WO2023074873A1 - Procédé de purification cellulaire - Google Patents

Procédé de purification cellulaire Download PDF

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WO2023074873A1
WO2023074873A1 PCT/JP2022/040488 JP2022040488W WO2023074873A1 WO 2023074873 A1 WO2023074873 A1 WO 2023074873A1 JP 2022040488 W JP2022040488 W JP 2022040488W WO 2023074873 A1 WO2023074873 A1 WO 2023074873A1
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cells
mrna
cell
switch
gene
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博英 齊藤
祥彦 藤田
萌 弘澤
魁人 正木
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国立大学法人京都大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N5/10—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
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to a cell purification method using an mRNA switch and a kit used therefor.
  • the tissues and organs of multicellular organisms are composed of many types of cells. There are about 411 types of cells that make up humans, including only mature cells. Techniques for selecting only target cells from a population of cell types with different properties are very important for regenerative medicine.
  • a method using a flow cytometer is known as a cell sorting technology.
  • cell sorting methods using a flow cytometer individual cells are identified and separated one by one. Therefore, it was difficult to perform mass processing for cell sorting.
  • RNA switch technology that uses mRNA that turns ON translation (miRNA-ON switch) or turns OFF translation (miRNA-OFF switch) in response to miRNA endogenous to the cell.
  • miRNA-ON switch mRNA that turns ON translation
  • miRNA-OFF switch turns OFF translation
  • the cell sorting technology using RNA switch technology has the problem that the sorting efficiency varies greatly depending on the cell type. This is thought to be due to differences in the amount of RNA switch introduced, translation efficiency, sensitivity to lethal gene products, and the like, depending on the cell type.
  • the amount of the switch In order to control cell fate with a single miRNA-OFF switch encoding a lethal gene, the amount of the switch must be tightly tuned so that the expression level of the lethal gene is below the threshold for undesired target cell death. (FIGS. 8A and 8B).
  • the miRNA-OFF switch alone cannot completely suppress the leak expression of the lethal gene when the target miRNA is detected. can't.
  • a miRNA-OFF switch that can regulate a pro-apoptotic protein (Bim).
  • Bim is leaky expressed in cells to be enriched, efficient cell purification has been an issue.
  • the Bax-encoding cell sorting circuit sensed miR-21-5p and selectively killed HeLa cells, but leaked expression of Bax resulted in non-target 293 cells. was also found to have died. Moreover, previous studies using the miR-21-5p response circuit selectively killed HeLa cells, but not 293FT cells. This may be due to differences in sensitivity to apoptotic genes resulting from differences in intracellular apoptotic pathways between the two cell types (Figs. 9A, B, C).
  • an mRNA switch that turns on translation in response to miRNAs activated in cells As a result of intensive studies, the present inventors found that an mRNA switch that turns on translation in response to miRNAs activated in cells, an mRNA switch that turns off translation in response to miRNAs, and an mRNA switch that is essential for cell survival.
  • the inventors have found that introduction of mRNA encoding a gene into a cell population makes it possible to select cells with high accuracy, and have completed the present invention.
  • the present invention includes the following aspects. [1] introducing a first mRNA, a second mRNA, a third mRNA, or a DNA encoding them into a cell population; and culturing the cell population in the presence of a drug, (1) the first mRNA is (1a) a nucleic acid sequence specifically recognized by miRNAs activated in the target cell or non-target cell; (1b) an OFF switch mRNA operably linked to a nucleic acid sequence encoding a lethal or anti-lethal gene; (2) the second mRNA is (2a) a nucleic acid sequence encoding an anti-lethal gene that inactivates the lethal gene of (1b) or a lethal gene that is inactivated by the anti-lethal gene of (1b); (2b) PolyA tail provided on the 3' side of (2a); (2c) a nucleic acid sequence that is specifically recognized by miRNA activated in the target cell or non-target cell provided on the 3' side of (2b); (2d) an
  • the purification method according to [1], wherein the step of introducing is a step of co-introducing the first mRNA, the second mRNA and the third mRNA.
  • the lethal gene is a gene that encodes an RNase, and the anti-lethal gene is a gene that encodes a protein that inactivates the RNase; or the lethal gene is a gene that encodes an apoptosis-inducing protein. and the anti-lethal gene is a gene encoding an apoptosis inhibitor protein.
  • the RNase is Barnase, and the protein that inactivates the RNase is Barstar; or the apoptosis-inducing protein is Bax or Bim, and the apoptosis-inhibiting protein is Bcl-2 or Bcl-
  • a method for producing a cell population enriched with the target cells comprising the step of purifying the target cells by the purification method according to any one of [1] to [6].
  • a target cell purification kit comprising a first mRNA, a second mRNA and a third mRNA, or DNAs encoding them, (1) the first mRNA is (1a) a nucleic acid sequence specifically recognized by miRNAs activated in the target cell or non-target cell; (1b) an OFF switch mRNA operably linked to a nucleic acid sequence encoding a lethal or anti-lethal gene; (2) the second mRNA is (2a) a nucleic acid sequence encoding an anti-lethal gene that inactivates the lethal gene of (1b) or a lethal gene that is inactivated by the anti-lethal gene of (1b); (2b) PolyA tail provided on the 3' side of (2a); (2c) a nucleic acid sequence that is specifically recognized by miRNA activated in the target cell or
  • the lethal gene is a gene that encodes an RNase
  • the anti-lethal gene is a gene that encodes a protein that inactivates the RNase; or the lethal gene is a gene that encodes an apoptosis-inducing protein. and the anti-lethal gene is a gene encoding an apoptosis inhibitor protein.
  • the RNase is Barnase
  • the protein that inactivates the RNase is Barstar
  • the apoptosis-inducing protein is Bax or Bim
  • the apoptosis-suppressing protein is Bcl-2 or Bcl- 10.
  • the activities of miRNAs in cells are different, it is possible to simultaneously select a large number of desired cell types. This allows the preparation of large amounts of cells required for transplantation into mammals, especially humans, in a short time and safely without the risk of contamination. Ultimately, this will lead to cost reductions in cell transplantation medicine.
  • FIG. 1A schematically shows an miRNA-responsive OFF switch and an ON switch, regarding the design and response of miRNA-responsive ON switches.
  • the OFF switch (left) is translated like normal mRNA (ON state) and inhibited in the presence of target miRNA (OFF state).
  • the ON switch (right) consists of a normal mRNA and a translational repression sequence downstream of the PolyA tail bound by a sequence complementary to the target miRNA. In the absence of the target miRNA, the hidden PolyA tail suppresses translation (OFF state). When the target miRNA is present, the miRNA removes the translational repressing sequence and promotes translation (ON state).
  • FIG. 1B shows fold-change in translational efficiency of ON switches in response to miR-21-5p.
  • miR-21 indicates an ON switch having a sequence complementary to miR-21-5p
  • “Shuffle” indicates an ON switch having a shuffled sequence complementary to miR-21-5p. All values are shown as the ratio of translation efficiency (EGFP/iRFP670) between 293FT and HeLa containing mRNAs with translational repression sequences (also called additional sequences, 495nt, 1250nt, or 30CAG repeats) downstream of the PolyA tail. There is Error bars represent mean ⁇ SD and data for each repeat are indicated by dots. ****P ⁇ 0.0005 (Welch's t-test). FIG.
  • 1C is a ratiometric image of cells co-transfected with iRFP670 mRNA and a miR-21-5p-responsive ON switch with a sequence complementary to or shuffled with miR-21-5p.
  • iRFP670 mRNA was used as an internal control.
  • the EGFP intensity at each pixel within the cell was divided by the iRFP670 intensity.
  • Red ratiometric pseudocolor indicates a high ratio of EGFP to iRFP670 (translational efficiency). Scale bar indicates 200 ⁇ m.
  • 2A shows miRNA-responsive ON-switch responses to various mimics, inhibitors, and endogenous miRNAs, co-introducing miR-21-5p-responsive ON-switches and OFF-switches with miR-21-5p mimics/inhibitors.
  • 1 is a ratiometric image of HeLa cells. The EGFP intensity at each pixel within the cell was calculated in the same manner as in Figure 1C. "miR-21-5p-ON” and “miR-21-5p-OFF” indicate miR-21-5p-responsive ON and OFF switches, respectively. "EGFP mRNA” indicates mRNA encoding EGFP that does not contain a sequence complementary to miR-21-5p. Scale bar indicates 200 ⁇ m. In FIG.
  • FIG. 2B is a ratiometric image of HeLa cells co-introduced with various mimics and their ON switches. The EGFP intensity at each pixel within the cell was calculated in the same manner as in Figure 1C. Columns indicate each miRNA-responsive switch and rows indicate miRNA mimics. Scale bar indicates 200 ⁇ m.
  • FIG. 2C is a ratiometric image of HeLa cells co-introduced with various mimics and their ON switches. The EGFP intensity at each pixel within the cell was calculated in the same manner as in Figure 1C. Columns indicate each miRNA-responsive switch and rows indicate miRNA mimics. Scale bar indicates 200 ⁇ m.
  • FIG. 2D shows the relative intensity of various miRNA-responsive ON switches and their mimics (EGFP/iRFP670). Error bars indicate mean ⁇ SD. Each value is indicated by a dot. *p ⁇ 0.05, **p ⁇ 0.001.
  • FIG. 2E is a ratiometric image of 293FT cells co-introduced with a miR-302-5p responsive switch. EGFP intensity at each pixel in the cell was calculated by the same method as in FIG. 1C. In 293FT cells, the translation efficiency of the miR-302-5p-responsive ON switch (EGFP/iRFP670) was suppressed. Scale bar indicates 200 ⁇ m.
  • 2F is a ratiometric image of 201B7 cells (human iPS cells: iPSCs) co-introduced with a miR-302-5p responsive switch. EGFP intensity at each pixel in the cell was calculated by the same method as in FIG. 1C. Translation efficiency was enhanced in 201B7 cells with high miR-302-5p activity. In cells transfected with an OFF switch, translation was repressed in iPSCs but not in 293FT. Scale bar indicates 200 ⁇ m.
  • FIG. 2G shows a representative flow cytometry dot plot. 293FT and iPSCs co-introduced with miR-302a-5p ON or OFF switches are shown in red and green, respectively.
  • FIG. 2H shows the relative intensity of 293FT and iPSCs co-introduced with miR-302a-5p responsive switch (EGFP/iRFP670). Error bars represent mean ⁇ SD. Data for each repeat are indicated by dots. **P ⁇ 0.01.
  • FIG. 3A Robustness of miRNA-responsive ON/OFF switches encoding Barnase and Barstar.
  • A A phase diagram showing the relationship between cell death and the expression levels of the lethal RNase Barnase and its inhibitor Barstar using single-switch or dual-switch selection. A region shown with fine dots indicates a region where cells die (Cell death). White areas indicate areas where cells survive (Cell Survive).
  • FIG. 3B is a schematic diagram of transfection for detecting intracellular Barnase activity through translation of EGFP to select specific cells.
  • miR-21-5p-responsive OFF switch encoding Barstar and miR-21-5p-responsive ON switch encoding Barnase are decreased or increased in expression due to high activity of miR-21-5p. , resulting in higher Barnase activity.
  • Barnase activated in HeLa cells suppresses the translation of EGFP from co-introduced EGFP-mRNA and kills HeLa cells.
  • miR-21-5p-Bn-ON and miR-21-5p-Bs-ON represent miR-21-5p-responsive ON switches encoding Barnase and Barstar, respectively.
  • -Bn-OFF and miR-21-5p-Bs-OFF indicate miR-21-5p-responsive OFF switches encoding Barnase and Barstar, respectively.
  • EGFP mRNA is degraded due to high Barnase activity, resulting in no EGFP signal.
  • FIG. 3D shows the dependence of barnase activity on the amount of transfected RNA. Values are the relative percentage of EGFP-positive cells co-transfected with RNA switch and EGFP mRNA normalized by the percentage of EGFP-positive cells transfected with EGFP mRNA alone.
  • miR-21-5p-Bn-ON and miR-21-5p-Bs-ON represent miR-21-5p-responsive ON switches encoding Barnase and Barstar, respectively
  • miR-21-5p-Bn-OFF and miR-21-5p-Bs-OFF represent miR-21-5p-responsive OFF switches encoding Barnase and Barstar, respectively.
  • Open circle is HeLa cell transfected with miR-21-5p responsive ON switch encoding Barnase and miR-21-5p responsive OFF switch encoding Barstar
  • Open triangle is miR-21-5p responsive encoding barnase HeLa cells transfected with an OFF switch and miR-21-5p-responsive ON switch encoding Barstar
  • Closed circle and Closed triangle represent 293FT cells cotransfected with the same switch as HeLa cells, respectively.
  • the percentage of EGFP-positive cells is largely unaffected by the total amount of RNA introduced. Error bars represent mean ⁇ SD.
  • FIG. 4A shows a schematic diagram showing the combination of switches to efficiently purify miR-21-5p-active (HeLa) or -inactive (293FT) cells for purification of cells by miRNA-ON and -OFF switches.
  • HeLa cells since miR-21-5p activity is high, the translation of the miR-21-5p-responsive ON switch encoding Barnase and the OFF switch encoding Barstar are increased and decreased, respectively, and barnase is activated. , resulting in accelerated cell death.
  • 293FT cells the translation of Barnase and Barstar decreased and increased, respectively, and 293FT cells survived.
  • FIG. 4B shows photomicrographs of each cell two days after transfection.
  • miR-21-5p-Bn-ON, miR-21-5p-Bs-ON, miR-21-5p-Bn-OFF, miR-21-5p-Bs-OFF are the same as in FIG. 3D.
  • miR-21-5p-Bs-ON and miR-21-5p-Bn-OFF (upper left)
  • 293FT cells exhibited an abnormal shape and detached from the bottom of the well.
  • miR-21-5p-Bs-OFF and miR-21-5p-Bn-ON bottom left
  • Both cell types transfected with either a single miR-21-5p-Bn-OFF or miR-21-5p-Bn-ON switch exhibited abnormal morphology.
  • Scale bar indicates 200 ⁇ m.
  • FIG. 4C shows the measurement results of cell viability by WST1 assay.
  • WST1 assay was performed 2 days after transfection. Absorbance was subtracted with the absorbance of blank wells and normalized with the absorbance of untransfected cells. Error bars represent the mean ⁇ SD, and each data point. *p ⁇ 0.05, ***p ⁇ 0.005.
  • FIG. 4D is a photomicrograph of co-cultured HeLa and 293FT cells 3 days after transfection. To distinguish between cell lines, HeLa cells stably expressing high hmAG1-M9 and 293FT cells stably expressing iRFP670-M9 were used.
  • FIG. 4E is a representative two-dimensional plot of flow cytometry, with squares indicating gates for each cell.
  • FIG. 4F shows the percentage of each cell. The number of each cell in the box in FIG. 4E was counted and the percentage of each cell line was calculated. Error bars represent the mean ⁇ SD, and each data point. ****P ⁇ 0.001.
  • 5A shows a schematic representation of double selection of iPSCs for purification of iPSCs from a mixture of HeLa cells and iPSCs.
  • miR-302-Bn-OFF and miR-302-Bs-ON represent miR-302a-5p-responsive OFF and ON switches encoding barnase and Barstar, respectively.
  • mRNAs encoding these switches and the blasticidin resistance gene (bsd) were co-introduced into a heterogeneous cell population containing the cells of interest.
  • bsd blasticidin resistance gene
  • FIG. 5B is a representative two-dimensional plot of flow cytometry.
  • iPS cells expressing GFP and HeLa cells expressing iRFP670-M9 were co-cultured and treated with miR-302-5p-responsive OFF and ON switches encoding Barnase and Barstar, respectively. After passaging the co-cultured cells, all cells were analyzed by flow cytometry. Squares indicate gates for iPS cells and HeLa cells, respectively.
  • FIG. 5C shows the ratio of 201B7 cells and HeLa cells after passaging after transfection. The number of each cell within the box in FIG. 5B was counted and the percentage of each cell line was calculated.
  • FIG. 5D shows merged fluorescence images of cells treated with switch followed by passage. 201B7-GFP and HeLa-iRFP670-M9 cells were colored green and red, respectively. Scale bar indicates 200 ⁇ m.
  • FIG. 6A shows a schematic representation of iPSC-cardiomyocyte double selection for purification of cardiomyocytes from differentiated heterologous cells. miR-208-Bn-OFF and miR-1-Bs-ON represent miR-208a-3p-responsive OFF and miR-1-3p-responsive ON switches encoding Barnase and Barstar, respectively.
  • FIG. 6B shows a representative two-dimensional plot of flow cytometry. Trapezoids indicate gates for cardiomyocytes expressing EGFP produced from the MYH6 promoter.
  • FIG. 6C shows microscopic merged images of heterogeneous cell populations treated with switch and G418. Hoechst-stained nuclei are shown in blue, and cardiomyocytes expressing GFP are shown in green. The composite image in the lower panel is the dashed square portion of the image in the upper panel. Red arrowheads indicate contractile cells. Areas indicated by red arrows are not occupied by cells due to the removal of non-target cells.
  • FIG. 6D shows the percentage of heterogeneous cardiomyocytes derived from 201B7 counted within the gates of FIG. 6B.
  • the sample names are the same as in FIG. 6B.
  • FIG. 7 is a scatter plot of miR-21a-5p responsive ON switches to endogenous miRNAs. Cells co-introduced with iRFP670 mRNA and a miR-21-5p responsive switch with complementary or shuffled sequences to miR-21-5p are shown. iRFP670 mRNA was used as an internal control. HeLa and 293FT cells are indicated by red and green dots, respectively.
  • FIG. 8A is a diagram schematically showing the distribution of gene expression in each cell type.
  • the level of lethal gene expression by the OFF switch is lower on average than in non-target cells (death) due to endogenous miRNAs in target cells (survive), but to some extent due to variations in RNA uptake and translational activity in individual cells. (Green and Orange distributions).
  • the amount of mRNA introduced into individual cells is variable and difficult to control precisely.
  • translational activity depends on cell conditions such as cell size, cell cycle, and health condition, the distribution of the expression level in each cell increases. Therefore, the expression level in target cells and the expression level in non-target cells overlap (overlap).
  • FIG. 8B is a diagram schematically showing the distribution of gene expression in each cell type when the amount of mRNA switch introduced into the cell is increased compared to FIG. 8A. Increasing the amount of mRNA switches shifts the distribution of target and non-target cells to higher expression levels. In this situation, although the purity of the target cells is increased, the expression level of the lethal gene in some of the target cells exceeds the lethal threshold, resulting in a low yield of the target cells.
  • FIG. 8B is a diagram schematically showing the distribution of gene expression in each cell type when the amount of mRNA switch introduced into the cell is increased compared to FIG. 8A. Increasing the amount of mRNA switches shifts the distribution of target and non-target cells to higher expression levels. In this situation, although the purity of the target cells is increased, the expression level of the lethal gene in some of the target cells exceeds the lethal threshold, resulting in a low yield of the target cells.
  • FIG. 9A shows the purification of HeLa cells with the miR-21a-5p responsive OFF-switch encoding the pro-apoptotic gene Bim
  • FIG. 9B shows dose dependence of the selective efficiency of the miR-21-5p-responsive Bim OFF switch.
  • Co-cultured HeLa and 293FT were treated with a miR-21-5p-responsive OFF-switch that encodes the apoptotic protein Bim. The number of each cell was counted in the same manner as in FIG. 4E, and the ratio of each cell line was calculated.
  • FIG. 9C is a merged fluorescence microscope image of co-cultured cells. To distinguish between cell lines, HeLa cells stably expressing high hmAG1-M9 and 293FT cells stably expressing iRFP670-M9 were used. Co-cultured cells were transduced with a miR-21-5p-responsive OFF switch encoding Bim EL. HeLa cells were expected to survive because highly active miR-21-5p downregulates Bim EL expression. However, both cells were eliminated by the switch. Scale bar indicates 200 ⁇ m. FIG.
  • FIG. 10A shows a representative two-dimensional plot of flow cytometry without passaging for the purification of iPSCs from a mixture of HeLa cells and iPSCs without passaging.
  • iPS cells expressing GFP and HeLa cells expressing iRFP670-M9 were co-cultured and treated with miR-302-responsive OFF and ON switches encoding Barnase and Barstar, respectively. After passaging the co-cultured cells, all cells were analyzed by flow cytometry. Squares indicate gates for iPS cells and HeLa cells, respectively.
  • FIG. 10B shows the ratio of 201B7 cells and HeLa cells after transfection without passaging. The number of each cell in the square in FIG. 5B was counted and the percentage of each cell line was calculated.
  • FIG. 10C shows merged fluorescence images of cells treated with switch followed by passage. 201B7-GFP and HeLa-iRFP670-M9 cells were colored green and red, respectively. Scale bar indicates 200 ⁇ m.
  • FIG. 11A is a graph depicting the results of purification of HeLa or 293FT cells from HeLa/293FT co-cultures.
  • FIG. 11B is a graph depicting the results of purification of HeLa or 293FT cells from HeLa/293FT co-cultures.
  • FIG. 12 is a graph showing the results of purification of iPS cells from HeLa/iPS co-culture.
  • the present invention relates to a method for purifying target cells, and includes the following steps (I) and (II). (I) introducing the first mRNA, the second mRNA and the third mRNA into the cell population; (II) Step of culturing the cell population in the presence of an agent Optionally, the following step (III) may be included. (III) culturing the cell population that has undergone step (II) in the absence of the drug
  • purification of target cells refers to selecting one type of target cells from a heterogeneous cell population that may contain two or more types of cells. In the above, it refers to selection so that the ratio of target cells in the cell population is large.
  • purification of target cells means killing cells other than the one type of target cells and maintaining the one type of target cells in a viable state, particularly using a sorting instrument such as a flow cytometer. It means to implement without
  • the cell population obtained as a result of performing the purification method is referred to as a purified cell population.
  • the ratio of target cells is increased compared to before the purification method.
  • the purified cell population may have a target cell ratio of 90% or more, preferably a target cell ratio of 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • the ratio of target cells can be measured, for example, by using a method known per se as the ratio of cells expressing the gene product to the total number of cells, using the expression of a target cell-specific gene product as an index.
  • Step Step (I) of the method for purifying target cells is a step of introducing the first mRNA, the second mRNA and the third mRNA into the cell population.
  • the term "cell” is not particularly limited and may be any cell. Moreover, the term “cell population” refers to a collection of two or more cells. There is no upper limit to the number of “cell populations”, but, for example, a population consisting of about 10 9 cells is referred to.
  • a “target cell” is a cell to be purified in the method of the present invention. In this specification, a target cell may be referred to as a “target cell”, and a cell other than the target cell may be referred to as a "non-target cell”.
  • Cells may be, for example, cells collected from multicellular organisms, or artificially manipulated cells (including cell lines). Cells derived from mammals (eg, humans, mice, monkeys, pigs, rats, etc.) are preferred, and cells derived from humans are most preferred. There are no particular restrictions on the degree of cell differentiation or the age of the animal from which the cells are collected, and the cells may be (A) stem cells, (B) progenitor cells, (C) terminally differentiated somatic cells, or (D) other cells. may
  • stem cells include, but are not limited to, embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells (“GS cells”). , embryonic germ cells (“EG cells”), induced pluripotent stem (iPS) cells, and the like. Among these, ES cells and iPS cells are preferred, and iPS cells are particularly preferred.
  • ES embryonic stem
  • ntES embryonic stem
  • GS cells spermatogonial stem cells
  • EG cells embryonic germ cells
  • iPS induced pluripotent stem
  • progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
  • tissue stem cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
  • Somatic cells include, for example, keratinizing epithelial cells (e.g., keratinizing epidermal cells), mucosal epithelial cells (e.g., tongue epithelial cells), exocrine gland epithelial cells (e.g., mammary gland cells), hormone secretion cells (e.g., adrenal medulla cells), cells for metabolism and storage (e.g., hepatocytes), luminal epithelial cells that make up the interface (e.g., type I alveolar cells), luminal epithelial cells of the inner chain duct ( vascular endothelial cells), ciliated cells with carrying capacity (e.g.
  • keratinizing epithelial cells e.g., keratinizing epidermal cells
  • mucosal epithelial cells e.g., tongue epithelial cells
  • exocrine gland epithelial cells e.g., mammary gland cells
  • hormone secretion cells
  • airway epithelial cells extracellular matrix-secreting cells (e.g. fibroblasts), contractile cells (e.g. smooth muscle cells), blood and Immune system cells (e.g. T lymphocytes), sensory cells (e.g. rod cells), central and peripheral nervous system neurons and glial cells (e.g. astrocytes), pigment cells (e.g. retinal pigment epithelium) cells), and their progenitor cells (tissue progenitor cells).
  • extracellular matrix-secreting cells e.g. fibroblasts
  • contractile cells e.g. smooth muscle cells
  • blood and Immune system cells e.g. T lymphocytes
  • sensory cells e.g. rod cells
  • central and peripheral nervous system neurons and glial cells e.g. astrocytes
  • pigment cells e.g. retinal pigment epithelium cells
  • progenitor cells tissue progenitor cells
  • Other cells include, for example, cells that have undergone differentiation induction, including progenitor cells and somatic cells that have undergone differentiation induction from pluripotent stem cells.
  • cells induced by so-called “direct reprogramming (also referred to as trans-differentiation)" in which somatic cells or progenitor cells are directly differentiated into desired cells without passing through an undifferentiated state, may be used.
  • the cell population may be a cell population that contains cells that have undergone induction of differentiation of cardiomyocytes, skeletal muscle cells, blood cells, and nerve cells as target cells, and that may contain undifferentiated cells. . It may be a cell population that contains undifferentiated cells as target cells and may contain cells that have undergone differentiation. Another example is a cell population that contains normal cells as target cells and may contain cancer cells. Cells of a specific subtype may be included as target cells, and cell populations composed of a plurality of subtypes may also be used.
  • the mRNAs to be introduced into the cell population are the following first mRNA, second mRNA and third mRNA.
  • the first mRNA is an OFF switch mRNA;
  • an mRNA switch in which translation is suppressed in the presence of a specific miRNA activity and translation is performed in the absence of miRNA activity is referred to as an OFF switch mRNA.
  • the first mRNA is the OFF switch mRNA.
  • OFF switch mRNA is sometimes abbreviated and referred to as OFF switch.
  • OFF switch mRNA and ON switch and mRNA to be described later may be collectively referred to as switch, miRNA switch, or switch mRNA.
  • the nucleic acid sequence of (1a) is present on the 5' side or 3' side of the nucleic acid sequence of (1b), and the (1a) and (1b) are operably linked.
  • a more specific structure of the first mRNA may be a structure in which a 5'-UTR, a coding region, and a 3'-UTR are linked in the 5' to 3' direction of the mRNA molecule.
  • the nucleic acid sequence of (1a) is a nucleic acid sequence specifically recognized by miRNAs activated in target cells or non-target cells.
  • Activated miRNA in target cells or non-target cells refers to miRNA that exists in a state where mature miRNA interacts with multiple predetermined proteins to form an RNA-induced silencing complex (RISC). shall mean.
  • RISC RNA-induced silencing complex
  • “Mature miRNA” is a single-stranded RNA (20-25 bases) generated from pre-miRNA by cleavage by Dicer outside the nucleus, and "pre-miRNA” is partially cleaved by an intranuclear enzyme called Drosha. It arises from pri-mRNA, a single-stranded RNA transcribed from DNA.
  • the miRNA in the present invention is appropriately selected from at least 10,000 types of miRNA.
  • miRNA registered in database information e.g., http://www.mirbase.org/ or http://www.microrna.org/
  • database information e.g., http://www.mirbase.org/ or http://www.microrna.org/
  • literature information described in the database are appropriately selected from the miRNAs described in . That is, in the present invention, miRNAs activated in target cells or non-target cells are not limited to specific miRNAs.
  • a specific miRNA is “activated” in a specific cell means that the activity level of the specific miRNA in a specific cell is compared with the activity level of the specific miRNA in other cells, for example, It means 1.1 times or more, preferably 1.5 times or more, more preferably 5 times or more, still more preferably 10 times or more.
  • the level of activity of a particular miRNA in a cell can be determined from the literature, or decreased fluorescence or luminescence from an mRNA that contains the complementary sequence of the particular miRNA and encodes a reporter protein such as a fluorescent protein, a luminescent protein, etc. It can also be quantified and confirmed by a method such as measuring the amount.
  • the nucleic acid sequence of (1a) is also referred to herein as the "miRNA target sequence".
  • a miRNA target sequence is a nucleic acid sequence capable of specifically binding to miRNAs that are activated in cells of interest or non-targets.
  • the miRNA target sequence is preferably, for example, a sequence that is fully complementary to the miRNA that is activated in the target cell.
  • the miRNA target sequence may have a mismatch (mismatch) with a completely complementary sequence as long as it can be recognized by miRNAs activated in target cells or non-target cells.
  • the mismatch from the sequence that is completely complementary to the miRNA may be a mismatch that can be normally recognized by the miRNA in the desired cell, and the original function in the cell in vivo is about 40 to 50% mismatch. It is said that it is acceptable to have Such mismatches include, but are not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases or 1%, 5, or 1% of the total recognition sequence. %, 10%, 20%, 30%, or 40% discrepancies are exemplified. Also, in particular, like the miRNA target sequence on the mRNA that cells have, in particular, the 5' side of the target sequence, which corresponds to about 16 bases on the 3' side of the miRNA, other than the seed region. A region may contain multiple mismatches, and a portion of the seed region may contain no mismatches, 1-, 2-, or 3-base mismatches.
  • Examples of combinations of typical cell types and typical miRNAs activated in the cells are shown below. However, the present invention is not limited to the specific miRNA targeting techniques described below.
  • the cells exemplified below can be both target cells for purification and non-target cells.
  • the nucleic acid sequence of (1b) is a nucleic acid sequence encoding a lethal gene or an anti-lethal gene.
  • Lethal genes include genes encoding RNA degrading enzymes such as barnase derived from Bacillus amyloliquefaciens, HokB, Fst, GhoT (membrane disruption), HipA (inhibition of nucleic acid elongation by phosphorylation), RelE, YafO, VapC, MazF, MqsR, Genes encoding toxins such as PemKHicA (endonuclease), FicT (Adenylation), Doc (Phosphorylation), CcdB, ParE (Gyrase inhibitor), Tact (Inhibitor of translation), cbtA (Inhibitor of cytoskeletal protein), Bax, Bim, etc.
  • RNA degrading enzymes such as barnase derived from Bacillus amyloliquefaciens, Hok
  • Genes encoding apoptosis-inducing proteins include, but are not limited to.
  • Anti-lethal genes are generally used in combination with lethal genes.
  • proteins that inactivate RNases such as barstar from Bacillus amyloliquefaciens.
  • toxins for proteins that inactivate toxins and for apoptosis-inducing proteins Bax ((BCL2 associated X, apoptosis regulator) and Bim (Bcl-2 interacting mediator, BCL2L11, BCL2 like 11) , Bcl-2 (B-cell/CLL lymphoma 2, also called BCL2 apoptosis regulator) and Bcl-xL (B-cell lymphoma-extra large, BCL2L1, BCL2 like 1), which are apoptosis inhibitor proteins.
  • Bcl-2 B-cell/CLL lymphoma 2
  • Bcl-xL B-cell lymphoma-extra large, BCL2L1, BCL2 like 1
  • RNase and proteins that inactivate RNase include barnase and barstar.
  • An example of the amino acid sequence of barnase is shown in SEQ ID NO: 42
  • an example of the amino acid sequence of barstar is shown in SEQ ID NO: 43, but other types of barnase and barstar can also be suitably used.
  • the barnase and barstar genes that can be encoded by the first mRNA or the second mRNA in this embodiment refer to DNAs encoding barnase and barstar proteins, respectively. Examples include DNAs having nucleotide sequences of the sequence represented by ID 75093094 (Barnase), or their transcript variants, splice variants and homologues.
  • it may be a DNA having a degree of complementarity that allows it to hybridize to nucleic acids having these sequences under stringent conditions.
  • Stringent conditions are melting of nucleic acids that bind complexes or probes, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on temperature (Tm). For example, the conditions for washing after hybridization are usually about "1 ⁇ SSC, 0.1% SDS, 37° C.”.
  • the complementary strand is preferably one that maintains a hybridized state with the target positive strand even after washing under such conditions.
  • such a complementary strand includes a strand consisting of a base sequence that is completely complementary to the base sequence of the target positive strand, and at least 90%, preferably 95% or more, more preferably 95% or more of the strand.
  • the Bax, Bim, Bcl-xL, and Bcl-2 genes that can be encoded by the first mRNA or the second mRNA in this embodiment contain DNAs encoding Bax, Bim, Bcl-xL, and Bcl-2 proteins, respectively.
  • sequences represented by NCBI-defined Gene ID 581 (Bax), Gene ID 10018 (BimEL), Gene ID 598 (Bcl-xL), Gene ID 596 (Bcl-2), or transcriptional variants thereof DNAs having transcript variants, splice variants and homologous nucleotide sequences are included.
  • it may be a DNA having a degree of complementarity that allows it to hybridize to nucleic acids having these sequences under stringent conditions. Stringent conditions are defined as above.
  • the nucleic acid sequence (1a) is a nucleic acid sequence that is specifically recognized by miRNA activated in the target cell
  • the nucleic acid sequence (1b) is preferably an anti-lethal gene.
  • the nucleic acid sequence (1a) is a nucleic acid sequence that is specifically recognized by miRNAs activated in non-target cells
  • the nucleic acid sequence (1b) is preferably a lethal gene.
  • (1a) and (1b) are operably linked, and (1a) is linked to the 5′ side of (1b) in the first embodiment, and (1a) is ( There can be a second embodiment linked to the 3' side of 1b) and an embodiment comprising both. Each aspect will be described.
  • the first mRNA has the nucleic acid sequence (1a) on the 5' side of the nucleic acid sequence (1b).
  • the 5'-UTR may be a structure in which [Cap structure or Cap analog] and [nucleic acid sequence of (1a)] are linked in order from the 5' end.
  • the Cap structure may be a 7-methylguanosine 5' phosphate.
  • the Cap analog is a modified structure recognized by eIF4E, which is a translation initiation factor similar to the Cap structure, and is manufactured by Ambion's Anti-Reverse Cap Analog (ARCA ), m7G(5')ppp(5')G RNA Cap Structure Analog from New England Biolabs, CleanCap from TriLink, and the like.
  • Cap analogs may be other modified structures recognized by translation initiation factors.
  • the 3' side of the Cap structure or Cap analog and the 5' side of the nucleic acid sequence of (1a) may contain an arbitrary nucleic acid sequence of, for example, about 0 to 50 bases, preferably about 0 to 30 bases. good. At least one nucleic acid sequence of (1a) may be included, but 2 repeats, 3 repeats, 4 repeats, or more repeats of the nucleic acid sequence of (1a) are included in the 5'-UTR good too.
  • the 3' end of the 5'-UTR, which is the 3' side of the nucleic acid sequence of (1a) 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 and do not specifically interact with the second and third mRNAs.
  • AUG which serves as an initiation codon, does not exist within the 5'-UTR.
  • frameshift can be avoided by adding one or two bases to the end of the sequence.
  • a termination codon sequence may be added outside of the nucleic acid sequence (1a) counted in units of 3 bases from the aforementioned AUG.
  • one or more bases of AUG can be converted into arbitrary bases for use as long as they do not affect the interaction with the protein.
  • the coding region of the first mRNA according to the first aspect comprises the nucleic acid sequence of (1b).
  • the nucleic acid sequence of (1b) is as described above.
  • a nucleic acid sequence encoding a fluorescent protein may be included.
  • the 3'-UTR of the first mRNA according to the first aspect contains PolyA tail.
  • PolyA tail may have a total length of A of 50 mer or more, and may contain nucleic acid bases other than A in the middle.
  • the 5' side of PolyA tail may also contain the nucleic acid sequence of (1a).
  • the first mRNA has the nucleic acid sequence (1a) on the 3' side of the nucleic acid sequence (1b).
  • the 5'-UTR consists of a nucleic acid sequence having a [Cap structure or Cap analogue] at the 5' end and a total base length of about 30 to 100 bases.
  • the nucleic acid sequence that constitutes the 5'-UTR in this case can be determined in the same manner as the arbitrary nucleic acid sequence of the first aspect.
  • the coding region of the first mRNA according to the second aspect may be the same as the coding region of the first mRNA according to the first aspect.
  • the 3'-UTR of the first mRNA according to the second aspect may have a structure in which [nucleic acid sequence of (1a)] and [Poly A tail] are linked in order from the 5' end.
  • the 3'-UTR of the first mRNA according to the second aspect may have a structure in which [nucleic acid sequence of (1a)] is inserted into Poly A tail.
  • the first mRNA may have a structure combining the first aspect and the second aspect. In this case, both the 5'-UTR and the 3'-UTR are provided with the [nucleic acid sequence of (1a)].
  • the second mRNA is an ON switch mRNA; (2a) a nucleic acid sequence encoding an anti-lethal gene that inactivates the lethal gene of (1b) or a lethal gene that is inactivated by the anti-lethal gene of (1b); (2b) PolyA tail provided on the 3' side of (2a); (2c) a nucleic acid sequence that is specifically recognized by miRNA activated in the target cell or non-target cell provided on the 3' side of (2b); (2d) a translation suppression sequence provided on the 3' side of (2c).
  • ON switch mRNA an mRNA switch whose translation is performed in the presence of a specific miRNA activity and whose translation is repressed in the absence of the miRNA activity is referred to as an ON switch mRNA.
  • the second mRNA is the ON switch mRNA.
  • ON switch mRNA may be abbreviated as ON switch or switch mRNA.
  • the nucleic acid sequences (2a), (2b), (2c), and (2d) are linked in this order from 5' to 3'.
  • a more specific structure of the second mRNA may be a structure in which a 5'-UTR, a coding region, and a 3'-UTR are linked in the 5' to 3' direction of the mRNA molecule.
  • the 5'-UTR of the second mRNA has a [Cap structure or Cap analog] at the 5' end and consists of a nucleic acid sequence with a total base length of about 30-100. This nucleic acid sequence does not have the nucleic acid sequence of (1a).
  • the 5'-UTR of the second mRNA can be determined similarly to any nucleic acid sequence that may be included in the 5'-UTR according to the second aspect of the first mRNA.
  • the coding region of the second mRNA contains the nucleic acid sequence of (2a).
  • the nucleic acid sequence of (2a) is determined in relation to the nucleic acid sequence of (1b) of the first mRNA.
  • the nucleic acid sequence of (2a) of the second mRNA is an anti-lethal gene that inactivates the lethal gene.
  • nucleic acid sequence (1b) of the first mRNA is a nucleic acid sequence encoding a specific anti-lethal gene
  • nucleic acid sequence (2a) of the second mRNA is inactivated by the anti-lethal gene. It is a nucleic acid sequence encoding a lethal gene.
  • the 3'-UTR of the second mRNA contains the nucleic acid sequences of (2b), (2c), (2d) in this order from 5' to 3'. It is preferred that no arbitrary nucleic acid sequence is included between the nucleic acid sequences (2b) and (2c). Any nucleic acid sequence, even if included, can be 10 bases or less, preferably 2 bases or less. Any nucleic acid sequence within 500 bases may be included between the nucleic acid sequences of (2c) and (2d).
  • the PolyA tail of (2b) may be the same as the polyA tail of the first mRNA.
  • the nucleic acid sequence of (2c) is a nucleic acid sequence specifically recognized by miRNAs activated in target cells or non-target cells.
  • the miRNAs that are active in target cells or non-target cells are identical to the miRNAs defined in the nucleic acid sequences of (1a).
  • the nucleic acid sequence of (2c) may be the same as (1a) or partially different from (1a) as long as it is specifically recognized by the miRNA.
  • the nucleic acid sequence of (1a) is identical to the nucleic acid sequence of (2c), preferably a complementary sequence to miRNAs that are activated in target or non-target cells.
  • the nucleic acid sequence of (2d) is bound downstream of the nucleic acid sequence of (2c), and can suppress translation of the second mRNA in the absence of miRNA that specifically recognizes the nucleic acid sequence of (2c). It is a simple translation repressing sequence.
  • This translation suppression sequence is sometimes referred to as an "additional sequence" in the present invention.
  • the translation suppression sequence is not particularly limited and may be any nucleic acid sequence.
  • the translation inhibitory sequence may be, for example, a nucleic acid sequence consisting of 5 or more bases, regardless of the type and sequence of the bases.
  • nucleic acid sequence (2d) consists of 5 or more bases
  • translation of the nucleic acid sequence (2a) is turned off in the absence of miRNA that specifically recognizes the nucleic acid sequence (2c).
  • Hold (translation repressed state) translation of the nucleic acid sequence of (2a) can be turned ON in the presence of miRNA that specifically recognizes the nucleic acid sequence of (2c), and the fold-change is 2 or more has been confirmed.
  • nucleic acid sequences consisting of 5 or more bases include nucleic acid sequences of 5 to 10 bases having the same nucleobase, and 10 to 20 bases in which two or more types of nucleic acids having different nucleobases are continuously linked.
  • nucleic acid sequence consisting of 5 or more bases may optionally consist 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, 1500 or more bases. It may be an array.
  • the third mRNA is an mRNA that contains a nucleic acid sequence encoding a drug-resistant gene and does not have the nucleic acid sequence of (1a). That is, the third mRNA is an mRNA that expresses a drug resistance protein independently of the activity of the introduced intracellular miRNA.
  • a more specific structure of the third mRNA may be a structure in which the 5'-UTR, the coding region, and the 3'-UTR are linked in order from the 5' side of the mRNA molecule.
  • the 5'-UTR of the third mRNA can be designed in the same configuration as the 5'-UTR of the second mRNA.
  • the third mRNA coding region contains a nucleic acid sequence encoding a drug resistance gene.
  • a drug-resistant gene can be determined in relation to the drug used for culturing the cell population in step (II).
  • the drug is not particularly limited as long as it is a drug commonly used in culturing cells derived from mammals. Therefore, examples of drug-resistant genes include blasticidin resistance gene, G418 (geneticin) resistance gene, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, gentamicin resistance gene, tetracycline resistance gene, chloram Examples include, but are not limited to, phenicol resistance gene and the like.
  • the 3'-UTR of the third mRNA preferably does not have a nucleic acid sequence specifically recognized by miRNA and has a PolyA tail.
  • the polyA tail may be similar to the polyA tail of the first mRNA.
  • the first mRNA, the second mRNA and the third mRNA contain modified bases such as 1-methylpseudouridine, pseudouridine, and 5-methylcytidine in place of normal uridine and cytidine to reduce cytotoxicity. may contain.
  • modified bases such as 1-methylpseudouridine, pseudouridine, and 5-methylcytidine in place of normal uridine and cytidine to reduce cytotoxicity. may contain.
  • the positions of the modified bases can be all or part of them independently, and when they are part, they can be random positions at any ratio.
  • the first mRNA, second mRNA and third mRNA can be synthesized by a person skilled in the art by any method known in genetic engineering, once the molecular structures and nucleic acid sequences are determined according to the above. 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. It is an advantage of the present invention that synthetic mRNA molecules as designed can be obtained in a convenient manner.
  • the first mRNA, second mRNA and third mRNA can be prepared as separate RNA molecules, or can be prepared as one or two mRNA molecules. That is, any two or more of the first mRNA, the second mRNA and the third mRNA may exist on the same molecule. DNAs encoding them can also be prepared as separate DNA molecules, or can be prepared as one or two DNA molecules. In the latter case, each mRNA may be controlled by one promoter.
  • the first mRNA, the second mRNA and the third mRNA are prepared as separate RNA molecules, at least the first mRNA and the third mRNA among the first mRNA, the second mRNA and the third mRNA
  • the mRNA of 2 is co-introduced into the cell population. It is further preferred to co-introduce the first mRNA, the second mRNA and the third mRNA 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.
  • RNA molecules can be introduced directly into cells.
  • Advantages of the introduction of synthetic RNA molecules include that there is no integration into the genome, and that cells after introduction of mRNA switches can be easily used for medical applications.
  • DNA constructs such as RNA expression vectors can be used to introduce the first mRNA, second mRNA and third mRNA into the cell population.
  • an expression vector that encodes an mRNA molecule can be designed, and the expression vector can be directly introduced into cells using the same introduction method as described above.
  • Expression vectors encoding the sequences of the first mRNA, the second mRNA and the third mRNA can be those well known and commonly used in the art, for example, virus vectors, artificial chromosome vectors, plasmid vectors, transposons The expression system used (sometimes called a transposon vector) and the like can be mentioned.
  • viral vectors examples include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, Sendai viral vectors and the like.
  • artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC, PAC) and the like.
  • a plasmid vector mammalian plasmids in general can be used, and for example, an episomal vector can be used.
  • transposon vectors include expression vectors using piggyBac transposons. For example, but not limited to, the vectors disclosed in US Pat. No. 10,378,070 by the present inventors, which is incorporated herein by reference. .
  • the mRNA switch transcribed from the expression vector and produced within the cell can function like a directly introduced synthetic mRNA molecule.
  • the introduction amount and introduction ratio of the first mRNA, second mRNA, and third mRNA into the cell population vary depending on the type of target cell and the structure of the mRNA, and are not limited to specific amounts.
  • the amounts of the first mRNA and the second mRNA to be introduced are determined by a preliminary experiment or the like so that the expression level of the lethal gene in (1b) or (2a) is below the threshold for undesirable target cell death. can be done.
  • the culturing step is a step of culturing the cell population introduced with the first mRNA, the second mRNA and the third mRNA in the presence of a drug.
  • a drug corresponding to the drug resistance gene encoded by the third mRNA is used. Cultivation conditions vary depending on the cell population containing the cells of interest, and can be cultured for about 1 to 4 days using an appropriate medium in an appropriate temperature and atmosphere.
  • Step (III) Culturing Step in the Absence of Drugs Step (III) is an optional step, and is a step of culturing the cell population that has undergone step (II) in the absence of drugs.
  • Culture conditions vary depending on the cell population containing the cells of interest, and can be carried out by an appropriate method.
  • the main culture step may be, for example, a subculture step.
  • Subculture may be performed for one passage only, or may be performed for two or more passages.
  • By subculturing dead cells and the like adhering to the culture vessel can be removed, and the target cells can be purified with higher accuracy.
  • the cell population that has undergone step (III) can be improved in purity by about 1 to 4%, and the purity of the target cells can be up to 99% or higher. Purification is possible.
  • target cells can be specifically survived and cells other than target cells can be specifically killed, and a large amount of target cells can be produced without using a device such as a cell sorter. can be purified all at once in a short period of time.
  • the present invention can also be regarded as a method for producing a cell population enriched with the target cells, including the step of purifying the target cells by the purification method described above.
  • Target cell purification kit The present invention, according to another embodiment, also relates to a target cell purification kit.
  • a purification kit comprises a first mRNA, a second mRNA and a third mRNA, and optionally an agent.
  • the first mRNA, second mRNA, third mRNA, and drug may be the mRNA and drug described in the purification method above.
  • a medium suitable for culturing the target cells and instructions for handling the purification kit may be included. According to the target cell purification kit according to the present embodiment, target cells can be obtained safely and in large quantities with high purity.
  • Barstar was amplified by fusion PCR of six synthetic oligo DNAs (YF472, YF473, YF474, YF475, YF476, YF477) and digested with NcoI and BglII. The digested fragment was cloned into the previously constructed pSM vector or pCM vector having the same sequence as the pSM vector except for the drug resistance gene, and the nucleotide sequence was determined. Barstar's ORF was then amplified using primers YF783 and YF804 and cloned into the same linear vector described above.
  • Tables 2A, 2B, and 2C show the base sequences of the oligo-DNAs synthesized in Examples, the peptide sequences of Barstar and Barnase used, and the translation inhibitory sequences, and Tables 3A and 3A show the combinations of PCR primers and DNA fragments.
  • 3B The template DNA sequences of Bcl-xL, Bcl2, BimEL, and Bax used in Examples are shown in SEQ ID NOS:61-64.
  • Template DNA for in vitro transcription was amplified by PCR (TOYOBO) using synthetic oligo DNA (Eurofin or Fasmac, listed in Tables 1 and 2).
  • An mRNA template for simple gene expression was amplified from a vector containing 5'-UTR, ORF and 3'UTR using primers with T7 promoter and polyA tail.
  • a miRNA-responsive OFF-switch template was amplified from the same plasmid using a primer containing a sequence complementary to the miRNA in the 5'-UTR.
  • GE healthcare SpeedBeads magnetic carboxylate modified particles
  • QIAGEN MinElute PCR purification Kit
  • ARCA Anti-Reverse Cap Analog
  • RNA was introduced into cells using Lipofectamine MessengerMax (Thermo Fisher Scientific) according to the manufacturer's protocol.
  • Cell clusters after differentiation into cardiomyocytes for at least 13 days were transferred to a conical tube, and harvested after the cell clusters naturally fell off. The collected cell aggregates were suspended in a 2 mg/mL collagenase I solution containing 10 ⁇ g/mL DNase I, and rotated at 37° C. for 2 hours or longer.
  • the supernatant was aspirated to resuspend the cell mass pellet, which was cultured at 37°C for about 30 minutes with AccuMax (Nacali tesque). Cell suspensions were then pipetted to dissociate cell clumps into single cells and then diluted with fresh differentiation medium. The dissociated cell suspension was centrifuged at 120 xg for 5 minutes at room temperature and the supernatant was aspirated. Cells were resuspended in differentiation medium and counted with Countess II (Thermo Fisher Scientific).
  • T302a-5p-responsive Barnase OFF switch and T302a-5p-responsive Barstar ON switch, and blasticidin-resistant mRNA (Bsd mRNA) were transfected using Lipofectamine Messenger MAX Transfection Reagent (ThermoFisher) according to the manufacturer's protocol. Four hours after transfection, the medium was changed to StemFit AK02N containing blasticidin (160 ⁇ g/mL). Before flow cytometry analysis, cells were washed with PBS and imaged with Cytell Cell Imaging System (GE Healthcare Life Science).
  • Embryoid bodies after differentiating cardiomyocytes for more than 14 days were collected and treated with collagenase (2 mg/mL collagenase I (SIGMA) and 10 ⁇ g/mL DNase I (EMD MilliPore)) at 37°C for 2 hours. , and treated with AccuMax at 37°C for 30 minutes.
  • Cells were dissociated by pipetting with differentiation-inducing medium and washed once with the same medium. Cells were reverse transfected with the RNAs listed in Table 4 and plated on fibronectin (Sigma) coated multiwell plates.
  • HeLa or 293FT cells from HeLa/293FT co-culture Purification of HeLa or 293FT cells from HeLa/293FT co-cultures was performed using a combination of the apoptotic gene as the lethal gene and the anti-apoptotic gene as the anti-lethal gene. Apoptotic gene/anti-apoptotic gene combinations were tested for Bax/Bcl-xL, Bax/Bcl-2, Bim/Bcl-xL. To distinguish between cells, HeLa cells (HeLa-hmAG1) constitutively expressing hmAG1-M9 (a green fluorescent protein linked to a nuclear localization signal) and iRFP670-M9 (a red fluorescent protein linked to a nuclear localization signal) were used.
  • miR-21-5p-responsive Bcl-xL ON switch and miR-21-5p-responsive Bax OFF switch, or miR-21-5p-responsive Bcl-xL OFF switch and miR-21-5p-responsive Bax ON switch were treated with Lipofectamine. Transfections were performed using MessengerMAX Transfection Reagent (ThermoFisher) according to the manufacturer's protocol.
  • miR-21-5p-responsive Bcl-2 ON switch and miR-21-5p-responsive Bax OFF switch were performed using Lipofectamine MessengerMAX Transfection Reagent (ThermoFisher) according to the manufacturer's protocol.
  • One day after transfection dead cells were washed away with medium, and cells adhering to the wells were observed under a fluorescence microscope (CQ1). After that, AccuMax (Funakoshi) was added to the cells and incubated at 37°C, 5% CO 2 for 10 minutes.
  • iPS cells from HeLa/iPS co-culture Purification of iPS cells from HeLa/iPS co-culture was performed using a combination of apoptotic genes as lethal genes and anti-apoptotic genes as anti-lethal genes. Apoptotic gene/anti-apoptotic gene combinations were tested for Bax/Bcl-2, Bim/Bcl-xL, Bax/Bcl-xL. To identify the cells, HeLa cells that constitutively express iRFP670 (HeLa-iRFP670) and iPS cells that constitutively express EGFP (iPS-EGFP) were mixed and seeded.
  • iRFP670 HeLa-iRFP670
  • iPS-EGFP iPS-EGFP
  • miR-302a-5p-responsive BclxL or Bcl-2 ON switch and miR-302a-5p-responsive Bax or Bim OFF switch were transfected using Lipofectamine Messenger MAX Transfection Reagent (ThermoFisher) according to the manufacturer's protocol.
  • the amount introduced for the ON switch was 133.4 ng
  • the amount introduced for the OFF switch was 66.6 ng.
  • Two days after transfection of the OFF switch dead cells were washed away with medium, and cells adhering to the wells were observed under a fluorescence microscope (CQ1). After that, AccuMax (Funakoshi) was added to the cells and incubated at 37°C, 5% CO 2 for 10 minutes.
  • miRNA-responsive ON-switch-mRNAs (miRNA-ON-switches) that upregulate gene expression in response to target miRNAs (Fig. 1A, right panel). It is known that mRNA in nature terminates with a polyA tail, and translation is promoted when the polyA tail is recognized by a polyA-binding protein.
  • anti-miRNA a sequence complementary to the target miRNA
  • Ex an extra sequence
  • a complementary sequence of miR-21-5p (miR-21-5p is highly expressed in cancer cells) is introduced downstream of the polyA tail of the mRNA encoding EGFP, and a translational repression sequence is further downstream of it.
  • miR-21-Ex495nt was introduced (switch-EGFP mRNA, Fig. 1B). Ex495nt means that the translational repression sequence consists of 495 bases.
  • HeLa and 293FT cells were cotransfected with iRFP670 mRNA and switch-EGFP mRNA as an internal control. HeLa and 293FT cells differed in their expression levels of miR-21-5p, with high activity of miR-21-5p in HeLa and low activity in 293FT.
  • EGFP expression from the switch mRNA was increased in HeLa cells, but not in 293FT cells, compared to the control mRNA with the miR-21-5p shuffle sequence (Fig. 1B, 1C and FIG. 7).
  • mRNA miR-21-Ex1250nt
  • it showed activity similar to that of miR-21-Ex 495nt, but the translational activity in HeLa cells was low. efficiency did not improve.
  • mRNA with a CAG repeat at the 5'-UTR and a GUC repeat downstream of the complementary sequence of miR-21-5p we designed an mRNA with a CAG repeat at the 5'-UTR and a GUC repeat downstream of the complementary sequence of miR-21-5p, and expected translational repression by the interaction between the CAG and GUC sequences.
  • the protein expression level of this mRNA was also increased in HeLa cells, although not as much as miR-21-495nt, but not in 293FT cells (Figs. 1B, 1C and 7). Based on the data obtained, a miRNA-ON switch with 495nt was used for the following studies. In addition, miRNA-ON switches using various other sequences were also tested as translation-repressing sequences.
  • a miRNA switch and a miRNA inhibitor or mimic were co-introduced into HeLa cells, and one day after introduction, the EGFP/iRFP670 ratio was confirmed using a fluorescence microscope and a flow cytometer (Fig. 2).
  • a miR-21-5p-responsive OFF switch in which a sequence completely complementary to miR-21-5p was inserted into the 5'-UTR of the mRNA, and suppressed translation in the presence of miR-21-5p. (Fig. 2A, middle row).
  • miRNA-ON switches function to detect cells for stem cell therapy (i.e., human iPSCs). Examined. The activity of miR-302a-5p is high in iPSCs and low in differentiated cell types, suggesting that miR-302a-5p contains a sequence complementary to miR-302a-5p after the polyA teil of the EGFP-encoding mRNA. Built -ON switch. In addition, miR302a-5p-OFF switch containing a sequence perfectly complementary to miR-21-5p in the 5'-UTR was constructed as a control.
  • miR-302a-5p-ON or -OFF switch When miR-302a-5p-ON or -OFF switch was introduced into human iPSCs (strain 201B7) and 293FT cells, miR-302a-5p-OFF switch suppressed EGFP expression in iPSCs but not in 293FT cells. didn't.
  • the miR-302a-5p-ON switch activated EGFP expression only in iPSCs (FIGS. 2E and 2F).
  • Flow cytometer two-dimensional plots observed iPSCs and 293FT cells as separate populations, confirming that miR-302a-5p-ON switch clearly distinguishes iPSCs (Fig. 2G).
  • the miR-302a-5p-ON and -OFF switches exhibited opposite behavior towards the iPSC target miR-302a-5p. This indicates that the ON switch senses miR-302a-5p and selectively activates translation, distinguishing iPSCs from other miR-302a-5p-inactive cells by fluorescent protein expression. (Fig. 2H).
  • the gene expression level of the introduced gene shows a wide distribution due to differences in the amount of introduced DNA/RNA and protein expression level in each cell. Therefore, if the lethal gene is controlled by a single OFF switch (e.g., the Bim-OFF switch), the group showing high expression levels of the lethal gene in the population of target cells to be purified is the non-target cell to be killed. Overlap population (Fig. 8A). Therefore, some non-target cells survive. This phenomenon causes contamination and affects the efficiency of cell purification. When the amount of transgene was adjusted to increase purity, leaky expression of the lethal gene resulted in a significant number of target cells in the region indicated by the fine dots (Fig. 3A left or green in the overlap region in Fig. 8B). distributions or dots) are eliminated. Therefore, it would be difficult to obtain a sufficient number of target cells using a single OFF switch that controls the level of a single pro-apoptotic gene.
  • a single OFF switch e.g., the Bim-OFF switch
  • the switch pair shown in Figure 4A was introduced into HeLa cells and 293FT cells, and the cells were observed under a microscope. As a result, cells with high Barnase activity detached from the bottom surface and exhibited an abnormal morphology (Fig. 4B). In contrast, cells with negligible or no Barnase activity exhibited normal morphology. As expected, either HeLa or 293FT cells were selectively killed when both ON and OFF switches were combined. In contrast to combinations of ON and OFF switches, introduction of either the Bn-OFF switch or the Bn-ON switch promoted cell death in both cell types, presumably due to leaky expression of Barnase. thought to be something.
  • HeLa cells used a combination of human thistle green (hmAG) and NLS (M9) (hmAG1-M9)
  • 293FT cells used a combination of iRFP670 and M9 (293FT-iRFP670-M9). Only one fluorescence (either HeLa or 293FT) was observed when cell purification was performed with the corresponding ON and OFF switch pairs (Fig. 4D).
  • the miR-21-5p-Bim-OFF switch encoding the apoptotic gene Bim was introduced into 293FT cells. and HeLa cells, and verified whether HeLa cells can be selected. Due to the low activity of miR-21-5p in 293FT cells, Bim expression is expected to lead to 293FT-specific cell death. However, we found that 293FT cells were not efficiently killed and HeLa cells were not purified, regardless of the amount of miR-21-5p-Bim-OFF switch (Fig. 9).
  • Bsd blasticidin resistance gene
  • iPSCs a stable expression strain expressing EGFP (green: iPSCs) and a stable expression strain expressing iRFP670 (red: HeLa) were used, respectively.
  • iPSCs green: iPSCs
  • iRFP670 red: HeLa
  • cardiomyocyte-specific miRNA-sensing ON/OFF switch human iPSCs were differentiated into cardiomyocytes and the cardiomyocytes were purified. For future applications, it will be important to purify cardiomyocytes differentiated from iPSCs without using a flow cytometer, as transplantation and cardiac therapy require a larger amount of cardiomyocytes.
  • the Bim-OFF switch which responds to miR-1 and miR-208, purified iPSC-derived cardiomyocytes with an efficiency of approximately 90%. Using the Bim-OFF switch also required optimizing the amount of mRNA introduced in each experiment, trading purity and yield.
  • FIG. 8A This is because the leak expression level of Bim in each cell varies, resulting in overlapping Bim expression levels in target iPSC-cardiomyocytes and non-target cell populations (FIGS. 8A and 8B).
  • Bn-OFF and Bs-ON switches that sense miR-1-3p and miR-208 (Fig. 6A). Cardiomyocytes differentiated from iPSCs were identified using GFP downstream of the MYH6 promoter. After iPSCs were differentiated into cardiomyocytes for more than two weeks, miR-208-Bn-OFF switch and miR-1-Bs-ON switch were introduced into the cell population. In addition, mRNA encoding the G418 resistance gene (aph) was introduced into the cells.
  • aph G418 resistance gene
  • GFP-positive cardiomyocytes were measured with a flow cytometer or fluorescence microscope (Fig. 6B, C, D). As a result, the purity of GFP-positive cells was improved to 91.6% by introducing an ON/OFF switch. Furthermore, by removing non-transfected cells and double-selecting non-target cells with G418, we successfully increased cardiomyocyte purity to over 95%.
  • Figures 11A, 11B depict the results of cell purification using miR-21-5p responsive ON and OFF switch combinations designed to purify HeLa or 293FT cells from HeLa/293FT co-cultures.
  • the combination of the miR-21-5p-responsive Bcl-xL OFF switch and the miR-21-5p-responsive Bax ON switch can purify 293FT to the extent that it contains more than 95% of the 293FT (Fig. 11A, middle).
  • the combination of the miR-21-5p-responsive Bcl-xL ON switch and the miR-21-5p-responsive Bax OFF switch could purify HeLa to include nearly 90% HeLa (Fig. 11A, right).
  • Figure 12 shows the results of purification of iPS cells using a combination of miR-302a-5p responsive ON and OFF switches designed to allow only iPS cells to survive from HeLa/iPS co-culture.
  • miR-302a-5p-responsive Bax OFF switch combined with miR-302a-5p-responsive Bcl-2 ON switch
  • miR-302a-5p-responsive Bim OFF switch combined with miR-302a-5p-responsive Bcl-xL ON switch
  • RNA transfection efficiency depends on various parameters such as cell type, cell number, and cell activity. 30 ng) can stably control Barnase activity.
  • the lethal RNase Barnase can self-regulate RNA-based circuits.
  • Co-introduced exogenous mRNA can be regulated by the activity of Barnase. Gene expression from co-introduced mRNA was almost completely suppressed in response to Barnase activity.
  • this system was applied to purify two types of cells: human iPSCs and iPSC-derived cardiomyocytes.
  • two types of cells could be efficiently selected from heterogeneous populations without using a cell sorter.
  • cells were approximately 95% pure after selection (FIGS. 4F, 10B, 6D), whereas iPSC purification yielded over 99% purity in a single passage. (Fig. 5C). It is believed that 5% of the population remained because moribund or dead cells adhering to the wells were not removed during washing (Fig. 6C). Thus, in cell culture, it is necessary to improve the purification efficiency of cells.
  • the cell purification system consisting of RNA only shown in this study has a low risk of inserting foreign RNA into the genome. Furthermore, large numbers of cells need to be processed simultaneously, as opposed to identifying cells with antibodies and analyzing them one by one with a flow cytometer, which is expensive and has the risk of contamination. No equipment required. Therefore, the cell sorting method according to the present invention is suitable for scale-up.
  • the RNA system according to the present invention can provide general-purpose techniques for preparing cells for transplantation and regenerative medicine in the future.

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Abstract

La présente invention concerne un procédé de purification cellulaire très précis pouvant être mis en œuvre sans utiliser de dispositif de tri cellulaire tel qu'un cytomètre de flux, afin de réduire au minimum l'influence des types cellulaires.　Un procédé de purification de cellules cibles selon la présente invention comprend les étapes suivantes : une étape d'introduction, dans une population cellulaire, d'un premier ARNm de type interrupteur OFF répondant aux miARN codant pour un gène létal ou un gène antilétal, d'un deuxième ARNm de type interrupteur ON répondant aux miARN codant pour un gène antilétal désactivant le gène létal ou un gène létal désactivé par le gène antilétal, et d'un troisième ARNm codant pour un gène pharmacorésistant ; et une étape de mise en culture de la population cellulaire en présence d'un médicament.
PCT/JP2022/040488 2021-10-29 2022-10-28 Procédé de purification cellulaire WO2023074873A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
WO2015141827A1 (fr) * 2014-03-20 2015-09-24 国立大学法人京都大学 Procédé de tri de cardiomyocytes
WO2016010119A1 (fr) * 2014-07-16 2016-01-21 国立大学法人京都大学 Procédé d'extraction de cellules différenciées
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

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
WO2015141827A1 (fr) * 2014-03-20 2015-09-24 国立大学法人京都大学 Procédé de tri de cardiomyocytes
WO2016010119A1 (fr) * 2014-07-16 2016-01-21 国立大学法人京都大学 Procédé d'extraction de cellules différenciées
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

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