WO2013146972A1 - Procédé pour réguler la fonction d'une cellule - Google Patents

Procédé pour réguler la fonction d'une cellule Download PDF

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WO2013146972A1
WO2013146972A1 PCT/JP2013/059173 JP2013059173W WO2013146972A1 WO 2013146972 A1 WO2013146972 A1 WO 2013146972A1 JP 2013059173 W JP2013059173 W JP 2013059173W WO 2013146972 A1 WO2013146972 A1 WO 2013146972A1
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nucleic acid
cell
cells
target
modified
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Japanese (ja)
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安田　賢二
英之 寺薗
賢徹 金
服部　明弘
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公益財団法人神奈川科学技術アカデミー
一般社団法人オンチップ・セロミクス・コンソーシアム
国立大学法人東京医科歯科大学
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Publication of WO2013146972A1 publication Critical patent/WO2013146972A1/fr

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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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
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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
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5032—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on intercellular interactions
    • G—PHYSICS
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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    • G01N33/5061—Muscle cells
    • G—PHYSICS
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/507—Pancreatic cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes

Definitions

  • the present invention relates to a method for producing and using a cell whose function is controlled using a nucleic acid chain that specifically binds to a target molecule on the surface of the target cell.
  • aptamers that have affinity for ions, sugar chains, proteins, molecules on the surface of living cells, and aptamers that determine the phosphorylation state of proteins. Aptamers are roughly classified into single-stranded DNA or single-stranded RNA, but other peptide aptamers have been reported. Among them, modified aptamers according to the purpose of use, such as aptamers using artificial bases and aptamers modified with organic molecules such as polyethylene glycol, have been developed.
  • aptamers are selected by two steps, SELEX (Systematic Evolution of Ligands by EXponential enrichment) proposed by C. Tuerk et al. And selection of the target nucleic acid molecules.
  • nucleic acid pool having a total length of about 60 to 140 bases consisting of a random sequence of 30 to 100 bases sandwiched between primer sequences of about 18 to 38 bases at both ends of 10 14 to 10 16 molecular species.
  • a sequence having affinity for the target molecule is selected from this nucleic acid pool.
  • aptamers having affinity for non-target molecules ("cross reactivity" referred to as antibodies) are also included. It is not unusual to exist. For this reason, aptamers that have affinity for target molecules but no affinity for non-target molecules in samples in which many types of molecules that are supposed to be used are mixed can be selected by the counter selection method (Counter Selection). ).
  • RNA aptamers to identify. This first selects RNA that binds to the phosphorylated protein ppERK2, and simultaneously introduces the selected RNA into the non-phosphorylated protein ERK2 as a non-target protein, and recovers the RNA that did not bind to it. To obtain the RNA aptamer of interest.
  • differentiated cells using human stem cells has also greatly advanced by the invention of human iPS cells, but as a problem there, differentiated cells produced by induction of differentiation by chemical substances are not the same population of cells, It is a heterogeneous cell population in which substances such as ion channels and cell surface proteins that are expressed are slightly different, and the limitation of cell differentiation control by such a differentiation induction technique at present is an issue.
  • aptamers prepared from conventional nucleic acids can be used as markers for identifying labeled antigenic substances on the cell surface or as probes for cell purification targeting these target substances. However, it is not used as a tool to control the function of cells used in in vitro measurements.
  • ion channel function control uses an agent that binds to a specific ion channel to control the operation and function of the ion channel.
  • this method is used, all cells in the cell culture medium to which the drug has been added will be controlled in the same way, for example. It was difficult to use controlled cells in the same culture.
  • cells that express the same function due to drugs that block specific ion channels, etc. coexist as cells that exhibit different functions in the same culture environment by the function control method of target molecules on different cell surfaces. It was difficult to make it happen.
  • the object of the present invention is to control the functional state of a cell to various states, which has been difficult with conventional techniques, and to allow the controlled cell to coexist as a heterogeneous functional cell in the same environment. It is to propose a method to use. Moreover, it is providing the method of changing the state of a cell during cell culture.
  • the present invention provides the following.
  • [1] (i) A target cell or a population of target cells is prepared, and a nucleic acid, a nucleic acid derivative, or a modified nucleic acid that specifically binds to a target molecule expressed on the cell surface of the target cell is provided on the surface of the target cell.
  • nucleic acid-modified cell by specifically binding to the target molecule expressed in (Ii) removing a nucleic acid that did not specifically bind to the target molecule expressed on the surface of the target cell; (Iii) A nucleic acid, a nucleic acid derivative or a nucleic acid modification that specifically binds to a target molecule different from the target molecule expressed on the cell surface of the target cell with respect to the nucleic acid-modified cell from the step (ii) Performing the above steps (i) and (ii) using a body, (Iv) Two or more types of different nucleic acid-modified cells prepared by repeating the operations of the above steps (i) and (ii) or the above step (iii) on a culture substrate in a common cell culture environment A step of constructing a cell network by controlling and arranging the spatial arrangement of each nucleic acid-modified cell, A cell function control method comprising: [2] Furthermore, (V) the nucleic acid that specifically binds to
  • nucleic acid is a single-stranded nucleic acid or an aptamer.
  • target cells are cardiomyocytes, nerve cells, fibroblasts, glial cells, liver cells, pancreatic cells, or stem cells.
  • a cell network comprising nucleic acid-modified cells produced by the cell function control method according to any one of [1] to [4] above.
  • a functionally controlled cell can be easily obtained by modifying a nucleic acid chain such as a DNA chain that binds to a specific target molecule of a specific cell in a short time.
  • target cells having different functions can be arranged on a substrate, and a nucleic acid chain such as a DNA chain used for controlling the function of the cell surface after the arrangement can be removed by a nuclease at an arbitrary time.
  • FIG. 1 is a flow diagram for explaining a cell function control method using a nucleic acid such as single-stranded DNA, a nucleic acid derivative or a nucleic acid modification having a function of specifically binding to a specific target molecule on the cell surface of the present invention.
  • the cells targeted in the present invention include, but are not limited to, various cells such as cardiomyocytes, nerve cells, fibroblasts, glial cells, liver cells, pancreatic cells, and stem cells.
  • various molecules such as a receptor protein, a surface antigen, and an ion channel protein exist as molecules expressed on the cell surface of interest in the present invention, but are not limited thereto.
  • an ion channel protein will be described as an example.
  • a nucleic acid such as single-stranded DNA or a nucleic acid derivative or a nucleic acid modified substance 1031, 1032, 1033 that specifically binds to a molecule such as a target ion channel protein 1021, 1022, 1023 on the surface of the cell 101 is modified to each cell. Then, after controlling the functions of the molecules such as the target ion channel proteins 1021, 1022, and 1023, these different cells 1011, 1012, and 1013 are arranged on the same cell culture substrate 104, so that the cells having different functions can be simply used.
  • 2 shows an example of the process of the method of the present invention in which the spatial distribution is arranged in the same cell culture environment.
  • nucleic acid includes DNA and RNA.
  • the nucleic acid chain is mainly described by taking the DNA chain as an example, but the target is not degraded by a proteolytic enzyme such as a DNA chain, an RNA chain, a nucleic acid derivative or a modified nucleic acid, but a nucleic acid chain degrading enzyme (nuclease) Can be disassembled.
  • a proteolytic enzyme such as a DNA chain, an RNA chain, a nucleic acid derivative or a modified nucleic acid, but a nucleic acid chain degrading enzyme (nuclease) Can be disassembled.
  • Nucleic acid chains such as aptamers are easily degraded by the action of nucleolytic enzymes present in the living body, and therefore various methods for modifying nucleic acid chains to impart degradation resistance to nucleic acid chains have been developed ( Example: JP 2010-115177).
  • a method in which an aptamer is obtained by the SELEX method and then chemically modified (eg, fluorination, PEGylation, etc.), or an artificial nucleic acid that has been previously chemically modified to a nucleic acid molecule is used.
  • nuclease resistance can be conferred by substituting S or N for part of the main chain at the 5 'or 3' end of the main chain of the nucleic acid chain (eg, DNA or RNA sugars). 4'-thio DNA or RNA in which O (oxygen) is replaced with S (sulfur). (Akira Matsuda "Development and Research of Nuclease-Resistant Chemically Modified Nucleic Acids" Pharmaceutical Journal, 2011, 131, 285-298. DOI: 10.1248 /yakushi.131.285).
  • nucleic acid derivative or “modified nucleic acid” refers to a nucleic acid chain that has been chemically modified to improve nuclease resistance.
  • a nucleic acid “specifically binds” to a protein or a fragment thereof means that the nucleic acid is different from the specific amino acid sequence of the protein or the fragment thereof. ) Means to bind irreversibly with an affinity substantially higher than the affinity for the amino acid sequence, which is the same as the binding of the antibody molecule to the antigen molecule in the antigen-antibody reaction.
  • substantially high affinity means that the specific amino acid sequence can be distinguished from other amino acid sequences and detected by a desired measurement device or method available to those skilled in the art.
  • Bins affinity typically the binding constant (K a ) is at least 10 7 M ⁇ 1 , preferably at least 10 8 M ⁇ 1 , more preferably 10 9 M ⁇ 1 , even more preferably It means a binding affinity such as 10 10 M ⁇ 1 , 10 11 M ⁇ 1 , 10 12 M ⁇ 1 or higher, for example up to 10 13 M ⁇ 1 or higher.
  • nucleic acid chains that specifically bind to a plurality of different types of molecules 1021, 1022, and 1023 expressed on the surface of a cell 101 (hereinafter referred to as aptamers).
  • Cells 1011, 1012, and 1013 having different functions can be formed by combining 1031, 1032, and 1033.
  • one type of aptamer is bound to each cell to create a cell in which one different type of cell surface molecule is functionally controlled.
  • another type of nucleic acid chain is added to each cell. It is also possible to create and use a cell in which the functions of both molecules are controlled by binding nucleic acid chains that specifically bind to the molecules 1021 and 1022, respectively, by repeating the same steps.
  • the cells 1011, 1012, and 1013 created from the cells 101 by this process are arranged on the chip 104, for example, by placing each type of cell in a band shape, or a cell sheet in which different functional cell regions and their boundaries exist or A cellular network can be created. Further, in this method, by adding an aptamer nucleolytic enzyme, the aptamer that has controlled the function of the cell can be decomposed to return the cell function to the original state.
  • the feature of this method is that cells having different function controls can coexist on the same chip 104. Even if drugs that specifically block specific ion channels are used, blocking of specific ion channel molecules can be realized, but in this case, all cells in contact with this solution are equally blocked. As shown in the chip 104 of FIG. 1, cells having different functional states cannot coexist on one chip.
  • FIG. 1 a state in which the cells 1011, 1012, and 1013 are distributed in a sheet shape is schematically shown.
  • FIG. 2 as an example of another cell arrangement technique, FIGS. In b) and (c), cardiomyocytes 2011 differentiated into ventricular myotypes and the membrane potential that blocked the K1 ion channel by an aptamer that specifically binds to the K1 ion channel of the cardiomyocyte 2011 increased and became unstable.
  • a network is constructed in units of one cell by combining the cardiomyocytes 2012.
  • the cell 2012 can be made from the same ventricular myocyte 2011 and used as a pseudo-heterogeneous cell in the same cell network.
  • FIG. 3 shows the use of this technique for a neural cell network. It is important to constitutively elucidate the function of chemical substance conduction between cells in a neuronal network, but it was difficult to transmit only specific secreted substances.
  • FIG. 3 schematically illustrates a method for solving this problem. For example, when there is a function of releasing two transmitters 304 and 305 existing on the surface of the synapse 303 that connects the two nerve cells 301 and 302, the transmitter is contained in the culture medium. By conducting an experiment of neurotransmission by mixing an aptamer that blocks 305, measuring the transmission characteristics of only the transmitter 304, and introducing the aptamer-degrading enzyme, the transmitter 305 that was originally supposed to be secreted is obtained. Released and its response can be measured.
  • heterogeneous cells can be easily obtained by modifying a nucleic acid chain such as a DNA chain that specifically binds to a target molecule on the cell surface, thus contributing to research development in the drug discovery and medical fields. Can do.
  • nucleic acid chains such as DNA strands that bind to the target molecules 2011, 2012 ... cardiomyocytes 301, 302 ... nerve cells 303 ... Synaptic connections 304, 305 ... Neurotransmitters

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Abstract

L'invention concerne un procédé permettant de purifier et de collecter un brin d'acide nucléique, tels qu'un brin d'ADN capable de se lier spécifiquement à une protéine cible présente sur une surface d'une cellule donnée. Selon le procédé, un brin d'acide nucléique, tel qu'un brin d'ADN, est lié à une protéine ou à une molécule similaire présente sur la surface d'une cellule. Ainsi, plusieurs différents types de cellules à fonction régulée sont produits et les cellules sont ménagées spatialement sur un substrat afin de permettre à différentes celules de co-exister spatialement.
PCT/JP2013/059173 2012-03-29 2013-03-28 Procédé pour réguler la fonction d'une cellule WO2013146972A1 (fr)

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JP2012077207A JP2013202018A (ja) 2012-03-29 2012-03-29 細胞機能制御方法
JP2012-077207 2012-03-29

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Citations (5)

* Cited by examiner, † Cited by third party
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JP2008005749A (ja) * 2006-06-28 2008-01-17 Onchip Biotechnologies Inc 細胞分離チップおよびこれを使用した細胞培養方法
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