WO2023281999A1 - 細胞から非侵襲的にマイクロrnaを取得する方法 - Google Patents
細胞から非侵襲的にマイクロrnaを取得する方法 Download PDFInfo
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
Definitions
- the present invention relates to a method for noninvasively obtaining microRNA from cells.
- MicroRNAs are small, single-stranded non-coding RNAs with a length of about 22 bases, and interact with mRNA to regulate mRNA expression. It has been clarified that miRNAs are involved in various biological processes such as cell proliferation, differentiation, apoptosis, and the onset and pathology of various diseases including cancer. Possibility of being a marker has been reported (Non-Patent Document 1). Since miRNA is contained in exosomes secreted from cells and is present in body fluids such as blood and urine, liquid biopsy can be used to noninvasively diagnose diseases by detecting and quantifying miRNA in body fluids. It's getting a lot of attention. In addition, since miRNA is closely related to the onset and pathology of various diseases, miRNA-containing exosomes are also expected as nucleic acid medicines.
- exosomes are formed by taking up the cytoplasm, miRNAs are contained in exosomes only at the concentration in the cytoplasm. Therefore, the amount of miRNA contained in exosomes is extremely small, and it is difficult to obtain a sufficient amount of miRNA for highly accurate diagnosis and drug development.
- Patent Document 1 In recent years, a method has been proposed to induce the production of exosome-like extracellular vesicles using artificially designed self-assembling protein nanocages (Patent Document 1, Non-Patent Document 2). According to this method, exosome-like vesicles containing the intracellularly expressed recombinant protein of interest can be obtained. However, since these extracellular vesicles are also formed by taking up the cytoplasm like exosomes, it is still difficult to obtain miRNAs, which are originally present in low abundance in the cytoplasm.
- the present invention aims to provide a method for non-invasively obtaining more miRNA from cells.
- the inventor succeeded in increasing the yield of miRNA by de novo designing a miRNA-binding protein of a size that can be included in exosome-like vesicles.
- the present invention provides a step of (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 is , a first portion consisting of the MID and PIWI domains of the Argonaute protein and a second portion consisting of the viral protein R, and wherein said vesicle-forming protein comprises a palmitoylation or myristoylation signal or a pleckstrin homology domain; comprising a self-assembling domain, an ESCRT or ESCRT-related factor-binding domain and a Gag p6 domain, thereby producing exosome-like vesicles containing microRNA, (2) collecting the extracellular fluid of the cell; 3) extracting the microRNA from the extracellular fluid.
- the microRNA-binding protein preferably contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1.
- the vesicle-forming protein preferably contains 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.
- the cells may be in vitro cells, and the extracellular fluid may be a culture supernatant.
- the cells may be in vivo cells, and the extracellular fluid may be a biological fluid.
- the present invention provides a microRNA comprising (a) a microRNA, (b) a first portion consisting of the MID domain and the PIWI domain of Argonaute protein and a second portion consisting of viral protein R. an exosome-like vesicle comprising a nanocage composed of an RNA binding protein and (c) a vesicle-forming protein comprising 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. It provides.
- the exosome-like vesicles preferably further contain (d) a membrane fusion protein.
- an extracellular fluid containing miRNA at a high concentration can be obtained. Therefore, the method according to the present invention allows miRNA analysis to be performed non-invasively, similarly to conventional exosome-based miRNA analysis, and with higher accuracy and sensitivity than that.
- the exosome-like vesicles according to the present invention contain high concentrations of miRNA and are useful for the development of nucleic acid medicines.
- FIG. 1 is a schematic diagram showing the domain structure of various Argonaute protein mutants.
- FIG. 2 shows the results of Western blot analysis confirming various Argonaute protein mutants contained in cells and extracellular vesicles.
- FIG. 3 shows the results of immunoprecipitation of Flag-Ago2-FL-Vpr or Flag-MID-PIWI-Vpr with anti-Flag antibody.
- FIG. 4 is a graph showing relative quantification of miRNA-let7a-5p co-precipitated with Flag-Ago2-FL-Vpr or Flag-MID-PIWI-Vpr.
- FIG. 5 shows the results of Western blot analysis confirming Flag-Ago2-FL-Vpr or Flag-MID-PIWI-Vpr contained in cells and extracellular vesicles.
- Figure 6 shows 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. It is a graph showing.
- FIG. 7 is a graph showing relative quantification of miR-92a-3p in extracellular vesicle fractions obtained from HEK293T cells co-expressing Flag-MID-PIWI-Vpr and EPN-01.
- FIG. 8 is a graph showing relative quantification of miR-191-5p in extracellular vesicle fractions obtained from HEK293T cells co-expressing Flag-MID-PIWI-Vpr and EPN-01.
- FIG. 9 is a graph showing relative quantification of miR-126-5p in extracellular vesicle fractions obtained from HEK293T cells co-expressing Flag-MID-PIWI-Vpr and EPN-01.
- FIG. 10 is a graph showing relative quantification of miR-10b-5p in extracellular vesicle fractions obtained from HEK293T cells co-expressing Flag-MID-PIWI-Vpr and EPN-01.
- FIG. 11 shows the results of Western blot analysis confirming changes in the expression levels of HIF1 ⁇ , EPN-01 and MID-PIWI-Vpr due to CoCl 2 treatment.
- FIG. 12 is a graph showing CoCl 2 treatment changes in miR-210 expression levels in HEK293T cells transfected or not with Flag-MID-PIWI-Vpr and EPN-01.
- FIG. 13 is a graph showing CoCl 2 treatment changes in miR-1303 expression levels in HEK293T cells transfected or not with Flag-MID-PIWI-Vpr and EPN-01.
- FIG. 14 shows the results of Western blot analysis confirming changes in the expression levels of HIF1 ⁇ , EPN-01 and MID-PIWI-Vpr due to CoCl 2 treatment.
- FIG. 12 is a graph showing CoCl 2 treatment changes in miR-210 expression levels in HEK293T cells transfected or not with Flag-MID-PIWI-Vpr and EPN-01.
- CoCl 2 treatment changes the content of miR-210 in the outer vesicle fraction from HEK293T cells transfected or not with Flag-MID-PIWI-Vpr and EPN-01.
- 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. It is a graph showing the particle size (average value) of.
- the present invention provides, according to a first embodiment, (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 is , a first portion consisting of the MID and PIWI domains of the Argonaute protein and a second portion consisting of the viral protein R, and wherein said vesicle-forming protein comprises a palmitoylation or myristoylation signal or a pleckstrin homology domain; comprising a self-assembling domain, an ESCRT or ESCRT-related factor-binding domain and a Gag p6 domain, thereby producing exosome-like vesicles containing microRNA, (2) collecting the extracellular fluid of the cell; 3) A method for non-invasively obtaining microRNAs from cells, comprising the step of extracting microRNAs from the extracellular fluid.
- a nucleic acid encoding a microRNA-binding protein and a nucleic acid encoding a vesicle-forming protein are introduced into cells.
- MicroRNA (also referred to as “miRNA”) is a small single-stranded non-coding RNA with a length of about 21 to 25 bases, and more than 20,000 types have been identified so far (http://mirbase. org/).
- the miRNA in this embodiment is not particularly limited, and may be any miRNA expressed in any cell.
- miRNAs in this embodiment may include not only known miRNAs but also unknown miRNAs.
- miRNA in the present embodiment means miRNA that is the final product, and does not include intermediate products such as pri-miRNA and pre-miRNA.
- the type of "cell” in the present embodiment is not particularly limited, and may be, for example, dendritic cells, T cells, B cells, nerve cells, stem cells, cancer cells, primary culture cells or cell lines derived therefrom. you can That is, the cells in this embodiment may be either in vivo or in vitro.
- the organism from which the cells are derived is not particularly limited, and may be any vertebrate, preferably mammals such as mice, rats, rabbits, pigs, cows, goats, monkeys, and humans, and particularly preferably mammals. is human.
- microRNA binding protein in this embodiment comprises a first portion consisting of the MID and PIWI domains of Argonaute protein and a second portion consisting of viral protein R.
- Ago RNA-induced silencing complex
- RISC RNA-induced silencing complex
- the Ago MID domain and PIWI domain used in this embodiment may be derived from any protein of the Ago family, preferably from Ago1, Ago2, Ago3 or Ago4, and particularly preferably from Ago2. be.
- the Ago MID domain and PIWI domain used in this embodiment may be derived from any vertebrate, preferably from mammals, and particularly preferably from humans.
- the amino acid sequence of human Ago2 (SEQ ID NO: 7) is shown below.
- Vpr Virus protein R
- HAV human immunodeficiency virus
- SIV simian immunodeficiency virus
- the Vpr used in this embodiment may be derived from any primate immunodeficiency virus, but is preferably derived from HIV, particularly preferably from HIV-1.
- the amino acid sequence of HIV-1 Vpr (SEQ ID NO: 8) is shown below.
- amino acid sequences of Ago and Vpr and the nucleic acid sequence information encoding them can be obtained from predetermined databases.
- NP_036286.2 GenBank
- NM_012154.5 GenBank
- HIV-1 Vpr NP_057852.2 (GenBank)
- NC_001802 GenBank 5105-5396 are available.
- the microRNA-binding protein in this embodiment most preferably contains an amino acid sequence (SEQ ID NO: 1) consisting of the MID and PIWI domains of human Ago2 and HIV-1-derived Vpr.
- microRNA binding protein SEQ ID NO: 1
- the microRNA-binding protein in this embodiment maintains miRNA-binding activity equivalent to Ago's MID domain and PIWI domain and Gag's p6 domain-binding activity equivalent to Vpr. and the amino acid sequence having 80% or more, preferably 90% or more, more preferably about 95% or more identity with the amino acid sequence of Vpr. Amino acid sequence identity can be calculated using sequence analysis software or using programs commonly used in the art (FASTA, BLAST, etc.).
- a "vesicle-forming protein” in this embodiment includes a palmitoylation or myristoylation signal or a pleckstrin homology (PH) domain, a self-assembling domain, an ESCRT or an ESCRT-related factor binding domain, and a Gag p6 domain.
- a vesicle-forming protein that can be used in this embodiment is disclosed as Enveloped Protein Nanocage (EPN) in WO 2016/138525, specifically, EPN-01 (SEQ ID NO: 2) , EPN-03 (SEQ ID NO:3), EPN-07 (SEQ ID NO:4), EPN-08 (SEQ ID NO:5), EPN-18 (SEQ ID NO:6), and the like.
- EPN-01 SEQ ID NO: 2
- EPN-03 SEQ ID NO:3
- EPN-07 SEQ ID NO:4
- EPN-08 SEQ ID NO:5
- EPN-18 SEQ ID NO:6
- the vesicle forming protein in this embodiment is
- EPN-03 (SEQ ID NO: 3)
- EPN-018 (SEQ ID NO: 6)
- the vesicle-forming protein in the present embodiment maintains an activity equivalent to that of the EPN subunit (that is, forms a nanocage by self-assembly to constitute an extracellular vesicle).
- Proteins consisting of amino acid sequences having 80% or more, preferably 90% or more, more preferably about 95% or more identity with the amino acid sequences of the EPN subunits disclosed in .
- microRNA-binding protein and vesicle-forming protein in this embodiment may have an epitope tag such as Myc, HA, FLAG added to their N-terminus and/or C-terminus.
- epitope tag such as Myc, HA, FLAG added to their N-terminus and/or C-terminus.
- Nucleic acids encoding microRNA-binding proteins and nucleic acids encoding vesicle-forming proteins can be prepared by any conventionally known genetic engineering method based on the sequences designed according to the above.
- those nucleic acids may be introduced into cells by methods well known in the art, for example, those nucleic acids may be cloned into expression vectors and introduced into cells.
- the type of expression vector is not particularly limited, and may be either a viral vector or a non-viral vector. It may be a plasmid vector such as pCAG.
- the microRNA-binding protein and the vesicle-forming protein are expressed in the cell, the microRNA-binding protein-miRNA complex is housed in a nanocage composed of the vesicle-forming protein, exosome-like vesicles are formed, and extracellular released to
- exosome-like vesicles in the present embodiment refers to nanoscale extracellular vesicles similar in structure and composition to exosomes.
- the extracellular fluid of the cells is collected. If the cells are cells in vitro, the extracellular fluid may be a culture supernatant, and if the cells are cells in vivo, the extracellular fluid may be a biological fluid.
- biological fluids include, but are not limited to, blood, plasma, serum, saliva, urine, and the like.
- miRNA is extracted from the extracellular fluid.
- miRNA can be extracted by already established procedures, for example, extracellular vesicles can be collected by ultracentrifugation, and miRNA can be isolated by a purification method such as the Boom method.
- a number of miRNA extraction kits based on the Boom method are commercially available, and such commercially available products can also be used in the method of the present embodiment.
- High Pure miRNA Isolation Kit (Roche Diagnostics), miRNeasy Mini Kit (Qiagen), mirVana (trademark) miRNA Isolation Kit (Thermo Fisher Scientific) and the like are preferred commercially available products.
- the method of the present embodiment can obtain extracellular fluid containing miRNA at a high concentration, enabling noninvasive, highly accurate, and highly sensitive diagnosis.
- the present invention provides a microRNA comprising (a) a microRNA, (b) a first portion consisting of the MID and PIWI domains of Argonaute protein and a second portion consisting of viral protein R.
- An exosome-like vesicle comprising a nanocage composed of an RNA binding protein and (c) a vesicle-forming protein comprising 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. be.
- Exosome-like vesicles are defined in the first embodiment is the same as
- the exosome-like vesicles of this embodiment can further contain (d) a membrane fusion protein.
- membrane fusion protein is meant a protein that causes fusion between homogeneous or heterologous cells or membrane vesicles.
- the membrane fusion protein in this embodiment is not particularly limited, but is preferably an enveloped virus-derived membrane fusion protein, such as vesicular stomatitis virus G protein (VSV-G), herpes simplex virus glycoprotein B (gB) and Recombinants thereof and the like are included.
- VSV-G vesicular stomatitis virus G protein
- gB herpes simplex virus glycoprotein B
- Exosome-like vesicles of this embodiment are obtained from the extracellular fluid of cells introduced with nucleic acids encoding microRNA-binding proteins and nucleic acids encoding vesicle-forming proteins by the procedure of the method of the first embodiment. can be done.
- the exosome-like vesicles of this embodiment can contain miRNA at a higher concentration than natural exosomes. Therefore, it is useful for diagnosis of diseases and development of nucleic acid drugs.
- EPN-01 SEQ ID NO: 2
- human Ago2 was used as the Argonaute protein. Since the nanocage formed by EPN-01 is 20 nm in diameter and it was assumed that it would be difficult for full-length Ago2 to be encapsulated in it, we prepared mutants with different sizes from Ago2 and examined whether they could be encapsulated into nanocages. was tested.
- Ago2 Full length of Ago2 (amino acid numbers 1-860), Ago2 PAZ domain-PIWI domain (amino acid numbers 227-860), L2 domain-PIWI domain (amino acid numbers 347-860), and MID domain-PIWI domain (amino acid numbers 446-446).
- 860 with a FLAG tag at the N-terminus and an HIV-1-derived Vpr (SEQ ID NO: 1) at the C-terminus (Flag-Ago2-FL-Vpr, Flag-PAZ-PIWI-Vpr, Flag- Expression vectors for L2-PIWI-Vpr and Flag-MID-PIWI-Vpr (Fig. 1) were prepared by the following procedure.
- Plasmid VB200705-1156knk (SEQ ID NO: 9) containing sequences encoding EPN-01 and Myc-EGFP-Vpr was synthesized by commissioning VectorBuilder.
- VB200705-1156knk as a template, inverse PCR was performed using primers 1 and 2 below. The resulting amplified product was cleaved at the EcoRI site and ligated to obtain pRP-Myc-EGFP-Vpr.
- PCR was performed using primers 3 and 4 below.
- the resulting amplification product was inserted into the XbaI site of pRP-Myc-EGFP-Vpr by In Fusion cloning to obtain plasmid pRP-Flag-Ago2-FL-Vpr containing the sequence encoding Flag-Ago2-FL-Vpr. rice field.
- PCR was performed using primers 5 and 6 below.
- 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.
- PCR was performed using the following primers 7 and 10, 8 and 10, or 9 and 10.
- the resulting amplified product was inserted into the NheI/XbaI site of pCMV-Flag-Ago2-FL-Vpr by In Fusion cloning, pCMV-Flag-PAZ-PIWI-Vpr, pCMV-Flag-L2-PIWI-Vpr, and pCMV-Flag-MID-PIWI-Vpr was obtained.
- EPN-01 expression vector In order to create an EPN-01 expression vector, inverse PCR was performed using VB200705-1156knk (SEQ ID NO: 9) as a template and primers 11 and 12 below. The resulting amplified product was cleaved at the KpnI site and ligated to obtain pRP-EPN-01. Using pRP-EPN-01 as a template, PCR was performed using primer 6 above and primer 13 below. The resulting amplified product was inserted into the NheI/HindIII site of pEGFP-C2 by In Fusion cloning to obtain pCMV-EPN-01.
- HEK293T cells ATCC were seeded in a 10 cm dish. The next day, each Ago2 mutant expression vector (5 ⁇ g) and EPN-01 expression vector (10 ⁇ g) were transfected with Lipofectamine2000 (Thermo Fisher Scientific), and after 6 hours, the medium was replaced with 10 ml of fresh medium. Twenty-four hours after transfection, the medium was harvested and centrifuged at 200 xg for 5 minutes at 4°C, 1,000 xg for 5 minutes at 4°C, and 10,000 xg for 30 minutes at 4°C. Clear was collected. 2 ml of 20% sucrose solution was placed in an ultracentrifugation tube, the supernatant was layered thereon, and ultracentrifugation was performed at 4° C.
- the extracellular vesicle solution and cell solution were subjected to SDS-PAGE (10% acrylamide gel), and after electrophoresis, proteins were transferred to a PDVF membrane.
- the PDVF membrane was blocked with 3.5% skim milk for 30 minutes, washed with TBS-T (Tris-HCl (25 mM), NaCl (150 mM), 0.1% Tween 20) three times (30 minutes in total), Incubated overnight at 4° C. in primary antibody solution.
- the primary antibody solution was removed, washed with TBS-T three times (total of 30 minutes), and then incubated in the secondary antibody solution for 1 hour at room temperature.
- Primary antibodies include anti-c-Myc antibody (anti-c-myc from mouse IgG1 ⁇ [9E10] (11667203001, Roche) (1:1000 dilution)); anti-Flag antibody (Monoclonal ANTI-FLAGTM M2 antibody produced in mouse (F3165, Sigma-Aldrich (1:1000 dilution)); and an anti- ⁇ -actin antibody ( ⁇ -Actin (13E5) Rabbit mAb (#4970, Cell Signaling Technology) (1:1000 dilution)). ECLTM anti-rabbit IgG (NA9340V, GE) (1:2000 dilution) and ECLTM anti-mouse IgG (NA9310V, GE) (1:2000 dilution) were used as secondary antibodies.
- Cell indicates the results for the cell solution
- Release indicates the results for the extracellular vesicle solution.
- Full-length Ago2 and all Ago2 mutants were expressed in cell solutions, but only the MID-PIWI-Vpr mutant was detected in extracellular vesicle solutions. This result confirmed that only the MID-PIWI-Vpr mutant could be incorporated into the EPN-01 nanocages.
- miRNA binding activity of MID-PIWI-Vpr mutant Next, in order to examine the miRNA-binding activity of the MID-PIWI-Vpr mutant, full-length Ago2 (Flag-Ago2-FL-Vpr) or Flag-MID-PIWI-Vpr was added to HEK293T cells by the same procedure as in 1 above. expressed. For controls, EGFP was expressed in place of the Ago2 mutant.
- Lysis Buffer HEPES (20 mM), pH 7.5, NaCl (150 mM), NaF (50 mM), Na 3 VO 4 (1 mM), 1% Digitonin, 1 mL of phenylmethylsulfonyl fluoride (1 mM), Leupeptin (5 ⁇ g/ml), Aprotinin (5 ⁇ g/ml), Pepstatin A (3 ⁇ g/ml) was added. The cell lysate was collected with a scraper, centrifuged at 4° C. and 15,000 rpm for 10 minutes, and the supernatant was collected.
- HEPES Lysis Buffer
- wash Buffer HEPES (10 mM) pH 7.5, NaCl (150 mM), 0.1% Triton-X), anti-Flag-M2 antibody (Sigma-Aldrich, F1804, 1:800 dilution) and 30 at room temperature.
- Dynabeads Protein G (Veritas) reacted for 1 minute was added to the supernatant and incubated for 1 hour at 4° C. with rotary mixing. After washing the beads three times with Wash Buffer, 120 ⁇ l of Elution Buffer was added, mixed, and incubated at 4° C.
- the miRNA was then purified from the immunoprecipitate using the mirVanaTM miRNA Isolation Kit (final volume 50 ⁇ l).
- a reverse transcription reaction was performed using 5 ⁇ l of miRNA solution, TaqManTM MicroRNA Reverse Transcription Kit (Thermo Fisher) and TaqManTM MicroRNA Assays (Thermo Fisher) to prepare cDNA.
- qPCR was performed using TaqManTM Universal Master Mix II, no UNG (Thermo Fisher) TaqManTM MicroRNA Assays (Thermo Fisher) to quantify miRNA-let7a-5p cDNA.
- the level of miRNA was a relative value when the amount of miRNA in the immunoprecipitate sample prepared from EGFP-expressing cells was set to 1.
- miRNA-let7a-5p in the extracellular vesicle solution was purified and quantified by the same procedure as in 2 above.
- the level of miRNA was a relative value when the amount of miRNA in the extracellular vesicle solution prepared from untransfected cells was set to 1.
- miR-210 in the cell solution and extracellular vesicle solution was purified and quantified by the same procedure as in 2 above.
- Cell solutions and extracellular vesicle fractions obtained from HEK293T cells without transfection but with medium exchange were used as controls.
- miR-1303 which has not been reported to be associated with hypoxia, was similarly purified and quantified.
- the quantification results of miR-210 in cells are shown in FIG. 12, and the quantification results of miR-210 in cells are shown in FIG.
- asterisks indicate p-values by one-way ANOVA with Tukey post-hoc test (**p ⁇ 0.01). Error bars indicate standard deviation.
- CoCl 2 treatment increased the expression level of miR-210 and did not significantly change the expression level of miR-1303. Also, the expression of either miRNA was not affected by co-expression of EPN-01 and MID-PIWI-Vpr.
- FIG. 14 shows the quantification results of miR-210 normalized based on the quantification results of miR-1303.
- asterisks indicate p-values by Student's t-test (***p ⁇ 0.001). Error bars indicate standard deviation. Extracellular vesicle fractions from control cells showed no significant increase in miR-210 abundance with CoCl 2 treatment.
- EPN-01/MID-PIWI-Vpr co-expression increases extracellular vesicle size and production>
- HEK293T cells co-expressing EPN-01 and Flag-MID-PIWI-Vpr and HEK293T cells co-expressing EPN-01 and EGFP were prepared and cultured by sucrose cushion centrifugation. An extracellular vesicle fraction was obtained from the supernatant. Non-transfected HEK293T cells were used as controls.
- Nanoparticle tracking analysis (NTA) was performed under the following conditions using Nanosite NS300 (Malvern Panalytical). Camera level was set to 16 for all recordings.
- the extracellular vesicle fraction was diluted 1:100 to 1:1000 with PBS to prepare a measurement sample having a particle number of 1 ⁇ 10 8 to 1 ⁇ 10 9 /ml.
- the camera focus was adjusted so that the particles appeared as sharp individual dots.
- Five 60-second images were recorded for each measured sample. All post-acquisition functions were set to automatic except the detection threshold was set to 8.
- Fig. 15 shows the particle size distribution and concentration of extracellular vesicles. Error bars indicate standard error.
- Both HEK293T cells co-expressing EPN-01 and Flag-MID-PIWI-Vpr and HEK293T cells co-expressing EPN-01 and EGFP had increased numbers of extracellular vesicles over non-transfected HEK293T cells.
- HEK293T cells co-expressing EPN-01 and Flag-MID-PIWI-Vpr and HEK293T cells co-expressing EPN-01 and EGFP had similar trends in the size distribution of extracellular vesicles.
- Fig. 16 shows the average particle size of extracellular vesicles.
- asterisks (*) indicate p-values by one-way ANOVA with Tukey post-hoc test (**p ⁇ 0.01). Error bars indicate standard error.
- Both HEK293T cells co-expressing EPN-01 and Flag-MID-PIWI-Vpr and HEK293T cells co-expressing EPN-01 and EGFP tended to increase the size of extracellular vesicles.
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Abstract
Description
本実施例では、小胞形成タンパク質としてEPN-01(配列番号2)を用い、アルゴノートタンパク質としてヒトAgo2を用いた。EPN-01により形成されるナノケージは直径20nmであり、全長Ago2がそれに内包されることは難しいことが想定されたため、Ago2からサイズの異なる変異体を調製し、それらがナノケージに内包され得るかどうかを試験した。
次に、MID-PIWI-Vpr変異体のmiRNA結合活性を調べるために、上記1と同様の手順により、HEK293T細胞に全長Ago2(Flag-Ago2-FL-Vpr)またはFlag-MID-PIWI-Vprを発現させた。対照には、Ago2変異体に代えてEGFPを発現させた。トランスフェクションから24時間後、細胞をPBSで1回洗浄し、Lysis Buffer(HEPES(20mM)、pH7.5、NaCl(150mM)、NaF(50mM)、Na3VO4(1mM)、1%Digitonin、フッ化フェニルメチルスルホニル(1mM)、Leupeptin(5μg/ml)、Aprotinin(5μg/ml)、Pepstatin A(3μg/ml))を1mL加えた。細胞溶解物をスクレーパーにより回収し、4℃、15,000rpmで10分間遠心分離を行い、上清を回収した。上清から50μlを分取し、2×SDSサンプルバッファーを50μl加えたものを、免疫沈降前の細胞溶液サンプルとした。予めWash Buffer(HEPES(10mM)pH7.5、NaCl(150mM)、0.1%Triton-X)で洗浄を行い、抗Flag-M2抗体(シグマアルドリッチ、F1804、1:800希釈)と室温で30分間反応させたDynabeads Protein G(ベリタス)を上清に加え、4℃で1時間、回転混和させながらインキュベートした。ビーズをWash Bufferで3回洗浄後、Elution Bufferを120μl加えて混合し、4℃で5分間インキュベートした。その後、上清100μlを回収し、2×SDSサンプルバッファーを100μl加えたものを免疫沈降物サンプルとした。免疫沈降前の細胞溶液(図3、「Input」)および免疫沈降物(図3、「IP:Flag」)について、上記1と同様の手順によりウェスタンブロッティングを行い、Flag-Ago2-FL-VprおよびFlag-MID-PIWI-Vpr変異体が免疫沈降されたことを確認した(図3)。
上記1と同様の手順により、全長Ago2(Flag-Ago2-FL-Vpr)またはFlag-MID-PIWI-VprおよびEPN-01を共発現するHEK293T細胞を調製し、細胞溶液および細胞外小胞溶液についてウェスタンブロッティングを行い、タンパク質の発現を確認した(図5)。図2の結果と同様、細胞外小胞溶液からはMID-PIWI-Vpr変異体のみが検出された。
次世代シークエンシングsmall RNA-seqを用いて、EPN-01およびFlag-MID-PIWI-Vprを共発現させたHEK293T細胞から得られた細胞外小胞画分に存在するmiRNAを網羅的に同定した。トランスフェクションを行わず培地交換のみを行ったHEK293T細胞から得られた細胞外小胞画分を対照とした。その結果、対照の細胞外小胞画分からは186種類、EPN-01/MID-PIWI-Vpr共発現細胞の細胞外小胞画分からは323種類のmiRNAが同定された。145種類のmiRNAが両者の細胞外小胞画分に共通して存在し、41種類のmiRNAが対照の細胞外小胞画分のみに存在し、178種類のmiRNAがEPN-01/MID-PIWI-Vpr共発現細胞の細胞外小胞画分のみに存在した。この結果から、miRNAがEPN-01/MID-PIWI-Vprの共発現により、細胞外小胞画分から検出可能なmiRNAの種類が増加することが確認された。
miR-210は、低酸素状態で発現が強く誘導されることが知られている。よく研究されている低酸素シグナル伝達経路の1つは、低酸素誘導因子(HIF)によって制御される。正常酸素状態では、HIF1αは水酸化され、E3リガーゼと結合してプロテアソームにより分解されるが、一方、低酸素状態ではHIF1αは安定化されており、分解されることなく核内に移行し、HIF1βと二量体を形成し、miR-210を含む標的遺伝子の転写を促進する(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.)。
上記1と同様の手順により、EPN-01およびFlag-MID-PIWI-Vprを共発現するHEK293T細胞と、EPN-01およびEGFPを共発現するHEK293T細胞を作製し、ショ糖クッション遠心分離により培養上清から細胞外小胞画分を得た。対照には、トランスフェクトされていないHEK293T細胞を用いた。ナノサイトNS300(マルバーンパナリティカル)を用いて、以下の条件によりナノ粒子トラッキング解析(NTA)を実施した。すべての録画についてカメラレベルを16に設定した。細胞外小胞画分をPBSにより1:100~1:1000希釈し、1×108~1×109/mlの粒子数になるように測定試料を調製した。粒子が鮮明な個々のドットとして見えるようにカメラの焦点を調整した。各測定試料につき、60秒の影像を5回記録した。検出しきい値を8に設定したことを除き、すべてのデータ取得後機能を自動に設定した。
Claims (7)
- (1)マイクロRNA結合タンパク質をコードする核酸および小胞形成タンパク質をコードする核酸を細胞に導入するステップと、ここで、前記マイクロRNA結合タンパク質が、アルゴノートタンパク質のMIDドメインおよびPIWIドメインからなる第1の部分ならびにウイルスタンパク質Rからなる第2の部分を含み、かつ、前記小胞形成タンパク質が、パルミトイル化もしくはミリストイル化シグナルまたはプレクストリン相同ドメイン、自己集合性ドメイン、ESCRTまたはESCRT関連因子結合ドメインおよびGag p6ドメインを含み、これにより、マイクロRNAを含むエクソソーム様小胞が産生され、
(2)前記細胞の細胞外液を回収するステップと、
(3)前記細胞外液からマイクロRNAを抽出するステップと
を含む、細胞から非侵襲的にマイクロRNAを取得する方法。 - 前記マイクロRNA結合タンパク質が、配列番号1のアミノ酸配列と少なくとも90%同一のアミノ酸配列を含む、請求項1に記載の方法。
- 前記小胞形成タンパク質が、配列番号2~6からなる群から選択されるアミノ酸配列と少なくとも90%同一のアミノ酸配列を含む、請求項1または2に記載の方法。
- 前記細胞がインビトロの細胞であり、前記細胞外液が培養上清液である、請求項1~3のいずれか1項に記載の方法。
- 前記細胞がインビボの細胞であり、前記細胞外液が生体液である、請求項1~3のいずれか1項に記載の方法。
- (a)マイクロRNA、
(b)アルゴノートタンパク質のMIDドメインおよびPIWIドメインからなる第1の部分ならびにウイルスタンパク質Rからなる第2の部分を含むマイクロRNA結合タンパク質、ならびに
(c)配列番号2~6からなる群から選択されるアミノ酸配列と少なくとも90%同一のアミノ酸配列を含む小胞形成タンパク質により構成されるナノケージ
を含んでなるエクソソーム様小胞。 - (d)膜融合タンパク質をさらに含む、請求項6に記載のエクソソーム様小胞。
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LIANG YUTING, HUANG SHENG, QIAO LONGWEI, PENG XIA, LI CHONG, LIN KUN, XIE GUOGANG, LI JIA, LIN LIHUI, YIN YUE, LIAO HUANJIN, LI QI: "Characterization of protein, long noncoding RNA and microRNA signatures in extracellular vesicles derived from resting and degranulated mast cells", JOURNAL OF EXTRACELLULAR VESICLES, TAYLOR & FRANCIS, UK, vol. 9, no. 1, 1 September 2020 (2020-09-01), UK , pages 1697583, XP093022364, ISSN: 2001-3078, DOI: 10.1080/20013078.2019.1697583 * |
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