present invention enables the preparation of extracellular fluid containing high concentrations of miRNA. Therefore, the method of the present invention enables miRNA analysis in a non-invasive manner similar to conventional exosome-based miRNA analysis, but with higher accuracy and sensitivity than conventional exosome-based miRNA analysis. Furthermore, the exosome-like vesicles of the present invention contain high concentrations of miRNA and are useful for the development of nucleic acid medicines. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram showing the domain structures of various Argonaute protein mutants. [Figure 2] FIG. 2 shows the results of Western blot analysis confirming the presence of various Argonaute protein mutants in cells and extracellular vesicles. [Figure 3] FIG. 3 shows the results of immunoprecipitation of Flag-Ago2-FL-Vpr or Flag-MID-PIWI-Vpr with an anti-Flag antibody. [Figure 4] FIG. 4 is a graph showing the relative quantification of miRNA-let7a-5p co-precipitated with Flag-Ago2-FL-Vpr or Flag-MID-PIWI-Vpr. [Figure 5] FIG. 5 shows the results of Western blot analysis confirming the presence of Flag-Ago2-FL-Vpr or Flag-MID-PIWI-Vpr in cells and extracellular vesicles. [Figure 6] Figure 6 is a graph showing the relative quantification of miRNA-let7a-5p in extracellular vesicle fractions obtained from HEK293T cells co-expressing Flag-Ago2-FL-Vpr or Flag-MID-PIWI-Vpr and EPN-01. [Figure 7]Figure 7 is a graph showing the relative quantification of miR-92a-3p in extracellular vesicle fractions obtained from HEK293T cells co-expressing Flag-MID-PIWI-Vpr and EPN-01. [Figure 8] Figure 8 is a graph showing the relative quantification of miR-191-5p in extracellular vesicle fractions obtained from HEK293T cells co-expressing Flag-MID-PIWI-Vpr and EPN-01. [Figure 9] Figure 9 is a graph showing the relative quantification of miR-126-5p in extracellular vesicle fractions obtained from HEK293T cells co-expressing Flag-MID-PIWI-Vpr and EPN-01. [Figure 10] Figure 10 is a graph showing the relative quantification of miR-10b-5p in extracellular vesicle fractions obtained from HEK293T cells co-expressing Flag-MID-PIWI-Vpr and EPN-01. [Figure 11] FIG. 11 shows the results of Western blot analysis confirming changes in the expression levels of HIF1α, EPN-01, and MID-PIWI-Vpr due to CoCl 2 treatment. [Figure 12] FIG. 12 is a graph showing changes in the expression level of miR-210 in HEK293T cells transfected or not with Flag-MID-PIWI-Vpr and EPN-01 by CoCl 2 treatment. [Figure 13] FIG. 13 is a graph showing changes in the expression level of miR-1303 by CoCl 2 treatment in HEK293T cells transfected or not with Flag-MID-PIWI-Vpr and EPN-01. [Figure 14] Figure 14 is a graph showing the change in miR-210 content in extravesicular fractions obtained from HEK293T cells transfected or not with Flag-MID-PIWI-Vpr and EPN-01 by CoCl2 treatment. [Figure 15]Figure 15 is a graph showing the vesicle concentration and particle size distribution in extracellular vesicle fractions obtained from HEK293T cells co-expressing Flag-MID-PIWI-Vpr and EPN-01, HEK293T cells co-expressing EGFP and EPN-01, or untransfected HEK293T cells. [Figure 16] Figure 16 is a graph showing the particle size (mean) of extracellular vesicles obtained from HEK293T cells co-expressing Flag-MID-PIWI-Vpr and EPN-01, HEK293T cells co-expressing EGFP and EPN-01, or untransfected HEK293T cells. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be described in detail below, but the present invention is not limited to the embodiments described in this specification.

[0019] According to a first embodiment, the present invention provides a method for non-invasively obtaining microRNA from cells, the method comprising: (1) introducing into cells a nucleic acid encoding a microRNA-binding protein and a nucleic acid encoding a vesicle-forming protein, wherein the microRNA-binding protein comprises a first portion consisting of the MID domain and PIWI domain of an Argonaute protein and a second portion consisting of viral protein R, and the vesicle-forming protein comprises a palmitoylation or myristoylation signal or a pleckstrin homology domain, a self-assembly domain, an ESCRT or ESCRT-associated factor-binding domain, and a Gag p6 domain, thereby producing exosome-like vesicles containing the microRNA; (2) recovering extracellular fluid from the cells; and (3) extracting the microRNA from the extracellular fluid.

[0020] In the method of this embodiment, a nucleic acid encoding a microRNA-binding protein and a nucleic acid encoding a vesicle-forming protein are introduced into a cell.

[0021] "MicroRNA" (also referred to as "miRNA") is a small, single-stranded, non-coding RNA approximately 21 to 25 bases in length, and more than 20,000 types have been identified to date (http: / / mirbase.org / ). The miRNA in this embodiment is not particularly limited and may be any miRNA expressed in any cell. Furthermore, miRNA in this embodiment may include not only known miRNAs but also unknown miRNAs. Note that miRNA in this embodiment refers to miRNAs that are final products, and does not include intermediate products such as pri-miRNA and pre-miRNA.

[0022] The type of "cell" in this embodiment is not particularly limited and may be, for example, dendritic cells, T cells, B cells, nerve cells, stem cells, cancer cells, or primary cultured cells or established cell lines derived therefrom. That is, the cells in this embodiment may be either in vivo or in vitro. Furthermore, the organism from which the cells are derived is also not particularly limited and may be any vertebrate, but is preferably a mammal such as a mouse, rat, rabbit, pig, cow, goat, monkey, or human, with human beings being particularly preferred.

[0023] The "microRNA-binding protein" in this embodiment comprises a first portion consisting of the MID domain and PIWI domain of the Argonaute protein, and a second portion consisting of the viral protein R.

[0024] "Argonaute protein" (hereinafter also referred to as "Ago") is a protein that binds to miRNA to form an RNA-induced silencing complex (RISC). It is composed of four characteristic domains: the N domain, the PAZ domain, the MID domain, and the PIWI domain, as well as two linker domains (L1 and L2). The MID and PIWI domains of Ago used in this embodiment may be derived from any protein in the Ago family, but are preferably derived from Ago1, Ago2, Ago3, or Ago4, and particularly preferably from Ago2. The MID and PIWI domains of Ago used in this embodiment may be derived from any vertebrate, but are preferably derived from mammals, and particularly preferably from humans. The amino acid sequence of human Ago2 (SEQ ID NO: 7) is shown below.

[0025] [ka]

[0026] "Viral protein R" (hereinafter also referred to as "Vpr") is a type of accessory protein specific to primate immunodeficiency viruses such as human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), and interacts with the p6 domain of the viral structural protein Gag. The Vpr used in this embodiment may be derived from any primate immunodeficiency virus, but is preferably derived from HIV, and particularly preferably from HIV-1. The amino acid sequence of HIV-1 Vpr (SEQ ID NO: 8) is shown below.

[0027] [ka]

[0028] The amino acid sequences of Ago and Vpr and the nucleic acid sequences encoding them are available from the appropriate databases. For example, NP_036286.2 (GenBank) and NM_012154.5 (GenBank) are available for human Ago2. NP_057852.2 (GenBank) and NC_001802 (GenBank) (5105-5396) are available for HIV-1 Vpr.

[0029] The microRNA-binding protein in this embodiment most preferably comprises an amino acid sequence (SEQ ID NO: 1) consisting of the MID domain and PIWI domain of human Ago2 and Vpr derived from HIV-1.

[0030] MicroRNA-binding protein (SEQ ID NO: 1) [ka]

[0031] In this embodiment, the microRNA-binding protein may include proteins whose amino acid sequences share 80% or more, preferably 90% or more, and more preferably about 95% or more identity with the amino acid sequences of the MID and PIWI domains of Ago and Vpr registered in a database, as long as they maintain the same miRNA-binding activity as the MID and PIWI domains of Ago and the same binding activity as Vpr to the p6 domain of Gag. The amino acid sequence identity can be calculated using sequence analysis software or programs commonly used in the art (e.g., FASTA, BLAST, etc.).

[0032] In this embodiment, the "vesicle-forming protein" comprises a palmitoylation or myristoylation signal or a pleckstrin homology (PH) domain, a self-assembly domain, an ESCRT or ESCRT-associated factor binding domain, and a Gag p6 domain. Vesicle-forming proteins that can be used in this embodiment are disclosed as Enveloped Protein Nanocages (EPNs) in International Publication No. 2016 / 138525, for example, and include, but are not limited to, EPN-01 (SEQ ID NO: 2), EPN-03 (SEQ ID NO: 3), EPN-07 (SEQ ID NO: 4), EPN-08 (SEQ ID NO: 5), and EPN-18 (SEQ ID NO: 6). The preferred vesicle-forming protein in this embodiment is EPN-01 (SEQ ID NO: 2).

[0033] EPN-01 (SEQ ID NO: 2) [ka]

[0034] EPN-03 (SEQ ID NO: 3) [ka]

[0035] EPN-07 (SEQ ID NO: 4) [ka]

[0036] EPN-08 (SEQ ID NO: 5) [ka]

[0037] EPN-018 (SEQ ID NO: 6) [ka]

[0038] In this embodiment, the vesicle-forming protein may include a protein consisting of an amino acid sequence that is 80% or more, preferably 90% or more, and more preferably about 95% or more identical to the amino acid sequence of the EPN subunit disclosed in the above-mentioned publication, as long as it maintains activity equivalent to that of the EPN subunit (i.e., it forms nanocages by self-assembly and constitutes extracellular vesicles).

[0039] The microRNA-binding protein and vesicle-forming protein in this embodiment may have an epitope tag such as Myc, HA, or FLAG attached to their N-terminus and / or C-terminus.

[0040] The nucleic acids encoding the microRNA-binding proteins and the vesicle-forming proteins can be prepared by any conventional genetic engineering method based on the sequences designed as described above. These nucleic acids can also be introduced into cells by methods well known in the art, such as cloning the nucleic acids into an expression vector and then introducing the vector into cells. The type of expression vector is not particularly limited and can be either a viral vector or a non-viral vector, such as a herpesvirus vector, adenovirus vector, lentivirus vector, or retrovirus vector, or a plasmid vector such as pCMV or pCAG.

[0041] When the microRNA-binding protein and the vesicle-forming protein are expressed in a cell, the microRNA-binding protein-miRNA complex is stored in a nanocage formed by the vesicle-forming protein, forming exosome-like vesicles that are released extracellularly. In this embodiment, "exosome-like vesicles" refer to nanoscale extracellular vesicles whose structure and composition are similar to those of exosomes.

[0042] The extracellular fluid of the cells is then collected. If the cells are in vitro, the extracellular fluid may be a culture supernatant. If the cells are in vivo, the extracellular fluid may be a biological fluid. Examples of biological fluids include, but are not limited to, blood, plasma, serum, saliva, and urine.

[0043] Next, miRNA is extracted from the extracellular fluid. MiRNA can be extracted using established procedures, such as ultracentrifugation to collect extracellular vesicles and isolating miRNA using a purification method such as the Boom method. Numerous Boom method-based miRNA extraction kits are commercially available, and these commercially available products can also be used in the method of this embodiment. Preferred commercially available products include the High Pure miRNA Isolation Kit (Roche Diagnostics), miRNeasy Mini Kit (Qiagen), and mirVana™ miRNA Isolation Kit (Thermo Fisher Scientific).

[0044] The method of this embodiment makes it possible to obtain extracellular fluid containing high concentrations of miRNA, enabling non-invasive, highly accurate, and highly sensitive diagnosis.

[0045] According to a second embodiment, the present invention provides an exosome-like vesicle comprising a nanocage composed of (a) a microRNA, (b) a microRNA-binding protein comprising a first portion consisting of the MID domain and PIWI domain of an Argonaute protein and a second portion consisting of viral protein R, and (c) a vesicle-forming protein comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 6.

[0046] In this embodiment, the terms "exosome-like vesicles," "microRNA," "Argonaute protein," "viral protein R," "microRNA-binding protein," and "vesicle-forming protein" are the same as those defined in the first embodiment.

[0047] The exosome-like vesicles of this embodiment may further comprise (d) a membrane fusion protein. The term "membrane fusion protein" refers to a protein that induces fusion between cells or membrane vesicles of the same or different species. The membrane fusion protein of this embodiment is not particularly limited, but is preferably a membrane fusion protein derived from an enveloped virus, such as vesicular stomatitis virus G protein (VSV-G), herpes simplex virus glycoprotein B (gB), and recombinant forms thereof.

[0048] The exosome-like vesicles of this embodiment can be obtained from the extracellular fluid of cells into which a nucleic acid encoding a microRNA-binding protein and a nucleic acid encoding a vesicle-forming protein have been introduced, by the procedure of the method of the first embodiment.

[0049] The exosome-like vesicles of the present embodiment can contain miRNA at a higher concentration than natural exosomes, and are therefore useful for disease diagnosis and the development of nucleic acid medicines. [Example]

[0050] The present invention will be further described below with reference to examples, which should not be construed as limiting the scope of the present invention.

[0051] 1. Search for Argonaute protein mutants stored in extracellular vesicles In this example, EPN-01 (SEQ ID NO: 2) was used as the vesicle-forming protein and human Ago2 was used as the Argonaute protein. Because the nanocages formed by EPN-01 have a diameter of 20 nm, it was expected that full-length Ago2 would be difficult to encapsulate within them. Therefore, we prepared Ago2 mutants of different sizes and tested whether they could be encapsulated within the nanocages.

[0052] Expression vectors for the full-length Ago2 (amino acids 1 to 860), the PAZ-PIWI domain (amino acids 227 to 860), the L2-PIWI domain (amino acids 347 to 860), and the MID-PIWI domain (amino acids 446 to 860) of Ago2 were constructed by adding a FLAG tag to the N-terminus and HIV-1-derived Vpr (SEQ ID NO: 1) to the C-terminus (Flag-Ago2-FL-Vpr, Flag-PAZ-PIWI-Vpr, Flag-L2-PIWI-Vpr, and Flag-MID-PIWI-Vpr, respectively; Figure 1).

[0053] VectorBuilder synthesized the plasmid VB200705-1156knk (SEQ ID NO: 9), which contains sequences encoding EPN-01 and Myc-EGFP-Vpr. Inverse PCR was performed using VB200705-1156knk as a template with primers 1 and 2. The resulting amplified product was digested at the EcoRI site and ligated to obtain pRP-Myc-EGFP-Vpr. PCR was performed using primers 3 and 4 with the plasmid pcDNA3.2-5'F-Ago2, which contains a sequence encoding full-length human Ago2 with an N-terminal Flag tag, as a template. The resulting amplified product was inserted into the XbaI site of pRP-Myc-EGFP-Vpr by in-fusion cloning to obtain the plasmid pRP-Flag-Ago2-FL-Vpr, which contains a sequence encoding Flag-Ago2-FL-Vpr. PCR was performed using pRP-Flag-Ago2-FL-Vpr as a template and the following primers 5 and 6. The resulting amplified product was inserted into the NheI / HindIII site of pEGFP-C2 (Clontech) by in-fusion cloning to obtain pCMV-Flag-Ago2-FL-Vpr.

[0054] PCR was performed using the following primers 7 and 10, 8 and 10, or 9 and 10, with pcDNA3.2-5'F-Ago2 as the template. The resulting amplified product was inserted into the NheI / XbaI site of pCMV-Flag-Ago2-FL-Vpr by in fusion cloning to obtain pCMV-Flag-PAZ-PIWI-Vpr, pCMV-Flag-L2-PIWI-Vpr, and pCMV-Flag-MID-PIWI-Vpr.

[0055] Table 1. Primer sets for constructing Ago2 mutant expression vectors [Table 1]

[0056] The amino acid sequence of the Flag-Argonaute protein mutant-Vpr expressed from the plasmid vector prepared as described above is shown below.

[0057] Flag-Ago2-FL-Vpr (SEQ ID NO: 23) [ka]

[0058] Flag-PAZ-PIWI-Vpr (SEQ ID NO: 24) [ka]

[0059] Flag-L2-PIWI-Vpr (SEQ ID NO: 25) [ka]

[0060] Flag-MID-PIWI-Vpr (SEQ ID NO: 26) [ka]

[0061] To prepare an expression vector for EPN-01, inverse PCR was performed using VB200705-1156knk (SEQ ID NO: 9) as a template and the following primers 11 and 12. The resulting amplified product was cleaved at the KpnI site and ligated to obtain pRP-EPN-01. PCR was performed using the above primer 6 and the following primer 13 as a template with pRP-EPN-01. The resulting amplified product was inserted into the NheI / HindIII site of pEGFP-C2 by in fusion cloning to obtain pCMV-EPN-01.

[0062] Table 2. Primer sets for constructing Ago2 mutant expression vectors [Table 2]

[0063] HEK293T cells (ATCC) were seeded in 10 cm dishes. The following day, each Ago2 mutant expression vector (5 μg) and EPN-01 expression vector (10 μg) were transfected using Lipofectamine 2000 (Thermo Fisher Scientific). Six hours later, the medium was replaced with 10 ml of fresh medium. 24 hours after transfection, the medium was collected and centrifuged at 200 × g for 5 minutes at 4 °C, 1,000 × g for 5 minutes at 4 °C, and 10,000 × g for 30 minutes at 4 °C. The supernatant was then layered on top of 2 ml of 20% sucrose solution in an ultracentrifuge tube and ultracentrifuged at 100,000 × g for 90 minutes at 4 °C. The supernatant was then discarded and gently washed with PBS. After removing the liquid, 100 μl of 1x SDS sample buffer (Tris-HCl (62.5 mM), pH 6.8, 20% glycerol, 2% SDS, 2.5% 2-mercaptoethanol) was added to the bottom of the tube without touching the wall, dissolving the pellet and obtaining an extracellular vesicle solution. The resulting solution was stored at -80°C. Meanwhile, after medium recovery, HEK293T cells were washed with PBS and collected, and 1 / 5 of the volume was dissolved in 200 μl of 1x SDS sample buffer to obtain a cell solution.

[0064] The extracellular vesicle solution and cell solution were subjected to SDS-PAGE (10% acrylamide gel). After electrophoresis, proteins were transferred to a PDVF membrane. The PDVF membrane was blocked with 3.5% skim milk for 30 minutes, washed three times with TBS-T (25 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20) for a total of 30 minutes, and then incubated overnight at 4°C in primary antibody solution. The primary antibody solution was removed, washed three times with TBS-T for a total of 30 minutes, and then incubated in secondary antibody solution at room temperature for 1 hour. The secondary antibody solution was removed, washed three times with TBS-T for a total of 30 minutes, and then detected using an ImmunoStar LD (Wako) and an LAS3000 (FUJIFILM). The primary antibodies used were anti-c-myc (anti-c-myc from mouse IgG1κ [9E10] (11667203001, Roche) at a dilution of 1:1000), anti-Flag (monoclonal ANTI-FLAG™ M2 antibody produced in mouse (F3165, Sigma-Aldrich) at a dilution of 1:1000), and anti-β-actin (β-Actin (13E5) Rabbit mAb (#4970, Cell Signaling Technology) at a dilution of 1:1000). The secondary antibodies used were ECL™ anti-rabbit IgG (NA9340V, GE) at a dilution of 1:2000 and ECL™ anti-mouse IgG (NA9310V, GE) at a dilution of 1:2000.

[0065] The results are shown in Figure 2. Hereafter, "Cell" refers to the cell solution, and "Release" refers to the extracellular vesicle solution. While expression of full-length Ago2 and all Ago2 mutants was observed in the cell solution, only the MID-PIWI-Vpr mutant was detected in the extracellular vesicle solution. This result confirmed that only the MID-PIWI-Vpr mutant could be incorporated into the EPN-01 nanocage.

[0066] 2. miRNA-binding activity of MID-PIWI-Vpr mutants To examine the miRNA-binding activity of the MID-PIWI-Vpr mutants, HEK293T cells were transfected with full-length Ago2 (Flag-Ago2-FL-Vpr) or Flag-MID-PIWI-Vpr using the same procedure as in 1 above. EGFP was expressed instead of the Ago2 mutants as a control. Twenty-four hours after transfection, cells were washed once with PBS and lysed in 1 mL of lysis buffer (HEPES (20 mM), pH 7.5, NaCl (150 mM), NaF (50 mM), Na3VO4 (1 mM), 1% digitonin, phenylmethylsulfonyl fluoride (1 mM), leupeptin (5 μg / ml), aprotinin (5 μg / ml), and pepstatin A (3 μg / ml)). The cell lysate was collected using a scraper and centrifuged at 15,000 rpm for 10 minutes at 4°C. The supernatant was then collected. A 50 μl aliquot of the supernatant was added to 50 μl of 2×SDS sample buffer to prepare the cell suspension sample before immunoprecipitation. The beads were washed with wash buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Triton-X). Dynabeads Protein G (Veritas) pre-incubated with anti-Flag-M2 antibody (Sigma-Aldrich, F1804, 1:800 dilution) for 30 minutes at room temperature was added to the supernatant and incubated for 1 hour at 4°C with rotational mixing. After washing the beads three times with wash buffer, 120 μl of elution buffer was added, mixed, and incubated at 4°C for 5 minutes. Afterwards, 100 μl of the supernatant was collected and 100 μl of 2×SDS sample buffer was added to prepare the immunoprecipitate sample. The cell solution before immunoprecipitation (Figure 3, "Input") and the immunoprecipitate (Figure 3, "IP:Flag") were subjected to Western blotting using the same procedure as in 1 above, confirming that Flag-Ago2-FL-Vpr and Flag-MID-PIWI-Vpr mutants had been immunoprecipitated (Figure 3).

[0067] Next, miRNA was purified from the immunoprecipitates using the mirVana™ miRNA Isolation Kit (final volume: 50 μl). Reverse transcription reactions were performed using 5 μl of the miRNA solution and the TaqMan™ MicroRNA Reverse Transcription Kit (Thermo Fisher) and TaqMan™ MicroRNA Assays (Thermo Fisher). Furthermore, qPCR was performed using TaqMan™ Universal Master Mix II, no UNG (Thermo Fisher) and TaqMan™ MicroRNA Assays (Thermo Fisher) to quantify miRNA-let7a-5p cDNA. The miRNA levels were expressed relative to the amount of miRNA in the immunoprecipitate sample prepared from EGFP-expressing cells (set at 1).

[0068] The results are shown in Figure 4. In the figure, an asterisk (*) indicates the p-value obtained by one-way ANOVA with Tukey's post-hoc test (**p<0.01). Error bars indicate standard deviation. The amount of miRNA-let7a-5p co-precipitated with Flag-MID-PIWI-Vpr was reduced compared to that co-precipitated with Flag-Ago2-FL-Vpr, but was significantly higher than that of the negative control, EGFP. These results demonstrate that the MID-PIWI-Vpr mutant binds to miRNA-let7a-5p.

[0069] 3. Amount of miRNA-let7a-5p in extracellular vesicles containing EPN-01 / MID-PIWI-Vpr mutants Using the same procedure as above, HEK293T cells co-expressing full-length Ago2 (Flag-Ago2-FL-Vpr) or Flag-MID-PIWI-Vpr and EPN-01 were prepared. Western blotting of the cell solution and extracellular vesicle solution was performed to confirm protein expression (Figure 5). Similar to the results in Figure 2, only the MID-PIWI-Vpr mutant was detected in the extracellular vesicle solution.

[0070] Next, miRNA-let7a-5p in the extracellular vesicle solution was purified and quantified using the same procedure as in 2. The miRNA level was expressed as a relative value, with the amount of miRNA in the extracellular vesicle solution prepared from untransfected cells set at 1.

[0071] The results are shown in Figure 6. In the figure, an asterisk (*) indicates a p-value from one-way ANOVA with Tukey's post-hoc test (**p<0.01). Error bars indicate standard deviation. The yield of miRNA-let7a-5p from extracellular vesicle solutions obtained from HEK293T cells coexpressing EPN-01 and Flag-MID-PIWI-Vpr was significantly increased. In contrast, coexpression of EPN-01 and Flag-Ago2-FL-Vpr did not significantly increase the yield of miRNA-let7a-5p compared to the negative control coexpression of EPN-01 and EGFP. These results demonstrate that coexpression of EPN-01 and MID-PIWI-Vpr mutants can increase the amount of miRNA-let7a-5p in the extracellular vesicle fraction.

[0072] 4. Types and quantities of miRNAs in extracellular vesicles containing EPN-01 / MID-PIWI-Vpr mutants Next-generation sequencing (Small RNA-seq) was used to comprehensively identify miRNAs present in extracellular vesicles (EVs) obtained from HEK293T cells co-expressing EPN-01 and Flag-MID-PIWI-Vpr. EVs obtained from untransfected HEK293T cells, which were subjected to medium exchange only, served as a control. 186 miRNAs were identified in the control EVs, while 323 miRNAs were identified in the EVs from EPN-01 / MID-PIWI-Vpr co-expressing cells. 145 miRNAs were present in both EVs, 41 miRNAs were present only in the control EVs, and 178 miRNAs were present only in the EVs from EPN-01 / MID-PIWI-Vpr co-expressing cells. These results confirmed that coexpression of EPN-01 / MID-PIWI-Vpr increases the number of miRNAs detectable in the extracellular vesicle fraction.

[0073] Next, miR-92a-3p, miR-191-5p, and miR-126-5p, which were commonly detected in the extracellular vesicle fractions of control and EPN-01 / MID-PIWI-Vpr co-expressing cells, were quantified by RT-qPCR using the same procedure as in 2 above. The results are shown in Figures 7 to 9. In the figures, asterisks (*) indicate p values ​​from the Student's t-test (***p<0.001). Error bars indicate standard deviation. It was confirmed that the yield of all miRNAs increased with co-expression of EPN-01 / MID-PIWI-Vpr.

[0074] Furthermore, the results for miR-10b-5p, which was confirmed to be present only in the extracellular vesicle fraction of EPN-01 / MID-PIWI-Vpr co-expressing cells by small RNA-seq analysis, are shown in Figure 10. In the figure, asterisks (*) indicate p-values ​​from Student's t-test (***p<0.001). Error bars indicate standard deviation. RT-qPCR confirmed the presence of miR-10b-5p in the control extracellular vesicle fraction, while the extracellular vesicle fraction of EPN-01 / MID-PIWI-Vpr co-expressing cells contained more than eight-fold more miR-10b-5p.

[0075] These results suggest that co-expression of EPN-01 and the MID-PIWI-Vpr mutant can increase the amount of a wide variety of miRNAs contained in extracellular vesicle fractions, potentially enabling the analysis of trace amounts of miRNAs that cannot be detected in normal exosomes.

[0076] 5. Non-destructive analysis of cells using extracellular vesicles containing EPN-01 / MID-PIWI-Vpr mutants miR-210 expression is known to be strongly induced under hypoxic conditions. One of the well-studied hypoxia signaling pathways is regulated by hypoxia-inducible factors (HIFs). Under normoxic conditions, HIF1α is hydroxylated, binds to E3 ligases, and is degraded by the proteasome. However, under hypoxic conditions, HIF1α is stabilized and translocates to the nucleus without degradation, where it dimerizes with HIF1β and promotes the transcription of target genes, including miR-210 (Genes Dev. 2004 Sep 15;18(18):2183-94. doi: 10.1101 / gad.1243304., Mol Cell. 2009 Sep 24;35(6):856-67. doi: 10.1016 / j.molcel.2009.09.006.).

[0077] Six hours after transfection of the MID-PIWI-Vpr and EPN-01 expression vectors, we added CoCl2 (50 μM) to induce hypoxia. Western blotting was performed on the cell and extracellular vesicle solutions to confirm protein expression. Anti-HIF-1α antibody [EP1215Y] (Abcam, ab51608, 1:1000 dilution) was used to detect HIF-1α.

[0078] The results are shown in Figure 11. CoCl2-treated cells showed accumulation of HIF1α. This result is consistent with a report that hypoxia caused by CoCl2 treatment leads to HIF1α accumulation (Biol Res. 2019 Mar 15;52(1):12. doi: 10.1186 / s40659-019-0221-z.). CoCl2-treated cells also showed decreased expression of EPN-01 and MID-PIWI-Vpr, which is consistent with a report that hypoxia inhibits protein production (Mol Cell Biol. 2006 May;26(10):3955-65. doi: 10.1128 / MCB.26.10.3955-3965.2006.). Similarly, the yields of EPN-01 and MID-PIWI-Vpr in the extracellular vesicle fraction were also reduced.

[0079] Next, miR-210 was purified and quantified in the cell solution and extracellular vesicle solution using the same procedure as in 2 above. Cell solution and extracellular vesicle fractions obtained from HEK293T cells that had undergone medium exchange without transfection served as controls. For comparison, miR-1303, which has not been reported to be associated with hypoxia, was also purified and quantified in the same manner.

[0080] The quantitative results of miR-210 in cells are shown in Figure 12, and the quantitative results of miR-210 in cells are shown in Figure 13. In the figures, asterisks (*) indicate p-values ​​from one-way ANOVA with Tukey's post-hoc test (**p<0.01). Error bars indicate standard deviation. CoCl2 treatment increased the expression level of miR-210, but no significant change was observed in the expression level of miR-1303. Furthermore, coexpression of EPN-01 and MID-PIWI-Vpr did not affect the expression of either miRNA.

[0081] As confirmed above, CoCl2 treatment reduced the expression levels of EPN-01 and MID-PIWI-Vpr in cells, which is expected to result in a decrease in extracellular vesicles and the miRNAs contained within them. Therefore, the miR-210 quantification results normalized based on the miR-1303 quantification results are shown in Figure 14. In the figure, an asterisk (*) indicates a p-value from Student's t-test (***p<0.001). Error bars indicate standard deviation. In the extracellular vesicle fraction from control cells, CoCl2 treatment did not significantly increase the amount of miR-210. In contrast, CoCl2 treatment significantly increased the amount of miR-210 in the extracellular vesicle fraction from cells coexpressing EPN-01 and MID-PIWI-Vpr. These results demonstrate that the coexpression of EPN-01 and MID-PIWI-Vpr enabled the detection of increased miR-210 expression with high accuracy.

[0082] These results demonstrate that co-expression of EPN-01 and MID-PIWI-Vpr in cells enables miRNA analysis with higher accuracy and sensitivity than conventional miRNA analysis using exosomes, without destroying the cells.

[0083] 6. Co-expression of EPN-01 / MID-PIWI-Vpr increases the size and production of extracellular vesicles. Using the same procedure as in 1 above, HEK293T cells co-expressing EPN-01 and Flag-MID-PIWI-Vpr and HEK293T cells co-expressing EPN-01 and EGFP were prepared, and extracellular vesicle fractions were obtained from the culture supernatant by sucrose cushion centrifugation. Non-transfected HEK293T cells were used as controls. Nanoparticle tracking analysis (NTA) was performed using a NanoSight NS300 (Malvern Panalytical) under the following conditions: the camera level was set to 16 for all recordings. The extracellular vesicle fraction was diluted 1:100–1:1000 with PBS, and 1 × 10 8 ~1×10 9 Measurement samples were prepared to achieve a particle count of 1000 / ml. The camera focus was adjusted so that particles appeared as sharp, individual dots. Five 60-second images were recorded for each measurement sample. All post-acquisition functions were set to automatic, except for the detection threshold, which was set to 8.

[0084] The particle size distribution and concentration of extracellular vesicles are shown in Figure 15. Error bars indicate standard error. The number of extracellular vesicles was increased in both HEK293T cells co-expressing EPN-01 and Flag-MID-PIWI-Vpr and HEK293T cells co-expressing EPN-01 and EGFP compared to untransfected HEK293T cells. Furthermore, the particle size distribution of extracellular vesicles tended to be similar in both HEK293T cells co-expressing EPN-01 and Flag-MID-PIWI-Vpr and HEK293T cells co-expressing EPN-01 and EGFP.

[0085] The average particle size of extracellular vesicles is shown in Figure 16. In the figure, an asterisk (*) indicates the p-value from one-way ANOVA with Tukey's post-hoc test (**p<0.01). Error bars indicate standard error. A tendency for the particle size of extracellular vesicles to increase was observed in both HEK293T cells co-expressing EPN-01 and Flag-MID-PIWI-Vpr and HEK293T cells co-expressing EPN-01 and EGFP.

[0086] These results demonstrate that extracellular vesicles larger than normal exosomes can be obtained at high concentrations from cells co-expressing EPN-01 and the MID-PIWI-Vpr mutant.

Claims

1. (1) introducing a nucleic acid encoding a microRNA-binding protein and a nucleic acid encoding a vesicle-forming protein into a cell (excluding cells in a human body), wherein the microRNA-binding protein comprises a first portion consisting of the MID domain and the PIWI domain of an Argonaute protein and a second portion consisting of a viral protein R, and the vesicle-forming protein comprises a palmitoylation or myristoylation signal or a pleckstrin homology domain, a self-assembly domain, an ESCRT or ESCRT-associated factor binding domain, and a Gag p6 domain, thereby producing exosome-like vesicles containing the microRNA; (2) collecting the extracellular fluid of the cells; (3) extracting microRNA from the extracellular fluid; A method for non-invasively obtaining microRNA from cells, comprising:

2. 2. The method of claim 1, wherein the microRNA-binding protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

1.

3. The method of claim 1 or 2, wherein the vesicle-forming protein comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 6.

4. The method of claim 1 , wherein the cells are in vitro cells and the extracellular fluid is a culture supernatant.

5. The method of claim 1 , wherein the cell is an in vivo cell and the extracellular fluid is a biological fluid.

6. (a) microRNA, (b) a microRNA-binding protein comprising a first portion consisting of the MID domain and the PIWI domain of an Argonaute protein and a second portion consisting of viral protein R; and (c) a nanocage composed of a vesicle-forming protein containing an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 6; An exosome-like vesicle comprising:

7. The exosome-like vesicle of claim 6, further comprising (d) a membrane fusion protein.

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