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WO2024242053A1 - Procédé de criblage de cellules souches hématopoïétiques, procédé de production de population de cellules souches hématopoïétiques et kit de criblage de cellules souches hématopoïétiques - Google Patents

Procédé de criblage de cellules souches hématopoïétiques, procédé de production de population de cellules souches hématopoïétiques et kit de criblage de cellules souches hématopoïétiques Download PDF

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WO2024242053A1
WO2024242053A1 PCT/JP2024/018347 JP2024018347W WO2024242053A1 WO 2024242053 A1 WO2024242053 A1 WO 2024242053A1 JP 2024018347 W JP2024018347 W JP 2024018347W WO 2024242053 A1 WO2024242053 A1 WO 2024242053A1
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hematopoietic stem
mirna
cells
hsa
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博英 齊藤
紘貴 小野
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Kyoto University NUC
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

  • the present invention relates to a method for selecting hematopoietic stem cells using an miRNA-responsive mRNA switch, a method for producing a hematopoietic stem cell population, and a kit for use therein.
  • hematopoietic stem cells are cells that have both the pluripotency to differentiate into all blood cells and the ability to self-replicate.
  • autologous and allogeneic transplants of hematopoietic stem cells are performed to treat leukemia, malignant lymphoma, multiple myeloma, aplastic anemia, and other diseases.
  • Non-Patent Document 1 In a study of gene therapy using hematopoietic stem cells, it has been reported that reporter vectors were used to quantify miRNA activity in hematopoietic stem cells, providing the basis for the future isolation of human hematopoietic stem cells that have miRNA as a functional biomarker (see, for example, Non-Patent Document 1).
  • RNA switch technology which uses mRNA that responds to endogenous miRNA and turns off the translation of the protein it encodes (see, for example, Patent Document 1).
  • Non-Patent Document 1 uses a reporter construct delivered by a vector. Such a reporter construct may be incorporated into the genome of the target cell and cause unexpected genome damage. This may cause problems in clinical application. Furthermore, the method disclosed in Patent Document 1 does not establish a specific process for selecting hematopoietic stem cells with high self-renewal ability and pluripotency.
  • a method for selecting hematopoietic stem cells comprising a step of introducing a first miRNA-responsive mRNA switch into a cell population, wherein the first miRNA-responsive mRNA switch is (a) a nucleic acid sequence specifically recognized by mmu-miR-126a-3p or hsa-miR-126-3p; (b) an mRNA molecule operably linked to a nucleic acid sequence encoding a first marker protein.
  • the step (i) further comprises a step of introducing a second miRNA-responsive mRNA switch into the cell population, the second miRNA-responsive mRNA switch being: (A) a nucleic acid sequence specifically recognized by any one of mouse miRNA sequences selected from mmu-miR-10a-5p, mmu-miR-126a-5p, mmu-miR-130a-3p, mmu-miR-155-5p, mmu-miR-196b-5p, and mmu-miR-223-3p, or any one of human miRNA sequences selected from hsa-miR-10a-5p, hsa-miR-126-5p, hsa-miR-130a-3p, hsa-miR-155-5p, hsa-miR-196b-5p, and hsa-miR-223-3p; (B) an mRNA molecule operably linked to a nucleic acid sequence
  • a method for producing a hematopoietic stem cell population comprising the step of obtaining a selected hematopoietic stem cell population by the selection method according to [1].
  • a kit for selecting hematopoietic stem cells comprising a first miRNA-responsive mRNA switch, comprising:
  • the first miRNA-responsive mRNA switch comprises: (a) a nucleic acid sequence specifically recognized by mmu-miR-126a-3p or hsa-miR-126-3p; (b) an mRNA molecule operably linked to a nucleic acid sequence encoding a first marker protein.
  • a hematopoietic stem cell population that is enriched for cells that are positive for mmu-miR-126a-3p or hsa-miR-126-3p activity and positive for mmu-miR-130a-3p or hsa-miR-130a-3p activity.
  • the hematopoietic stem cell population according to [12] which is hsa-miR-126-3p activity-positive and hsa-miR-130a-3p activity-positive and is for use in human living donor transplantation.
  • the hematopoietic stem cell selection method or the method for producing a selected hematopoietic stem cell population according to the present invention makes it possible to obtain hematopoietic stem cells with high self-renewal ability and pluripotency with high accuracy.
  • FIG. 1 shows an outline of an experiment in which miRNA activity in mouse hematopoietic stem cells cultured in vitro is measured using an “miRNA-responsive mRNA switch” capable of detecting miRNA activity in cells.
  • Figure 2A shows the results of measuring miR-126a-3p activity in mouse hematopoietic stem cells cultured in vitro for two weeks, and is a plot of EGFP vs TagBFP and a histogram with EGFP/TagBFP values on the horizontal axis for cells transfected with control EGFP mRNA that does not contain a miRNA target sequence (top row) and cells transfected with a miR-126a-3p-responsive miRNA switch (bottom row).
  • Figure 3A is a histogram showing the EGFP/TagBFP values on the horizontal axis in cells into which EGFP mRNA or a miR-126a-3p-responsive miRNA switch was introduced together with a miR-126a-3p inhibitor.
  • Figure 3B shows the percentage of miR-126a-3p positive cells in cells that were transfected with a miR-126a-3p inhibitor simultaneously with transfection of EGFP mRNA or a miR-126a-3p-responsive miRNA switch.
  • FIG. 4 shows the results of an analysis of cell surface markers expressed by miR-126a-3p activity-positive and -negative cells.
  • FIG. 5A is a schematic diagram showing that expanded mouse hematopoietic stem cells are contained in the EPCR + KSL fraction.
  • FIG. 5B shows the gating for FACS sorting of the EPCR + KSL fraction.
  • FIG. 6 shows the percentage of miR-126a-3p activity-positive/-negative cells in the EPCR + KSL fraction.
  • FIG. 7 shows an outline of an experiment for evaluating the function of miR-126a-3p activity-positive/-negative cells by transplantation assay.
  • FIG. 8A is a graph showing the progress of peripheral blood chimerism (PB chimerism %) over the number of weeks since transplantation.
  • FIG. 8B is a graph showing peripheral blood chimerism (PB chimerism %) 16 weeks after transplantation in unsorted, miR-126a-3p + cells, and miR-126a-3p ⁇ cells.
  • FIG. 9A illustrates the experimental scheme.
  • FIG. 9B is a graph showing the progress of peripheral blood chimerism (PB chimerism %) over the number of weeks since transplantation.
  • Figure 9C is a graph showing peripheral blood chimerism (PB chimerism %) 16 weeks after transplantation in EPCR + KSL miR-126a-3p + cells and EPCR + KSL miR-126a-3p - cells.
  • FIG. 10 shows gating used to isolate EPCR + KSL, EPCR - KSL, and Sca-1 - Lineage - fractions from mouse hematopoietic stem cells cultured in vitro for 2 weeks in a quantitative experiment of miRNA expression levels in the EPCR + KSL, EPCR - KSL, and Sca-1 - Lineage - fractions using the Small RNA-seq method.
  • FIG. 11A shows the results of quantification of miRNA expression levels in EPCR + KSL, EPCR ⁇ KSL, and Sca-1 ⁇ Lineage ⁇ fractions using the Small RNA-seq method.
  • FIG. 11A shows the results of quantification of miRNA expression levels in EPCR + KSL, EPCR ⁇ KSL, and Sca-1 ⁇ Lineage ⁇ fractions using the Small RNA-seq method.
  • FIG. 11B shows the results of quantification of miRNA expression levels in the EPCR + KSL, EPCR ⁇ KSL, and Sca-1 ⁇ Lineage ⁇ fractions using the Small RNA-seq method.
  • FIG. 12A shows the results of quantification of miRNA expression levels in EPCR + KSL, EPCR ⁇ KSL, and Sca-1 ⁇ Lineage ⁇ fractions using the Small RNA-seq method.
  • FIG. 12B shows the results of quantification of miRNA expression levels in the EPCR + KSL, EPCR ⁇ KSL, and Sca-1 ⁇ Lineage ⁇ fractions using the Small RNA-seq method.
  • Figure 13A shows histograms with the EGFP/TagBFP values on the vertical axis obtained in an experiment to measure the activity of miRNAs selected from miRNA expression level analysis using the Small RNA-seq method, and shows representative histograms of cells into which control EGFP mRNA or the miR-126a-3p-responsive miRNA switch was introduced.
  • Figure 13B shows a bar graph showing the coefficient of variation (CV) of EGFP/TagBFP values in cell populations transfected with each of multiple candidate miRNAs.
  • Figure 13B shows miRNAs that were more highly expressed in EPCR + KSL than in EPCR ⁇ KSL.
  • Figure 13C shows a bar graph showing the coefficient of variation (CV) of EGFP/TagBFP values in cell populations transfected with each of multiple candidate miRNAs.
  • Figure 13C shows miRNAs that were more highly expressed in EPCR ⁇ KSL than in EPCR + KSL.
  • FIG. 14A shows a plot obtained as a result of an experiment of simultaneously measuring the activities of two types of miRNA, with the vertical axis representing the EGFP/TagBFP value and the horizontal axis representing the mT-Sapphire/TagBFP value.
  • FIG. 14B shows a plot obtained as a result of an experiment of simultaneously measuring the activities of two types of miRNA, with the vertical axis representing the EGFP/TagBFP value and the horizontal axis representing the mT-Sapphire/TagBFP value.
  • Figure 15A is a graph comparing the proliferation of cells when an miRNA switch was introduced into cultured hematopoietic stem cells, cells positive or negative for miR-126a-3p activity were sorted, and then the cells were cultured again in medium containing PVA. The graph shows the results of measuring the number of cells recovered from a 250-cell derived culture.
  • FIG. 15B is a graph comparing the proliferation of cells when an miRNA switch was introduced into cultured hematopoietic stem cells, cells positive or negative for miR-126a-3p activity were sorted, and then the cells were cultured again in medium containing PVA. The graph shows the results of measuring the number of cells recovered from a 500-cell derived culture.
  • FIG. 16A is a diagram explaining the experimental scheme for selecting mouse hematopoietic stem cells using the activities of two types of miRNA as indicators.
  • FIG. 16B shows gating for FACS sorting of miR-126a-3p + miR-130a-3p + and miR-126a-3p + miR-130a-3p ⁇ fractions.
  • FIG. 16C shows an outline of an experiment evaluating the function of miR-126a-3p + miR-130a-3p + cells and miR-126a-3p + miR-130a-3p ⁇ cells by transplantation assay.
  • FIG. 16D shows the results of peripheral blood analysis after transplantation.
  • Figure 16E is a graph showing peripheral blood chimerism (PB chimerism %) 12 weeks after transplantation in host mice transplanted with miR-126a-3p + miR-130a-3p + cells or miR-126a-3p + miR-130a-3p - cells.
  • Figure 17A shows an outline of an experiment using miRNA switches to measure miRNA activity in human hematopoietic stem progenitor cells (HSPCs) cultured in vitro.
  • HSPCs human hematopoietic stem progenitor cells
  • FIG. 17B shows the results of an experiment measuring the activity of hsa-miR-126-3p, hsa-miR-126-5p, hsa-miR-130a-3p, hsa-miR-223-3p, or hsa-miR-10a-5p in human hematopoietic stem progenitor cells.
  • FIG. 18A shows an outline of an experiment for simultaneously measuring the activities of two types of miRNAs in human hematopoietic stem and progenitor cells.
  • FIG. 18B shows the results of an experiment in which the activities of two types of miRNAs were simultaneously measured in human hematopoietic stem and progenitor cells.
  • the present invention provides a method for selecting a hematopoietic stem cell population, comprising the following step (i): (i) introducing a first miRNA-responsive mRNA switch into a cell population
  • the method for selecting a hematopoietic stem cell population may optionally comprise the following steps (ii) and/or (iii). (ii) selecting hematopoietic stem cells based on the expression level of a first marker protein encoded by the first miRNA-responsive mRNA; and (iii) culturing the hematopoietic stem cells selected in the step (ii) in a hematopoietic stem cell proliferation medium.
  • the method for producing a hematopoietic stem cell population may optionally comprise, prior to step (i), the following step: Obtaining a hematopoietic stem cell population selected based on cell surface markers
  • the selection of hematopoietic stem cells refers to selecting hematopoietic stem cells from a heterogeneous cell population that includes hematopoietic stem cells and may include other cells, and making them distinguishable from other cells. Therefore, it means that the ratio of hematopoietic stem cells in the cell population after selection is higher than that of the cell population before selection.
  • the method for selecting hematopoietic stem cells according to this embodiment can also be said to be a method for producing a selected hematopoietic stem cell population.
  • the selection of hematopoietic stem cells refers to presenting detectable signal information for hematopoietic stem cells from a heterogeneous cell population that includes hematopoietic stem cells and may include other cells, which is different from other cell types, and in particular, can refer to presenting visually recognizable information.
  • visually recognizable information is not limited to the emission of a signal that can be directly seen by the cells, but refers to information in which the signal emitted by the cells is converted into visually recognizable information using numerical values, charts, images, etc., and refers to information that is visually recognizable by a person skilled in the art.
  • selecting may include, after selection, recognizing a hematopoietic stem cell population, distinguishing a hematopoietic stem cell population, identifying a hematopoietic stem cell population, classifying a hematopoietic stem cell population, isolating a hematopoietic stem cell population, removing cell types other than hematopoietic stem cells, determining whether hematopoietic stem cells are viable or dead, detecting or quantifying specific biological signals of hematopoietic stem cells, and fractionating hematopoietic stem cells based on specific physical or chemical signals.
  • Hematopoietic stem cells are cells that have the pluripotency to differentiate into all blood cells and can self-renew.
  • self-renewal means producing cells with the same functions and properties as the cells themselves through cell division.
  • cells produced by self-renewal of hematopoietic stem cells have the pluripotency to differentiate into all blood cells and the ability to self-renew. Because of these functions, hematopoietic stem cells have the ability to reconstruct bone marrow and continuously produce all blood cells when transplanted into an animal whose bone marrow has been destroyed.
  • Hematopoietic stem cells are generally identified by cell surface markers and cluster of differentiation (CD).
  • Mouse hematopoietic stem cells can generally be determined as CD150 + , CD34 - , CD135 - , CD48 - , EPCR + , c-Kit + , Sca-1 + , and Lineage - .
  • Human hematopoietic stem cells can generally be defined as CD34 + , CD49f + , CD45RA - , CD90/Thy1 + , CD38 low/- , c-Kit -/low and Lineage - . Based on these markers, hematopoietic stem cell populations can be separated by fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • a step of obtaining a hematopoietic stem cell population selected based on a cell surface marker can be carried out before or after the step of introducing the miRNA-responsive mRNA switch into the cell population.
  • the selection of the hematopoietic stem cell population based on a cell surface marker is as described above, and the mouse hematopoietic stem cell population can be obtained by selecting cells having a characteristic selected from CD150 + , CD34 - , CD135 - , CD48 - , EPCR + , c-Kit + , Sca-1 + , and Lineage - by fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • the mouse hematopoietic stem cell population can be defined as a cell population identified using a set of CD150 + , CD48 - , CD34 - , CD135 - , c-Kit + , Sca-1 + , and Lineage -, for example, with reference to Nature. 2018 Jan 24; 553(7689): 418-426.
  • the mouse hematopoietic cell population immediately after collection from bone marrow can be defined as a cell population identified using a set of CD150 + , CD34 - , c-Kit + , Sca-1 + , and Lineage - or a set of CD150 + , CD48 - , c-Kit + , Sca-1 + , and Lineage - .
  • a human hematopoietic stem cell population can be obtained by selecting cells having a characteristic selected from CD34 + , CD49f + , CD45RA - , CD90/Thy1 + , CD38 low/- , c-Kit -/low , and Lineage - by fluorescence activated cell sorting (FACS).
  • the cell surface markers that identify a hematopoietic stem cell population may vary depending on the state of the target cell population, for example, whether or not it has been cultured, and a person skilled in the art can refer to the literature to identify a combination of cell surface markers that select a given hematopoietic stem cell population.
  • Step Step (i) is a step of introducing the first miRNA-responsive mRNA switch into a cell population.
  • the "cell population" that is the target of selection and into which the first miRNA-responsive mRNA switch is introduced refers to a collection of two or more cells that may include hematopoietic stem cells.
  • the cell population may be a cell population collected from a multicellular organism. For example, it may be a cell population collected from umbilical cord blood, peripheral blood, or bone marrow of a mammal, particularly a human or mouse, or a cell population that is further cultured and expanded.
  • the genome-edited cell may be, for example, a cell after genome editing of hematopoietic stem cells immediately after collection from a living body or after a certain period of culture and amplification for the purpose of treating a hereditary blood disease.
  • the selection of the present invention can be carried out for the purpose of selecting cells that retain the function of hematopoietic stem cells from among such cells. It is also possible to introduce a miRNA-responsive mRNA switch into a cell population in which it is unclear whether or not it contains hematopoietic stem cells.
  • the cell population may be, for example, a group of cells induced to differentiate from stem cells into hematopoietic stem cells.
  • stem cells include, but are not limited to, embryonic stem (ES) cells, cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transfer, spermatogonial stem cells ("GS cells”), embryonic germ cells (“EG cells”), and induced pluripotent stem (iPS) cells.
  • ES embryonic stem
  • ntES cloned embryo-derived embryonic stem
  • GS cells spermatogonial stem cells
  • EG cells embryonic germ cells
  • iPS induced pluripotent stem
  • the method of inducing differentiation of hematopoietic stem cells from pluripotent stem cells can be carried out by any known method, such as the method disclosed in Sugimura, R., Jha, D., Han, A. et al. Haematopoietic stem and progenitor cells from human pluripotent stem cells. Nature 545, 432-438 (2017) or Tan, Y.-T., et al. Respecifying human iPSC-derived blood cells into highly engraftable hematopoietic stem and progenitor cells with a single factor. Proc. Natl. Acad. Sci. USA 115, 2180-2185 (2016).
  • the miRNA-responsive mRNA switch introduced into a cell population refers to an mRNA molecule in which the translation of a protein encoded by the mRNA is suppressed or activated in the presence of a specific miRNA activity.
  • miRNA switch may be abbreviated to "miRNA switch.”
  • an miRNA-responsive mRNA switch in which the translation of a protein encoded by the mRNA is suppressed in the presence of a specific miRNA activity and the translation of a protein encoded by the mRNA is performed in the absence of a specific miRNA activity may be referred to as an "OFF switch”
  • an miRNA-responsive mRNA switch in which the translation of a protein encoded by the mRNA is activated in the presence of a specific miRNA activity and the translation of a protein encoded by the mRNA is suppressed in the absence of a specific miRNA activity may be referred to as an "ON switch.”
  • the first miRNA-responsive mRNA switch is an mRNA molecule in which the following nucleic acid sequence (a) and nucleic acid sequence (b) are operably linked, and is preferably an OFF switch.
  • the first miRNA-responsive mRNA switch is hereinafter referred to as the first miRNA switch.
  • the nucleic acid sequence of (a) is preferably located on the 5' or 3' side of the nucleic acid sequence of (b), and (a) and (b) are operably linked.
  • a more specific structure of the first miRNA switch may be a structure in which the 5'-UTR, a coding region encoding a first marker protein, and the 3'-UTR are linked in the 5' to 3' direction of the mRNA molecule.
  • mmu-miR-126a-3p is known as one of the miRNAs that is activated in mouse hematopoietic stem cells, and is represented by ucguaccgugaguaauaaugcg (SEQ ID NO: 1).
  • the miRNA that is activated in human hematopoietic stem cells is hsa-miR-126-3p, and its sequence is identical to that of mmu-miR-126a-3p (SEQ ID NO: 2).
  • "Activated miRNA" in hematopoietic stem cells refers to miRNA that exists in a state where mature miRNA interacts with multiple specific proteins to form an RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • “Mature miRNA” is a single-stranded RNA (20-25 bases) that is generated from pre-miRNA by cleavage by Dicer outside the nucleus, and "pre-miRNA” is generated from pri-miRNA, a single-stranded RNA transcribed from DNA, by partial cleavage by a nuclear enzyme called Drosha.
  • the nucleic acid sequence of (a) is also referred to as "miRNA target sequence" in this specification.
  • the miRNA target sequence is preferably a sequence that is completely complementary to mmu-miR-126a-3p or hsa-miR-126-3p.
  • the miRNA target sequence may have a mismatch with a completely complementary sequence as long as it can be recognized by mmu-miR-126a-3p or hsa-miR-126-3p. It is considered that a mismatch from a sequence that is completely complementary to the miRNA may be about 40 to 50% in terms of the original function in cells in vivo.
  • Such a mismatch is not particularly limited, but examples include a mismatch of 1 base, 2 bases, 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, or 10 bases, or 1%, 5%, 10%, 20%, 30%, or 40% of the entire recognition sequence.
  • the miRNA target sequence on the mRNA possessed by the cell particularly in the portion other than the seed region, i.e., in the 5' region of the target sequence corresponding to about 16 bases on the 3' side of mmu-miR-126a-3p or hsa-miR-126-3p, multiple mismatches may be included, and the seed region portion may contain no mismatches or may contain mismatches of 1, 2, or 3 bases.
  • a miRNA switch having a human miRNA target sequence can be used for introduction into a human cell population
  • a miRNA switch having a mouse miRNA target sequence can be used for introduction into a mouse cell population.
  • the nucleic acid sequence of (b) is a nucleic acid sequence encoding a first marker protein.
  • the marker protein is a protein that is expressed from the miRNA switch, functions as a marker in a cell, and can identify the cell.
  • An example of a marker protein is a protein that can be visualized and quantified by fluorescence, luminescence, color, or by assisting fluorescence, luminescence, or color.
  • fluorescent proteins examples include blue fluorescent proteins such as Sirius and EBFP; cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, and CFP; green fluorescent proteins such as TurboGFP, AcGFP, TagGFP, Azami-Green (e.g., hmAG1), ZsGreen, EmGFP, EGFP, GFP2, and HyPer; and yellow fluorescent proteins such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, and mBanana.
  • blue fluorescent proteins such as Sirius and EBFP
  • cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, and CFP
  • green fluorescent proteins such as TurboGFP, AcGFP, TagGFP, Azami-Green (e.g., hmAG1), ZsGreen, EmGFP,
  • Photoproteins include, but are not limited to, orange fluorescent proteins such as KusabiraOrange (e.g., hmKO2) and mOrange; red fluorescent proteins such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, and mStrawberry; and near-infrared fluorescent proteins such as TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed (e.g., hdKeimaRed), mRasberry, and mPlum.
  • orange fluorescent proteins such as KusabiraOrange (e.g., hmKO2) and mOrange
  • red fluorescent proteins such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, and mStrawberry
  • near-infrared fluorescent proteins such as TurboFP60
  • a luminescent protein is aequorin, but is not limited to this.
  • an example of a protein that assists fluorescence, luminescence, or color development is an enzyme that decomposes a fluorescent, luminescent, or color precursor, such as luciferase, phosphatase, peroxidase, beta-galactosidase, beta-lactamase, etc., but is not limited to these.
  • the desired cell can be identified by contacting the cell with the corresponding precursor, or by introducing the corresponding precursor into the cell.
  • marker proteins include proteins that directly affect cell function. Examples include, but are not limited to, cell proliferation proteins, cell death proteins, cell signaling factors, drug resistance genes, transcription factors, translation factors, differentiation factors, reprogramming inducers, RNA-binding protein factors, chromatin regulators, and membrane proteins.
  • cell proliferation proteins function as markers by proliferating only cells that express them and identifying the proliferated cells.
  • Cell death proteins cause cell death in the cells that express them, killing the cells themselves and functioning as markers indicating the live or dead state of the cells. Examples of cell death proteins include, for example, Barnase, an RNA-degrading enzyme, and Bax or Bim, which are apoptosis-promoting proteins.
  • Cell signaling factors are proteins that function as markers by identifying the signals that cells that express them emit specific biological signals, and examples of such proteins include CD4, a transmembrane protein, low affinity nerve growth factor receptor (LNGFR), and its loss-of-function mutant ( ⁇ LNGFR). These cell signaling factors can be advantageously used as marker proteins for the purpose of fluorescently labeling cells using antibodies or biotin, and for the purpose of cell separation using magnetic beads.
  • LNGFR low affinity nerve growth factor receptor
  • ⁇ LNGFR loss-of-function mutant
  • the first miRNA switch has a nucleic acid sequence of (a) located on the 5' side of a nucleic acid sequence of (b).
  • the 5'-UTR may have a structure in which a [Cap structure or Cap analog] and a [nucleic acid sequence of (a)] are linked in this order from the 5' end.
  • the Cap structure may be 7-methylguanosine 5'-phosphate
  • the Cap analog is a modified structure that is recognized by the translation initiation factor eIF4E in the same manner as the Cap structure, and examples of such modified structures include, but are not limited to, Anti-Reverse Cap Analog (ARCA) manufactured by Ambion, m7G(5')ppp(5')G RNA Cap Structure Analog manufactured by New England Biolabs, and CleanCap (registered trademark) manufactured by TriLink.
  • ARCA Anti-Reverse Cap Analog
  • m7G(5')ppp(5')G RNA Cap Structure Analog manufactured by New England Biolabs and CleanCap (registered trademark) manufactured by TriLink.
  • CleanCap® AG and AU cap mRNA with the natural 5'-N7-methylguanosine 2'-O-methyl structure
  • CleanCap® AG (3'-OMe) caps mRNA with the 5'-N7-methyl-3'-O-methylguanosine structure found in mRNA capped using the ARCA method.
  • Cap analogs may also be other modified structures recognized by translation initiation factors.
  • the 3' side of the Cap structure or Cap analog, the 5' side of the nucleic acid sequence of (a) may contain an arbitrary nucleic acid sequence of, for example, about 0 to 50 bases, preferably about 0 to 30 bases. At least one nucleic acid sequence of (a) may be contained, but 2 repeats, 3 repeats, 4 repeats, or more of the nucleic acid sequence of (a) may be contained in the 5'-UTR.
  • the 3' end side of the 5'-UTR which is the 3' side of the nucleic acid sequence of (a), may contain an arbitrary nucleic acid sequence of, for example, about 0 to 50 bases, preferably about 10 to 30 bases.
  • nucleic acid sequences are preferably nucleic acid sequences that do not form secondary structures.
  • the 5'-UTR does not have an AUG, which is the start codon.
  • a frameshift can be avoided by adding one or two bases to the end of the sequence.
  • a stop codon sequence may be added outside the nucleic acid sequence of (a) counted in 3-base units from the AUG.
  • one or more of the AUG bases can be converted to any base as long as it does not affect the interaction with the protein.
  • the coding region of the first miRNA switch comprises the nucleic acid sequence of (b).
  • the 3'-UTR of the first miRNA switch includes a PolyA tail.
  • the total length of As in the PolyA tail may be 50 mer or more, and may contain nucleic acid bases other than As along the way.
  • the 5' side of the PolyA tail may also contain the nucleic acid sequence of (a).
  • the first miRNA switch has a nucleic acid sequence (a) present on the 3' side of a nucleic acid sequence (b).
  • the 5'-UTR is provided with a [Cap structure or Cap analog] at the 5' end and is composed of a nucleic acid sequence having a total base length of about 30 to 100.
  • the nucleic acid sequence constituting the 5'-UTR in this case can be determined in the same manner as the arbitrary nucleic acid sequence of the first embodiment.
  • the coding region of the first mRNA according to the second embodiment may be the same as the coding region of the first miRNA switch according to the first embodiment.
  • the 3'-UTR of the first miRNA switch according to the second embodiment may have a structure in which the [nucleic acid sequence of (a)] and the [Poly A tail] are linked in this order from the 5' end.
  • the 3'-UTR of the first miRNA switch according to the second embodiment may have a structure in which the [nucleic acid sequence of (a)] is inserted into the Poly A tail.
  • the first miRNA switch may have a structure that combines the first and second embodiments. In this case, both the 5'-UTR and the 3'-UTR have the nucleic acid sequence (a).
  • the first miRNA switch may have a modified sugar residue (ribose) of each nucleotide for the purpose of reducing cytotoxicity, etc.
  • the sites of modification in the sugar residue include those in which the hydroxyl groups or hydrogen atoms at the 2', 3', and/or 4' positions of the sugar residue are replaced with other atoms.
  • the types of modification include fluorination, alkoxylation (e.g., methoxylation, ethoxylation), O-allylation, S-alkylation (e.g., S-methylation, S-ethylation), S-allylation, and amination (e.g., -NH2 ).
  • the sugar residues of the first miRNA switch can also be bridged at the 2' and 4' positions to form a BNA (bridged nucleic acid) (LNA: linked nucleic acid).
  • BNA bridged nucleic acid
  • LNA linked nucleic acid
  • the first miRNA switch may also have a nucleic acid base (e.g., purine, pyrimidine) modified (e.g., chemically substituted). Examples of such modifications include modification of the 5-position pyrimidine, modification of the 6- and/or 8-position purine, modification with an exocyclic amine, substitution with 4-thiouridine, and substitution with 5-bromo- or 5-iodo-uracil.
  • the first miRNA switch may contain modified bases such as pseudouridine ( ⁇ ), N1-methylpseudouridine (N1m ⁇ ), and 5-methylcytidine (5mC) instead of normal uridine and cytidine.
  • the positions of the modified bases can be all or part of the uridine and cytidine independently, and if part of the modified bases, they can be at random positions in any proportion.
  • the phosphate group (e.g., terminal phosphate residue) contained in the first miRNA switch may be modified to improve resistance to nucleases and hydrolysis.
  • the P(O)O group which is a phosphate group, may be substituted with P(O)S (thioate), P(S)S (dithioate), P(O) NR2 (amidate), P(O)R, R(O)OR', CO, or CH2 (formacetal), or 3'-amine (-NH- CH2 - CH2- ) (wherein each R or R' is independently H, or substituted or unsubstituted alkyl (e.g., methyl, ethyl)).
  • linking groups include -O-, -N-, or -S-, and the adjacent nucleotide may be bound through these linking groups.
  • the molecular structure and nucleic acid sequence of the first miRNA switch have been determined as described above, a person skilled in the art can synthesize it by any method known in genetic engineering. For example, it can be obtained as a synthetic mRNA molecule by an in vitro synthesis method using a template DNA containing a promoter sequence as a template.
  • One advantage of the present invention is that a synthetic mRNA molecule as designed can be obtained by a simple method.
  • the introduction of the first miRNA switch into the cell population can be carried out using any method commonly used for introducing RNA molecules into cells.
  • methods for directly introducing RNA molecules into cells include lipofection, liposome, electroporation, calcium phosphate coprecipitation, DEAE-dextran, microinjection, and gene gun methods.
  • the advantage of introducing synthetic RNA molecules is that they are not integrated into the genome, and the cells after the introduction of the mRNA switch are easy to use for medical applications.
  • the amount and ratio of the first miRNA switch introduced into the cell population are not limited to a specific amount.
  • the first miRNA switch introduced into the cell population suppresses the translation of the marker protein in cells in which mmu-miR-126a-3p or hsa-miR-126-3p is activated, and allows the translation of the marker protein in cells in which mmu-miR-126a-3p or hsa-miR-126-3p is not activated.
  • the selection step which is an optional step following the introduction step, is a step of selecting hematopoietic stem cells based on the expression level of the first marker protein.
  • the expression level of the marker protein may include information on the presence or absence of translation of the marker protein, or quantitative information such as the expression level due to translation.
  • the marker protein is a protein that can be visualized and quantified by fluorescence, luminescence, color, or the assistance of fluorescence, luminescence, or color
  • specific cells can be selected by isolating them using, for example, a flow cytometer.
  • the marker protein is a protein that directly affects the function of a cell, the presence or absence of translation of the marker protein can be detected as a change in the function of the cell.
  • a cell population consisting of a plurality of cells into which an mmu-miR-126a-3p-responsive mRNA switch or an hsa-miR-126-3p-responsive mRNA switch has been introduced can be separated into two populations: cells with mmu-miR-126a-3p or hsa-miR-126-3p activity (mmu-miR-126a-3p or hsa-miR-126-3p positive cells) and cells with little or no mmu-miR-126a-3p or hsa-miR-126-3p activity (mmu-miR-126a-3p or hsa-miR-126-3p negative cells) based on the translation information of the marker protein, and these cell populations with different characteristics can be identified.
  • the marker protein is an apoptosis protein
  • the apoptosis protein is translated and expressed in cells in which mmu-miR-126a-3p or hsa-miR-126-3p is not activated. This makes it possible to kill cells in which mmu-miR-126a-3p or hsa-miR-126-3p is not activated, i.e., cells that are presumed not to be hematopoietic stem cells, and to select hematopoietic stem cells without using equipment such as a cell sorter.
  • the selection process can generally be performed using a first miRNA switch based on the expression of a first marker protein, but selection can also be performed using an additional miRNA ON switch.
  • the first miRNA switch may be an OFF switch (hereinafter referred to as a lethal OFF switch) that has a miRNA target sequence that specifically recognizes mmu-miR-126a-3p or hsa-miR-126-3p and encodes a lethal protein as the first marker protein.
  • the additional miRNA ON switch is an mRNA molecule (hereinafter referred to as an anti-lethal ON switch) that includes a miRNA target sequence that specifically recognizes mmu-miR-126a-3p or hsa-miR-126-3p and a nucleic acid sequence that codes for a protein, and in cells in which these miRNAs are activated, the expression of the encoded protein is activated.
  • an anti-lethal ON switch an mRNA molecule that includes a miRNA target sequence that specifically recognizes mmu-miR-126a-3p or hsa-miR-126-3p and a nucleic acid sequence that codes for a protein, and in cells in which these miRNAs are activated, the expression of the encoded protein is activated.
  • the antilethal ON switch is an ON switch mRNA, and is an mRNA molecule comprising (a) a nucleic acid sequence encoding an antilethal gene that inactivates a lethal gene, (b) a PolyA tail provided on the 3' side of (a), (c) a nucleic acid sequence specifically recognized by mmu-miR-126a-3p or hsa-miR-126-3p provided on the 3' side of (b), and (d) a translational repression sequence provided on the 3' side of (c).
  • the translational repression sequence is a nucleic acid sequence consisting of five or more bases that can suppress translation of the antilethal gene in the absence of a miRNA that specifically recognizes the nucleic acid sequence of (c).
  • the translation inhibitory sequence may be, for example, a nucleic acid sequence of 5 to 10 bases having the same nucleic acid base, a nucleic acid sequence of 10 to 20 bases in which two or more types of nucleic acids with different nucleic acid bases are consecutively linked, a nucleic acid sequence of 30 to 50 bases in which two or more types of nucleic acids with different nucleic acid bases are consecutively linked, a nucleic acid sequence containing a repeat of a specific nucleic acid sequence of about 3 to 10 bases, a nucleic acid sequence of 20 or more bases that specifically recognizes the PolyA tail, or a nucleic acid sequence that specifically recognizes the 5'-UTR.
  • nucleic acid sequence of 5 or more bases may be a nucleic acid sequence of 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more, 300 or more, 500 or more, 1000 or more, or 1500 or more bases, depending on the case.
  • the structure of a general miRNA ON switch and a method for producing the same are disclosed in the prior art WO2018/003779 by the present inventors, and a person skilled in the art can produce a miRNA ON switch based on the disclosure.
  • a lethal protein and an anti-lethal protein is not particularly limited.
  • a lethal protein that is an RNA-degrading enzyme, barnase, derived from Bacillus amyloliquefaciens can be combined with an anti-lethal protein, barstar, a protein that inactivates RNA-degrading enzymes, derived from Bacillus amyloliquefaciens.
  • apoptosis-inducing proteins such as Bax (BCL2 associated X, apoptosis regulator) and Bim (Bcl-2 interacting mediator, also known as BCL2L11, BCL2 like 11
  • Bcl-2 B-cell/CLL lymphoma 2, also known as BCL2 apoptosis regulator
  • Bcl-xL B-cell lymphoma-extra large, also known as BCL2L1, BCL2 like 1
  • Bcl-2 B-cell lymphoma-extra large, also known as BCL2L1, BCL2 like 1
  • Bcl-2 B-cell lymphoma-extra large, also known as BCL2L1, BCL2 like 1
  • the first miRNA-responsive mRNA, which is the lethal OFF switch, and the antilethal ON switch are introduced into the cell substantially simultaneously in the introduction step.
  • the expression of the lethal protein encoded by the lethal OFF switch is suppressed.
  • the expression of the antilethal protein encoded by the antilethal ON switch is activated.
  • the hematopoietic stem cell population can be distinguished and selected from non-hematopoietic stem cell populations by translation of a marker protein selected to suit the purpose of selection.
  • the culturing step which is an optional step following the selection step (ii), is a step of expanding the selected hematopoietic stem cells without substantially differentiating them.
  • the selected hematopoietic stem cells are expanded by culturing them in a hematopoietic stem cell proliferation medium.
  • the hematopoietic stem cell proliferation medium is not particularly limited as long as it is a medium that can proliferate hematopoietic stem cells without differentiating them.
  • a medium containing polyvinyl alcohol and not containing serum components or albumin can be used.
  • a commercially available product can also be used as the hematopoietic stem cell proliferation medium.
  • the culture period can be from about 7 days to about 2 months.
  • step (iii) By carrying out step (iii) following step (ii), the highly pure hematopoietic stem cell population can be further expanded. Therefore, compared to conventional techniques, a large amount of a highly pure hematopoietic stem cell population can be obtained more efficiently. Since hematopoietic stem cells are undifferentiated cells and prone to fluctuations, there is a great advantage to expanding them at a stage immediately prior to clinical use, such as transplantation.
  • the method for selecting hematopoietic stem cells or the method for producing a hematopoietic stem cell population according to the first embodiment of the present invention makes it possible to obtain a cell population with a high purity of hematopoietic stem cells, compared to conventional methods using cell surface markers.
  • it is a method for introducing RNA molecules, there is no risk of genome damage, making it safer than introducing a DNA construct.
  • the present invention provides a method for producing a hematopoietic stem cell population, comprising the following step (i): (i) introducing a first miRNA-responsive mRNA switch and a second miRNA-responsive mRNA switch into a cell population;
  • the method may include steps (ii) and/or (iii) below.
  • steps (iii) culturing the hematopoietic stem cells selected in step (ii) in a hematopoietic stem cell proliferation medium may include steps (ii) and/or (iii) below.
  • the method for selecting hematopoietic stem cells according to the second embodiment relates to a method for selecting hematopoietic stem cells using at least two types of miRNA-responsive mRNA switches.
  • the first miRNA-responsive mRNA used in the second embodiment may be the same as the first miRNA-responsive mRNA described in the first embodiment.
  • the second miRNA-responsive mRNA switch is (A) a nucleic acid sequence specifically recognized by any one of mouse miRNA sequences selected from mmu-miR-10a-5p, mmu-miR-126a-5p, mmu-miR-130a-3p, mmu-miR-155-5p, mmu-miR-196b-5p, and mmu-miR-223-3p, or any one of human miRNA sequences selected from hsa-miR-10a-5p, hsa-miR-126-5p, hsa-miR-130a-3p, hsa-miR-155-5p, hsa-miR-196b-5p, and hsa-miR-223-3p; (B) An mRNA molecule operably linked to a nucleic acid sequence encoding a second marker protein, the second marker protein being different from the first marker protein.
  • the second miRNA-responsive mRNA switch is referred to as a second miRNA switch.
  • a miRNA switch having a human miRNA target sequence can be used for introduction into a human cell population
  • a miRNA switch having a mouse miRNA target sequence can be used for introduction into a mouse cell population.
  • Step (i) is a step of introducing the first miRNA switch and the second miRNA switch into a cell population.
  • the definition of the cell population to be introduced may be the same as in the first embodiment.
  • the sequence of (A) can be designed in the same manner as the first miRNA switch.
  • the target sequence of the miRNA is preferably completely complementary to any of the six miRNAs described above, but may have a degree of mismatch as defined in the first embodiment.
  • the sequence encoding the marker protein (B) can be selected from the same options as those for (b) in the first embodiment.
  • the second marker protein is a protein different from the first marker protein.
  • the 5'-UTR, coding region, and 3'-UTR of the second miRNA switch can be designed in the same manner as the first miRNA switch in the first embodiment, except that the sequences (A) and (B) are different from the sequences (a) and (b), and modifications of sugar residues, cross-linked structures, and phosphate groups may also be similar to those in the first embodiment.
  • any method generally used for introducing an RNA molecule into a cell, as described in the first embodiment, can be used. It is preferable to co-introduce the first miRNA switch and the second miRNA switch into the cell population. This is because the activity ratio of proteins expressed from two or more co-introduced mRNAs is constant within the cell population.
  • the selection step which is an optional step following the introduction step, is a step of selecting hematopoietic stem cells based on the translation of the first and second marker proteins.
  • a lethal protein can be used as a marker protein, and selection can be performed using an anti-lethal ON switch in combination.
  • the first and second marker proteins may be the same lethal gene or different lethal genes.
  • a cell population consisting of multiple cells into which two types of switches have been introduced can be separated and identified into four different populations based on the translation information of the first and second marker proteins. More specifically, mmu-miR-126a-3p or hsa-miR-126-3p is defined as the first miRNA, and any one of mouse miRNA sequences selected from mmu-miR-10a-5p, mmu-miR-126a-5p, mmu-miR-130a-3p, mmu-miR-155-5p, mmu-miR-196b-5p, and mmu-miR-223-3p, or hsa-miR-10a-5p, hsa-miR-126-5p, hsa-miR-130a-3p, hsa-miR-155-5p, hsa-miR-196b-5p, When any one of human miRNA sequences selected from miR-1
  • Step (iii) Culturing step using hematopoietic stem cell proliferation medium
  • the culturing step which is an optional step following the selection step, is a step of expanding the selected hematopoietic stem cells without substantially differentiating them.
  • Step (iii) according to the second embodiment can be carried out in the same manner as step (iii) according to the first embodiment.
  • the hematopoietic stem cell population obtained through steps (i), (ii), and optionally step (iii) of this embodiment can also be called a hematopoietic stem cell population in which cells that are positive for the first miRNA activity and positive or negative for the second miRNA activity are enriched.
  • the definitions of the first miRNA and the second miRNA are as explained in the selection step (ii) above.
  • the second miRNA is mmu-miR-223-3p (mouse) or hsa-miR-223-3p (human)
  • a cell population in which cells that are positive for the first miRNA activity and negative for the second miRNA activity are enriched (first miRNA single positive: SP) can be called a hematopoietic stem cell population.
  • mmu-miR-223-3p or hsa-miR-223-3p is low in hematopoietic stem cells.
  • the second miRNA is mmu-miR-10a-5p, mmu-miR-126a-5p, mmu-miR-130a-3p, mmu-miR-155-5p, or mmu-miR-196b-5p (mouse), or hsa-miR-10a-5p, hsa-miR-126-5p, hsa-miR-130a-3p, hsa-miR-155-5p, or hsa-miR-196b-5p (human), a cell population in which cells positive for the first miRNA activity and positive for the second miRNA activity are enriched (double positive: DP) can be said to be a hematopoietic stem cell population.
  • the hematopoietic stem cell population is a mouse or human hematopoietic stem cell population that is positive for mmu-miR-126a-3p or hsa-miR-126-3p activity and is enriched for cells that are positive for mmu-miR-130a-3p or hsa-miR-130a-3p activity, and most preferably, is a human hematopoietic stem cell population that is positive for hsa-miR-126-3p activity and is enriched for cells that are positive for hsa-miR-130a-3p activity.
  • Such a cell population has different characteristics from other cell populations and can be used for transplantation in living organisms.
  • a mouse or human hematopoietic stem cell population that is positive for miR-126a-3p activity and is enriched for cells that are positive for miR-130a-3p activity exhibits a high engraftment rate and is capable of producing blood even after transplantation into a living organism. Therefore, this hematopoietic stem cell population is suitable for transplantation into patients with blood-related diseases.
  • the method for selecting hematopoietic stem cells or the method for producing a hematopoietic stem cell population according to the second embodiment of the present invention enables more detailed selection that focuses on the activity of different miRNAs compared to the method according to the first embodiment.
  • the present invention relates to a kit for selecting hematopoietic stem cells.
  • the selection kit includes a first miRNA-responsive mRNA switch and optionally includes a second miRNA-responsive mRNA switch.
  • the first miRNA-responsive mRNA switch and the second miRNA-responsive mRNA switch may be the miRNA molecule described in the previous selection method.
  • the kit may include a medium used in selecting hematopoietic stem cells, a hematopoietic stem cell growth medium such as a medium containing PVA used in expanding hematopoietic stem cells, and/or an instruction manual for handling the selection kit.
  • hematopoietic stem cells can be safely and accurately selected and used for transplantation, etc.
  • Bone marrow was harvested from mouse tibias, femurs, and pelvises. Bone marrow cells were stained with APC-conjugated c-Kit antibody (Thermo Fisher Scientific, 17-1171-83) for 30 min, washed, and then treated with Anti-APC MicroBeads (Miltenyi Biotec, 130-090-855) for 15 min. c-Kit positive cells were then enriched using an LS column (Miltenyi Biotec, 130-042-401) and a QuadroMACS Separator (Miltenyi Biotec).
  • Enriched c-Kit positive cells were stained with Lineage antibody cocktail [biotinylated CD4 antibody (Thermo Fisher Scientific, 13-0042-81), biotinylated CD8 (Thermo Fisher Scientific, 13-0081-81), biotinylated CD45R (Thermo Fisher Scientific, 13-0452-81), biotinylated TER119 (Thermo Fisher Scientific, 13-5921-81), biotinylated Ly-6G/Ly-6C (Thermo Fisher Scientific, 13-5931-81), biotinylated CD11b (Thermo Fisher Scientific, 13-0112-81), and biotinylated CD127 antibody (Thermo Fisher Scientific, 13-1271-81)] for 30 min.
  • Lineage antibody cocktail [biotinylated CD4 antibody (Thermo Fisher Scientific, 13-0042-81), biotinylated CD8 (Thermo Fisher Scientific, 13-0081-81), biotinylated CD
  • the cells were cultured in a fibronectin-coated 96-well plate (Corning, 354409) containing 200 ⁇ L of medium in a 37°C, 5% CO2 incubator. The medium was replaced 5 days after sorting, and thereafter, the medium was replaced every 2 days.
  • the human hematopoietic stem cells used were commercially available human umbilical cord blood-derived CD34 positive cells (STEMCELL Technologies, 70008.2). Cells were cultured in IMDM w/o Glutamine (Sigma, 3390-500ML) containing 1X Penicillin-Streptomycin solution (Wako, 168-23191), 1X ITS-X (Thermo Fisher Scientific, 51500056), 4 mM L-Glutamine (Thermo Fisher Scientific, 25030149), 0.1 mg/mL Soluplus (BASF, 50539897), 1 ⁇ M UM729 (STEMCELL Technologies, 72332), 5 ⁇ M 740 YP (MedChemExpress, HY-P0175), 0.2 ⁇ M butyzamide (Shionogi), and 10 ng/mL Human Flt3-Ligand (Shenandoah Biotechnology, 100-21-10UG). The cells were cultured in a Corn
  • Template DNA for in vitro transcription was synthesized by PCR using a plasmid vector containing the 5'-UTR, an ORF encoding a fluorescent protein, and the 3'-UTR.
  • the PCR product was treated with Dpn I (TOYOBO, DPN-101) and purified using Monarch PCR & DNA Cleanup Kit (NEB, T1030).
  • mRNA was transcribed using MEGAscript T7 Transcription Kit (Thermo Fisher Scientific, AM1334).
  • the reaction mixture was incubated at 37 °C for 10 h with 1 ⁇ Reaction Buffer (Thermo Fisher Scientific), 6 mM CleanCap AG (3' Ome) (TriLink Biotechnologies, N-7413), 7.5 mM GTP (Thermo Fisher Scientific), 7.5 mM ATP (Thermo Fisher Scientific), 7.5 mM CTP (Thermo Fisher Scientific), 7.5 mM N1- ⁇ TP (TriLink Biotechnologies, N-1081), 1 ⁇ T7 Enzyme mix (Thermo Fisher Scientific), and IVT template.
  • TURBO DNase Thermo Fisher Scientific was added and incubated at 37 °C for 30 min, and purified using Monarch RNA Cleanup Kit (NEB, T2040).
  • Dephosphorylation was then performed by adding Antarctic Phosphatase (NEB, M0289) and Antarctic Phosphatase Buffer (NEB) and incubating for 30 min. Finally, the product was purified using Monarch RNA Cleanup Kit (NEB, T2040).
  • the primers used for the synthesis of mRNA are shown in Table 2.
  • the sequences of the plasmid vectors used as templates are shown in SEQ ID NOs: 17 to 40 in the sequence listing.
  • the cells were transferred to a Fibronectin-coated 24-well plate (Corning, 354411) containing 0.5-1 mL of medium and cultured in an incubator at 37 °C and 5% CO2 .
  • miRNA inhibitors were purchased from GeneDesign. In experiments using miRNA inhibitors, 12.5 pmol of miRNA inhibitor per 1 ⁇ 105 cells was electroporated simultaneously with mRNA.
  • the cells were transferred to a Corning CellBIND 24-well plate (Corning, 3337) containing 1 mL of medium and cultured in a 37°C, 5% CO2 incubator.
  • miRNA inhibitors were purchased from GeneDesign. In experiments using miRNA inhibitors, 20 pmol or 40 pmol of miRNA inhibitors were electroporated simultaneously with mRNA.
  • the cells were stained with Lineage antibody cocktail [biotinylated CD4 antibody (Thermo Fisher Scientific, 13-0042-81), biotinylated CD8 (Thermo Fisher Scientific, 13-0081-81), biotinylated CD45R (Thermo Fisher Scientific, 13-0452-81), biotinylated TER119 (Thermo Fisher Scientific, 13-5921-81), biotinylated Ly-6G/Ly-6C (Thermo Fisher Scientific, 13-5931-81), and biotinylated CD127 antibody (Thermo Fisher Scientific, 13-1271-81)] for 30 min.
  • Lineage antibody cocktail [biotinylated CD4 antibody (Thermo Fisher Scientific, 13-0042-81), biotinylated CD8 (Thermo Fisher Scientific, 13-0081-81), biotinylated CD45R (Thermo Fisher Scientific, 13-0452-81), biotinylated TER119 (Thermo Fisher Scientific
  • CD45.1 mouse-derived hematopoietic stem cells were cultured and the desired fraction was sorted using a FACS Aria II (BD).
  • Host CD45.2 mice were lethally irradiated (4.9 Gy per dose, 2 doses with a 4-hour interval) using Gamma Cell 40 (Best Theratronics), and then hematopoietic stem cells and 5 ⁇ 105 bone marrow cells derived from CD45.1/CD45.2 mice were simultaneously transplanted into the tail vein of the host.
  • the reaction mixture containing 0.25 ⁇ M 3' randomized adapter (5'- App/NNNNTGGAATTCTCGGGTGCCAAGG/ddC -3', purchased from IDT (sequence number 41)), 200 units of T4 RNA ligase 2 truncated KQ (NEB, M0373), 1X T4 RNA ligase reaction buffer (NEB), 20% PEG 8000 (NEB), 10 units of SUPERase In RNase Inhibitor (Thermo Fisher Scientific, AM2694) and total RNA was incubated at 25°C for 16 hours to ligate the 3' random adapter to the small RNA.
  • the ligated RNA was then electrophoresed on a 15% denaturing polyacrylamide gel, and the unreacted adapter was removed by cutting out the 40-55 base fraction.
  • the excised gel pieces were placed in a Gel breaker tube (Cosmo Bio) and crushed by centrifugation at 20,400 g for 10 minutes.
  • the crushed gel pieces were soaked in 0.3 M NaCl (Nacalai tesque, 06900-14) and stirred at 25°C at 1,500 rpm overnight to elute small RNAs.
  • the eluted RNA was purified by ethanol precipitation using sodium acetate (Nippon Gene, 316-90081) and GlycoBlue Coprecipitant (Thermo Fisher Scientific, AM9515).
  • the reaction mixture contained 0.18 ⁇ M 5' randomized adapter (5'- G*T*T*C*A*G*A*G*T*T*C*T*A*C*A*G*T*C*C*G*A*C*rGrArUrCr(N:25252525)r(N)r(N)r(N) -3', purchased from IDT (SEQ ID NO: 42), 14 units of T4 RNA ligase 1 (NEB, M0204), 1X T4 RNA ligase reaction buffer (NEB), 20% PEG 8000 (NEB), 1 mM ATP (NEB), 14 units of SUPERase ⁇ In RNase Inhibitor (Thermo Fisher Scientific, AM2694) and purified RNA.
  • the adapter was ligated at 37°C for 1 hour, and then the enzyme was inactivated by incubation at 65°C for 15 minutes. Then, cDNA was synthesized by incubating the reaction mixture containing 200 units of SuperScript III reverse transcriptase (Thermo Fisher Scientific, 18080085), 1X first-strand buffer (Thermo Fisher Scientific), 0.2 ⁇ M RT primer (RTP, GCCTTGGCACCCGAGAATTCCA (SEQ ID NO: 43)), 0.5 mM dNTPs (included in TruSeq Small RNA kit, Illumina), 5 mM DTT (Thermo Fisher Scientific) and adapter-ligated RNA at 37°C for 1 h and 70°C for 15 min.
  • SuperScript III reverse transcriptase Thermo Fisher Scientific, 18080085
  • 1X first-strand buffer Thermo Fisher Scientific
  • 0.2 ⁇ M RT primer RTP, GCCTTGGCACCCGAGAATTCCA (SEQ ID NO:
  • the reaction mixture contained cDNA, 1 unit of Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, F530), 1X Phusion HF buffer (Thermo Fisher Scientific), 0.5 ⁇ M primers (RP1 forward primer and RPI reverse primer from TruSeq Small RNA kit from Illumina), and 0.2 mM dNTPs (TAKARA) and was reacted with the following settings to amplify the cDNA library.
  • PCR reaction cycle 98°C 30 sec, (98°C 10 sec, 60°C 30 sec, 72°C 15 sec), 72°C 10 min, (repeated 15 times in parentheses).
  • the amplified cDNA library was electrophoresed on a 6% non-denaturing polyacrylamide gel, and products of about 150 bp were excised.
  • the cDNA was purified according to the ethanol precipitation method described above.
  • the purified cDNA was used as a template in a reaction solution containing 1 unit of Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, F530), 1X Phusion HF buffer (Thermo Fisher Scientific), 0.5 ⁇ M primers (RP1 forward primer and RPI reverse primer from TruSeq Small RNA kit from Illumina), and 0.2 mM dNTPs (TAKARA) to amplify the cDNA library again under the following conditions: PCR reaction cycle: 98°C 30 sec, (98°C 10 sec, 60°C 30 sec, 72°C 15 sec), 72°C 10 min, (repeated 10 times in parentheses).
  • the amplified cDNA library was electrophoresed on a 6% non-denaturing polyacrylamide gel, and products of about 150 bp were excised.
  • the cDNA was purified according to the ethanol precipitation method described above.
  • the size of the cDNA library was confirmed using an Agilent 2100 Bioanalyzer (Agilent).
  • the concentration of the cDNA library was quantified using KAPA Library Quantification Kits (KAPA Biosystems). Equal amounts of the cDNA libraries were mixed and pooled, and sequencing was performed on the NextSeq 500 platform (Illumina). NextSeq 500/550 High Output Kit v2.5 (75 cycles, Illumina) was used.
  • FIG. 1 shows an outline of an experiment to measure miRNA activity in mouse hematopoietic stem cells cultured in vitro using a "miRNA-responsive mRNA switch" capable of detecting miRNA activity in cells.
  • hematopoietic stem cells Lineage - c-Kit + Sca-1 + CD150 + CD34 -
  • FACS fluorescence-activated cell sorting
  • a miR-126a-3p-responsive miRNA switch encoding EGFP and mRNA encoding TagBFP were introduced into the cells cultured for 2 weeks by electroporation. 24 hours after the introduction of the mRNA, the cells were analyzed using a flow cytometer.
  • a mouse miRNA-responsive switch was used as the miRNA-responsive switch. In the following examples, unless mmu- or hsa- is specified, it refers to mouse miRNA.
  • Figure 2A shows the results of measuring miR-126a-3p activity in mouse hematopoietic stem cells cultured in vitro for 2 weeks, with EGFP vs TagBFP plots and histograms of EGFP/TagBFP values on the horizontal axis for cells transfected with control EGFP mRNA that does not contain a miRNA target sequence (top row) and cells transfected with the miR-126a-3p-responsive miRNA switch (bottom row).
  • EGFP/TagBFP is the EGFP fluorescence intensity divided by the TagBFP fluorescence intensity to correct for differences in electroporation efficiency between cells. EGFP expression is suppressed in a portion of cells transfected with the miR-126a-3p-responsive miRNA switch.
  • a gate was set to define miR-126a-3p-positive cells (cells in which EGFP expression is suppressed) based on the histogram of cells transfected with control EGFP mRNA.
  • the percentage of miR-126a-3p-positive cells was reduced by the introduction of the miR-126a-3p inhibitor.
  • n 2.
  • the black bar indicates the average value of two independent experiments.
  • Cells transfected with control EGFP mRNA or the miR-126a-3p-responsive miRNA switch were stained with fluorescent antibodies and analyzed by flow cytometry. Sca-1 vs c-Kit plots and FSC-A vs EPCR plots for the miR-126a-3p-responsive miRNA switch-transfected cells are shown in Figure 4.
  • the miR-126a-3p activity-positive cells were enriched in fractions that are considered to be enriched for hematopoietic stem cells, as defined by the expression patterns of cell surface antigens, i.e., c-Kit + Sca-1 + Lineage - : KSL and EPCR + KSL fractions, compared with the unseparated bulk population and the miR-126a-3p activity-negative cells.
  • mouse HSCs expanded in PVA-containing medium were found to be contained in the c-Kit + Sca-1 + Lineage - EPCR + (EPCR + KSL) fraction.
  • Figure 5A shows a schematic diagram of expanded mouse HSCs contained in the EPCR + KSL fraction.
  • Figure 5B shows the gating for FACS sorting of the EPCR + KSL fraction. As shown in the right panel of Figure 5B, functional HSCs are enriched in the EPCR + KSL fraction.
  • FIG. 6 shows the percentage of miR-126a-3p activity-positive and -negative cells in the EPCR + KSL fraction. It was confirmed that the miR-126a-3p activity in the EPCR + KSL fraction was heterogeneous, and that it contained both miR-126a-3p activity-positive and -negative cells.
  • Figure 7 shows an outline of the experiment to evaluate the function of miR-126a-3p activity positive/negative cells by transplantation assay.
  • Hematopoietic stem cells collected from CD45.1 B6 mice were cultured in vitro for 2 weeks, and mRNA was introduced by electroporation.
  • Figure 8A is a graph showing the change in peripheral blood chimerism (PB chimerism%) over the number of weeks since transplantation. Mice transplanted with miR-126a-3p activity-positive cells (miR-126a-3p + ) showed higher peripheral blood (PB) chimerism than mice transplanted with cells not separated by miR-126a-3p activity positive/negative (Unsorted) or miR-126a-3p activity-negative cells (miR-126a-3p - ).
  • Figure 8B is a graph showing peripheral blood chimerism (PB chimerism%) 16 weeks after transplantation in Unsorted, miR-126a-3p + cells, and miR-126a-3p - cells.
  • mice that received unsorted, miR-126a-3p + , or miR-126a-3p - transplants it was confirmed that three lineages of cells derived from the donor cells, myeloid, B cells, and T cells, were produced in the peripheral blood.
  • EPCR + KSL cells positive for miR-126a-3p activity or negative for miR-126a-3p activity were sorted by FACS.
  • FIG. 9A is a diagram explaining the experimental scheme.
  • FIG. 9B is a graph showing the change in peripheral blood chimerism (PB chimerism%) over the number of weeks since transplantation. Mice transplanted with EPCR + KSL miR-126a-3p + showed higher peripheral blood chimerism than mice transplanted with EPCR + KSL miR-126a-3p - .
  • FIG. 9A is a diagram explaining the experimental scheme.
  • FIG. 9B is a graph showing the change in peripheral blood chimerism (PB chimerism%) over the number of weeks since transplantation. Mice transplanted with EPCR + KSL miR-126a-3p + showed higher peripheral blood chimerism than mice transplanted with EPCR + KSL miR-126a-3p - .
  • FIG. 9C is a graph showing peripheral blood chimerism (PB chimerism%) 16 weeks after transplantation in EPCR + KSL miR-126a-3p + cells and EPCR + KSL miR-126a-3p - cells. It was confirmed that three lineages of cells derived from donor cells, myeloid, B cells, and T cells, were produced in the peripheral blood.
  • FIG. 10 shows gating for isolating EPCR + KSL, EPCR - KSL, and Sca-1 - Lineage - fractions from mouse hematopoietic stem cells cultured in vitro for 2 weeks in a quantitative experiment of miRNA expression levels in EPCR + KSL, EPCR - KSL, and Sca-1 - Lineage - fractions using the Small RNA-seq method.
  • a comparative analysis of miRNA expression levels in EPCR + KSL and EPCR - KSL was performed, and miRNAs whose expression levels were significantly altered between EPCR + KSL and EPCR - KSL were selected.
  • AQ-seq libraries were prepared according to a modified AQ-seq protocol. Differentially expressed miRNAs between EPCR+KSL and EPCR-KSL were analyzed to select candidate miRNAs for downstream screening by miRNA switch. The selection criteria were as follows: (1) The average RPM of triplicate biological experiments must be ⁇ 1,000 for at least one of the three fractions. (2) miRNAs significantly expressed between EPCR+KSL and EPCR-KSL (adjp ⁇ 0.1)
  • miRNAs whose expression levels were significantly altered between EPCR + KSL and EPCR - KSL and their sequence numbers are shown in Table 3.
  • miRNAs whose expression levels were higher in EPCR + KSL compared to EPCR - KSL are indicated as UP, and miRNAs whose expression levels were lower in EPCR + KSL compared to EPCR - KSL are indicated as DOWN.
  • FIG. 11A Quantification of miRNA expression levels in EPCR + KSL, EPCR - KSL, and Sca-1 - Lineage - fractions using the small RNA-seq method is shown in Figure 11A and Figure 11B.
  • the vertical axis indicates miRNA expression levels (RPM: Reads per million mapped reads). A higher RPM indicates a higher relative expression level of miRNA. Dots represent individual data points, and bars indicate the average of three biological experiments.
  • hematopoietic stem cells Lineage - c-Kit + Sca-1 + CD150 + CD34 -
  • FACS fluorescence-activated cell sorting
  • Figure 13A shows a histogram with the EGFP/TagBFP values on the vertical axis obtained in an experiment to measure the activity of miRNAs selected from miRNA expression level analysis using the Small RNA-seq method. Representative examples of histograms are shown for cells into which control EGFP mRNA or the miR-126a-3p-responsive miRNA switch was introduced ( Figure 13A). Bar graphs showing the coefficient of variation (CV) of EGFP/TagBFP values in cell populations into which each of multiple candidate miRNAs was introduced are shown ( Figures 13B and 13C). Among the multiple candidate miRNAs, miRNAs showing high CV values are thought to be miRNAs with high heterogeneity in activity in mouse hematopoietic stem cells expanded in vitro ( Figures 13B and 13C).
  • hematopoietic stem cells Lineage - c-Kit + Sca-1 + CD150 + CD34 -
  • FACS fluorescence-activated cell sorting
  • a miRNA switch encoding EGFP (responsive to miR-126a-3p, miR-155-5p, miR-196b-5p, miR-130a-3p, miR-10a-5p, miR-126a-5p, or miR-223-3p), a miR-126a-3p-responsive miRNA switch encoding mT-Sapphire, and mRNA encoding TagBFP were introduced into the cells by electroporation. The cells were analyzed using a flow cytometer 24 hours after mRNA introduction.
  • FIG. 15A shows the results of cells harvested after culturing 250 cells
  • Figure 15B shows the results of cells harvested after culturing 500 cells, diluted 2-fold with trypan blue, and counted using Countess III (Thermo Fisher Scientific).
  • miR-126a-3p activity-positive cells showed higher proliferation than miR-126a-3p activity-negative cells (miR-126a-3p - ), untreated cells without mRNA introduction (Untreated), and cells introduced with control EGFP mRNA and TagBFP mRNA (EGFP+TagBFP).
  • Mouse bone marrow-derived hematopoietic stem cells (Lineage - c-Kit + Sca-1 + CD150 + CD34 - ) isolated by fluorescence-activated cell sorting (FACS) were cultured in medium containing PVA for 2 weeks. Then, the miR-126a-3p-responsive miRNA switch encoding mT-Sapphire, the miR-130a-3p-responsive miRNA switch encoding EGFP, and the mRNA encoding TagBFP were introduced into the hematopoietic stem and progenitor cells by electroporation. An overview is shown in Figure 16A. FACS analysis and cell sorting were performed 24 hours after mRNA introduction.
  • Figure 16B shows gating for FACS sorting of miR-126a-3p + miR-130a-3p + and miR-126a-3p + miR-130a-3p - fractions. Gates were set to separate positive and negative populations for each miRNA based on controls that contained inhibitors for miR-126a-3p and miR-130a-3p.
  • FIG. 16C shows an outline of an experiment to evaluate the function of miR-126a-3p + miR-130a-3p + cells and miR-126a-3p + miR-130a-3p - cells by transplantation assay.
  • Hematopoietic stem cells collected from CD45.1 B6 mice were cultured in vitro for 2 weeks, and mRNA was introduced by electroporation.
  • Figure 16D is a graph showing the change in peripheral blood chimerism (PB chimerism%) over the number of weeks since transplantation. Mice transplanted with miR-126a-3p + miR-130a-3p + cells showed higher peripheral blood (PB) chimerism than mice transplanted with miR-126a-3p + miR-130a-3p - cells.
  • FIG. 16E is a graph showing peripheral blood chimerism (PB chimerism%) 12 weeks after transplantation in host mice transplanted with miR-126a-3p + miR-130a-3p + cells or miR-126a-3p + miR-130a-3p - cells.
  • Mice transplanted with miR-126a-3p + miR-130a-3p + cells showed higher peripheral blood (PB) chimerism than mice transplanted with miR-126a-3p + miR-130a-3p - cells.
  • PB peripheral blood
  • mice transplanted with either type of cells it was confirmed that three lineages of cells derived from donor cells, myeloid, B cells, and T cells, were produced in the peripheral blood.
  • FIG. 17A shows an outline of an experiment to measure miRNA activity in human hematopoietic stem and progenitor cells (HSPCs) cultured in vitro using the miRNA switch.
  • HSPCs human hematopoietic stem and progenitor cells
  • Human miRNA switch encoding EGFP (responsive to hsa-miR-126-3p, hsa-miR-126-5p, hsa-miR-130a-3p, hsa-miR-223-3p, or hsa-miR-10a-5p) and mRNA encoding TagBFP were introduced into the cells by electroporation. The cells were analyzed using a flow cytometer 24 hours after mRNA introduction.
  • Figure 17B shows the results of an experiment to measure the activity of hsa-miR-126-3p, hsa-miR-126-5p, hsa-miR-130a-3p, hsa-miR-223-3p, or hsa-miR-10a-5p in human hematopoietic stem progenitor cells.
  • the plot shows the expression level of EGFP on the vertical axis and the expression level of TagBFP on the horizontal axis.
  • the plot in the upper left shows cells transfected with EGFP mRNA that does not contain a miRNA target sequence.
  • FIG. 18A shows an outline of an experiment to simultaneously measure the activity of two types of miRNAs in human hematopoietic stem and progenitor cells.
  • human umbilical cord blood-derived CD34 + cells were cultured in vitro for 2 weeks.
  • an hsa-miR-126-3p-responsive miRNA switch encoding mT-Sapphire an EGFP-encoding miRNA switch responsive to a second miRNA (responsive to hsa-miR-130a-3p, hsa-miR-10a-5p, hsa-miR-126-5p, or hsa-miR-223-3p), and an mRNA encoding TagBFP were introduced by electroporation. Analysis by FACS was performed 24 hours after mRNA introduction.
  • Figure 18B shows plots of EGFP/TagBFP values on the vertical axis and mT-Sapphire/TagBFP values on the horizontal axis, obtained as a result of an experiment to simultaneously measure the activity of two types of miRNAs in human hematopoietic stem and progenitor cells.
  • the top row shows plots of cells transfected with a negative control miRNA inhibitor.
  • the bottom row shows plots of cells transfected with both an inhibitor against hsa-miR-126a-3p and an inhibitor against a second miRNA (hsa-miR-130a-3p, hsa-miR-10a-5p, hsa-miR-126-5p, or hsa-miR-223-3p) for gate setting.
  • hsa-miR-130a-3p hsa-miR-10a-5p
  • hsa-miR-126-5p hsa-miR-223-3p

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Abstract

Procédé permettant de cribler, à un haut degré de pureté, des cellules souches hématopoïétiques vivantes présentant une capacité d'autoréplication et une pluripotence élevées, selon un procédé ne présentant pas de risques d'endommagement du génome.　Ce procédé de criblage de cellules souches hématopoïétiques comprend (i) une étape d'introduction d'un premier commutateur d'ARNm sensible au miARN dans une population de cellules. Le premier commutateur d'ARNm sensible aux miARN est une molécule d'ARNm dans laquelle (a) une séquence nucléotidique reconnue spécifiquement par mmu-miR-126a-3p ou hsa-miR-126-3p, et (b) une séquence nucléotidique codant pour une première protéine de marquage sont liées de manière fonctionnelle. Ce procédé de production de population de cellules souches hématopoïétiques comprend une étape permettant d'obtenir, selon ledit procédé de criblage, une population de cellules souches hématopoïétiques par criblage.
PCT/JP2024/018347 2023-05-19 2024-05-17 Procédé de criblage de cellules souches hématopoïétiques, procédé de production de population de cellules souches hématopoïétiques et kit de criblage de cellules souches hématopoïétiques Pending WO2024242053A1 (fr)

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Citations (5)

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WO2015105172A1 (fr) * 2014-01-10 2015-07-16 国立大学法人京都大学 MÉTHODE PERMETTANT D'IDENTIFIER UN TYPE DE CELLULE RECHERCHÉ FAISANT APPEL À L'EXPRESSION D'UN mi-ARN À TITRE D'INDICATEUR
WO2017073600A1 (fr) * 2015-10-28 2017-05-04 国立大学法人京都大学 MÉTHODE DE CONCEPTION D'ARNm
WO2018003779A1 (fr) * 2016-06-27 2018-01-04 国立大学法人京都大学 Procédé d'expression d'un gène de protéine en réponse à l'expression de miarn
WO2019031595A1 (fr) * 2017-08-10 2019-02-14 国立大学法人京都大学 Procédé de criblage de cellules rénales progénitrices
JP2022112522A (ja) * 2019-05-31 2022-08-03 国立大学法人京都大学 標的遺伝子を編集する蛋白質を細胞特異的に制御する方法

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