WO2013146972A1 - Method for regulating function of cell - Google Patents

Method for regulating function of cell 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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    • 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
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    • G01N33/507—Pancreatic cells
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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

The purpose of the present invention is to purify and collect a nucleic acid strand, such as a DNA strand, that can bind specifically to a target protein present on the surface of a given cell. For achieving the purpose, a nucleic acid strand, such as a DNA strand, is bound to a protein or the like present on the surface of a cell. In this manner, multiple different types of function-regulated cells are produced, and the cells are spatially arranged on a substrate to thereby allow the different cells to co-exist spatially.

Description

細胞機能制御方法Cell function control method
　本発明は、標的細胞表面の標的分子に特異的に結合する核酸鎖を用いて、細胞の機能を制御した細胞を製造し利用する方法に関する。 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.
　一本鎖核酸はその高次構造により、特定の標的分子に対して結合能を有することが、１９９０年にC. Tuerkらのグループによって報告（非特許文献１：Science, 249, 505-510 (1990)）され、それらの核酸は「アプタマー」と命名された。これまでイオンや糖鎖、タンパク質、生細胞表面上の分子に親和性のあるアプタマー、さらにはタンパク質のリン酸化状態を判別するアプタマーなどが先行研究により多数報告されている。アプタマーは大別して一本鎖ＤＮＡまたは一本鎖ＲＮＡから成るが、他にもペプチドアプタマーなどが報告されている。またそれらの中には人工塩基を用いたアプタマー、ポリエチレングリコールなどの有機分子により修飾されたアプタマーなど、使用目的に合わせた修飾アプタマーも開発されている。 It was reported in 1990 by a group of C. Tuerk et al. That non-patent literature 1: Science, 249, 505-510 ( 1990)) and these nucleic acids were named “aptamers”. Previous studies have reported many 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.
　これらアプタマーは、C. Tuerkらによって提案されたＳＥＬＥＸ（Systematic Evolution of Ligands by EXponential enrichment）という、目的の核酸分子の選別と、それら分子の増幅という２つのステップによって選択される。 These 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.
　一般的には、まず、１０１４～１０１６分子種の、両端が１８～３８塩基ほどのプライマー配列に挟まれた３０～１００塩基のランダム配列からなる全長が約６０～１４０塩基の核酸プールを準備する。次に、この核酸プールから、標的分子に親和性をもつ配列を選別するが、この操作にはこれまで様々な方法が開発されている。一般に、標的分子のみを用いて親和性をもつ核酸を選別する場合、最終的に得られたアプタマーの中には、非標的分子にも親和性を持つ（抗体で言う“cross reactivity”）アプタマーが存在することも珍しくない。そのため、用いることが想定される多種類の分子が混合されているようなサンプルにおいて、標的分子には親和性をもつが非標的分子に親和性を持たないというアプタマーを、対向選別法（Counter Selection）によって選別することも行われている。 Generally, first, a 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. prepare. Next, a sequence having affinity for the target molecule is selected from this nucleic acid pool. Various methods have been developed for this operation. In general, when nucleic acids having affinity are selected using only target molecules, among the aptamers finally obtained, 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). ).
　その例として、２０００年にScott D Seiwertらによって報告（非特許文献２：Chemistry & Biology, 7, 833-843 (2000)）された、同じタンパク質のリン酸化状態を１０倍の親和性の違いで識別するアプタマーがある。これは、はじめリン酸化状態のタンパク質ppERK2に結合するＲＮＡを選別し、同時に非標的タンパク質として、リン酸化されていないタンパク質ERK2に選別されたＲＮＡを導入して、それに結合しなかったＲＮＡを回収する、という目的のＲＮＡアプタマーを取得するものである。 As an example, the phosphorylation state of the same protein reported in 2000 by Scott D Seiwert et al. (Non-Patent Document 2: Chemistry & Biology, 7, 833-843 (2000)) with a 10-fold affinity difference. There are 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.
　他方、ヒト幹細胞を用いた分化細胞の製造も、ヒトiPS細胞の発明によって大きく前進したが、そこでの課題として、化学物質による分化誘導で産生した分化細胞は、決して同一の細胞の集団ではなく、発現しているイオンチャンネルや細胞表面タンパク質等の物質が少しずつ異なるヘテロな細胞集団であり、現状におけるこのような分化誘導の手法での細胞分化制御についての限界が課題となっている。 On the other hand, the production of 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.
　このように、従来の核酸から作成したアプタマーは、細胞表面の標識抗原物質の同定をするためのマーカーとして用いたり、あるいは、この標的物質をターゲットにした細胞精製のためのプローブとして用いられて来たが、in vitro 計測で用いる細胞の機能を制御するためのツールとしての利用はなされていない。 Thus, 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.
　また、分化誘導によって作成したヘテロな細胞集団の状態を制御する技術として、従来は、たとえばイオンチャンネルの機能制御には、特定のイオンチャンネルに結合する薬剤を用いてイオンチャンネルの動作、機能を制御することがなされて来たが、この手法を用いると、薬剤が添加された細胞培養液中のすべての細胞の状態を同一にブロックする等の制御となってしまい、異なる制御操作によって異なる状態に制御された細胞を同じ培養液中で用いることは難しかった。 In addition, as a technology for controlling the state of heterogeneous cell populations created by induction of differentiation, conventionally, for example, ion channel function control uses an agent that binds to a specific ion channel to control the operation and function of the ion channel. However, if 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.
　あるいは、上記特定のイオンチャンネル等をブロックする薬物による同一の機能を発現している細胞の状態をそれぞれ異なる細胞表面のターゲット分子の機能制御手法によって同一培養環境内で、異なる機能を示す細胞として共存させることは難しかった。 Alternatively, 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.
　したがって、本発明の目的は、従来の手法では困難であった、細胞の機能状態をさまざまな状態に制御して、この制御された細胞を同一の環境内で、異種の機能細胞として共存させて利用する方法を提案することである。
　また、細胞培養中に、細胞の状態を変化させる手法を提供することである。 Therefore, 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.
　したがって、本発明は、以下を提供する。
［１］（ｉ）標的細胞または標的細胞の集団を準備し、上記標的細胞の細胞表面に発現している標的分子に特異的に結合する核酸または核酸誘導体もしくは核酸修飾体を上記標的細胞の表面に発現している上記標的分子に特異的に結合させて、核酸修飾細胞を作製する工程、
　（ｉｉ）上記標的細胞の表面に発現している上記標的分子に特異的に結合しなかった核酸を除去する工程、
　（ｉｉｉ）上記工程（ｉｉ）からの核酸修飾細胞に対して、上記標的細胞の細胞表面に発現している、上記標的分子とは異なる標的分子に特異的に結合する核酸または核酸誘導体あるいは核酸修飾体を用いて、上記工程（ｉ）および（ｉｉ）を行う工程、
　（ｉｖ）上記工程（ｉ）および（ｉｉ）の操作、または上記工程（ｉｉｉ）の操作を繰り返すことによって作製した２種類以上の異なる核酸修飾細胞を、共通の細胞培養環境中の培養基板上で、上記各核酸修飾細胞の空間配置を制御して配列して細胞ネットワークを構築する工程、
を含む、細胞機能制御方法。
［２］さらに、
　（ｖ）上記工程（ｉｖ）で培養している上記核酸修飾細胞の上記標的分子に特異的に結合する上記核酸または上記核酸誘導体もしくは上記核酸修飾体を分解させる核酸分解酵素を上記細胞培養環境中に導入する工程、を含む、上記［１］に記載の方法。
［３］上記核酸が一本鎖核酸またはアプタマーである、上記［１］または［２］に記載の細胞機能制御方法。
［４］上記標的細胞が、心筋細胞、神経細胞、線維芽細胞、グリア細胞、肝臓細胞、膵臓細胞、または幹細胞である、上記［１］～［３］のいずれかに記載の細胞機能制御方法。［５］上記［１］～［４］のいずれか一項に記載の細胞機能制御方法により作製された核酸修飾細胞を含む細胞ネットワーク。
［６］上記［５］に記載の細胞ネットワークを使用して細胞機能を検査する方法。 Accordingly, 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. Producing a 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 the target molecule of the nucleic acid-modified cell cultured in the step (iv) or the nucleolytic enzyme that degrades the nucleic acid derivative or the modified nucleic acid in the cell culture environment The method according to [1] above, which comprises a step of introducing into the above.
[3] The cell function control method according to [1] or [2] above, wherein the nucleic acid is a single-stranded nucleic acid or an aptamer.
[4] The cell function control method according to any one of the above [1] to [3], wherein the target cells are cardiomyocytes, nerve cells, fibroblasts, glial cells, liver cells, pancreatic cells, or stem cells. . [5] A cell network comprising nucleic acid-modified cells produced by the cell function control method according to any one of [1] to [4] above.
[6] A method for examining cell function using the cell network according to [5] above.
　本発明によれば、特定の細胞の特定のターゲット分子に結合するＤＮＡ鎖等の核酸鎖を短時間で修飾する事で、機能制御を行った細胞を簡便に得ることができる。また、異なる機能を持った標的細胞を基板上に配置し、配置後に細胞表面の機能制御に用いたＤＮＡ鎖等の核酸鎖を任意の時間にヌクレアーゼにより除去することができる。 According to the present invention, 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. In addition, 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.
　図１は本発明の細胞表面の特定の標的分子に特異的に結合する機能を有する１本鎖ＤＮＡ等の核酸または核酸誘導体あるいは核酸修飾体を用いた細胞機能制御方法を説明するフロー図である。本発明において対象とする細胞には、心筋細胞、神経細胞、線維芽細胞、グリア細胞、肝臓細胞、膵臓細胞、幹細胞等種々の細胞が含まれるがこれらに限定されない。また、本発明において対象とする細胞表面に発現する分子には、受容体タンパク質、表面抗原、イオンチャンネルタンパク質等種々の分子が存在するがこれらに限定されない。ここではイオンチャンネルタンパク質を例に説明する。すなわち、細胞１０１表面の標的イオンチャンネルタンパク質１０２１，１０２２，１０２３等の分子に特異的に結合する１本鎖ＤＮＡ等の核酸または核酸誘導体あるいは核酸修飾体１０３１，１０３２，１０３３を、それぞれの細胞に修飾して、標的イオンチャンネルタンパク質１０２１，１０２２，１０２３等の分子の機能を制御した後、これら異なる細胞１０１１，１０１２，１０１３を、同一の細胞培養基板１０４上に配置して、簡便に機能の異なる細胞を同一の細胞培養環境に空間分布を制御して配置する本発明の方法の過程の一例を示している。本明細書中、「核酸」は、ＤＮＡおよびＲＮＡを含むものとする。ここで核酸鎖については、主にＤＮＡ鎖を例に説明を行うが、対象はＤＮＡ鎖、ＲＮＡ鎖、または核酸誘導体あるいは核酸修飾体などのタンパク質分解酵素によって分解されないが核酸鎖分解酵素（ヌクレアーゼ）によって分解することができるものである。 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. In addition, 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. Here, an ion channel protein will be described as an example. That is, 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. In the present specification, “nucleic acid” includes DNA and RNA. Here, 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.
　なお、アプタマーのような核酸鎖は、生体中に存在する核酸分解酵素の作用を受けて分解しやすいことから、核酸鎖を修飾して核酸鎖に分解耐性を与える方法が種々開発されている（例：特開２０１０－１１５１７７）。例えば、ヌクレアーゼ耐性の向上については、SELEX法でアプタマーを得た後で化学修飾（例：フッ素化、PEG化、etc.）を施すという方法、あるいは予め核酸分子に化学修飾を施した人工核酸のライブラリの中からアプタマーを選別する方法等が開発されている（特開２００４－２３８３５３；ＷＯ２０１０／０２１３４４；Kuwahara et al., Nucleic Acids Res., (2008), 36, 4257）。あるいは、核酸鎖の主鎖の五員環や5'または3'末端での主鎖の一部をSやNで置換することによってヌクレアーゼ耐性を付与することもできる（例：DNAやRNAの糖部分のO (酸素) をS (硫黄) で置き換えた4’-チオDNAやRNA）（松田　彰「ヌクレアーゼ抵抗性化学修飾核酸の開発研究」薬学雑誌, 2011, 131, 285-298. DOI:10.1248/yakushi.131.285）。 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). For example, to improve nuclease resistance, 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. A method for selecting aptamers from a library has been developed (JP 2004-238353; WO 2010/021344; Kuwahara et al., Nucleic Acids Res., (2008), 36, 4257). Alternatively, 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).
　本明細書中、「核酸誘導体」または「核酸修飾体」とは、ヌクレアーゼ耐性を向上させるための化学修飾が施された核酸鎖のことをいうものとする。 In this specification, “nucleic acid derivative” or “modified nucleic acid” refers to a nucleic acid chain that has been chemically modified to improve nuclease resistance.
　本明細書中、ある核酸があるタンパク質またはその断片に「特異的に結合する」とは、その核酸が、そのタンパク質またはその断片の特定のアミノ酸配列に対して、他の（タンパク質またはその断片の）アミノ酸配列に対する親和性よりも、実質的に高い親和性で不可逆的に結合することを意味し、これは抗原抗体反応において抗体分子が抗原分子に結合する事と同意である。ここで、「実質的に高い親和性」とは、当業者に利用可能な所望の測定装置または方法によって、その特定のアミノ酸配列を他のアミノ酸配列から区別して検出することが可能な程度に高い親和性を意味し、典型的には、結合定数（Ｋａ）が少なくとも１０７Ｍ－１、好ましくは、少なくとも１０８Ｍ－１、より好ましくは、１０９Ｍ－１、さらにより好ましくは、１０１０Ｍ－１、１０１１Ｍ－１、１０１２Ｍ－１または
それより高い、例えば、最高で１０１３Ｍ－１またはそれより高いものであるような結合親和性を意味する。 In the present specification, 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. Here, “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. Means 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.
　図１に示すように、本発明の方法によれば、細胞１０１の表面に発現した複数の異なる種類の分子１０２１，１０２２，１０２３に対してそれぞれ特異的に結合する核酸鎖（以降アプタマーと呼ぶ）１０３１，１０３２，１０３３を結合させて、機能が異なる細胞１０１１，１０１２，１０１３を作ることができる。ここで、本実施例では、細胞にそれぞれ１種類のアプタマーを結合させ、異なる一種類の細胞表面分子が機能制御された細胞を作成しているが、それぞれの細胞について別の種類の核酸鎖を用いて同様の工程を繰り返す事で、たとえば分子１０２１と１０２２にそれぞれ特異的に結合する核酸鎖を結合させて２つの分子をともに機能制御した細胞を作成して用いても良い。 As shown in FIG. 1, according to the method of the present invention, 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. Here, in this example, 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. However, 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.
　次に、この工程によって細胞１０１から作成した細胞１０１１，１０１２，１０１３をチップ１０４上に、たとえばそれぞれの種類の細胞を帯状に配置して異なる機能細胞の領域とこれらの境界が存在する細胞シートあるいは細胞ネットワークを作成することができる。また、本手法では、アプタマーの核酸分解酵素を添加することで、細胞の機能を制御していたアプタマーを分解して、細胞の機能をもとの状態に戻す事ができる。 Next, 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.
　このように本手法の特徴は同一のチップ１０４上に異なる機能制御を行われた細胞を共存させることができることにある。特定のイオンチャンネルを特異的にブロックする薬剤等を用いても、特定のイオンチャンネル分子のブロックは実現できるが、その場合には、この溶液と接するすべての細胞が等しくイオンチャンネルブロックをされてしまい、図１のチップ１０４に示したような、１つのチップ上に機能状態の異なる細胞を共存させることはできない。 As described above, 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.
　図１のチップ１０４では、細胞１０１１、１０１２、１０１３をシート状に分布させた状態を模式的に示したが、図２では、その他の細胞の配置技術の例として、図２（ａ）、（ｂ）、および（ｃ）では、心室筋型に分化した心筋細胞２０１１と、心筋細胞２０１１のK１イオンチャンネルに特異的に結合するアプタマーによってK１イオンチャンネルをブロックした膜電位が上昇して不安定化した心筋細胞２０１２を組み合わせて１細胞単位でネットワークを構築したものである。このように一般の心室筋細胞では豊富に発現しているK１イオンチャンネルをブロックする事で、擬似的にプルキンエ細胞あるいはＨｉｓ細胞のような細胞膜のインピーダンスが高く自律拍動能を持つ異なる特性を持った細胞２０１２を、同一の心室筋細胞２０１１から作り、かつ、同一の細胞ネットワークの中で擬似的に異種細胞として用いることができる。 In the chip 104 of FIG. 1, a state in which the cells 1011, 1012, and 1013 are distributed in a sheet shape is schematically shown. However, in 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. In this way, by blocking the K1 ion channel that is abundantly expressed in general ventricular myocytes, the impedance of the cell membrane such as Purkinje cells or His cells is high, and it has different characteristics with autonomous pulsation ability. The cell 2012 can be made from the same ventricular myocyte 2011 and used as a pseudo-heterogeneous cell in the same cell network.
　図３は、本手法を神経細胞ネットワークに用いたものである。神経細胞ネットワークにおいて細胞間の化学物質の伝導の機能を構成的に解明する事は重要であるが、特定の分泌物質だけを伝達させることは難しかった。図３では、模式的に、本課題を解決する手法を説明する。たとえば結合した２つの神経細胞３０１、３０２について、その２つの細胞を結合するシナプス３０３の表面に存在する２つの伝達物質３０４、３０５を放出する機能を有しているとき、培養液中に伝達物質３０５をブロックするアプタマーを混入させて神経伝達の実験を行い、伝達物質３０４のみの伝達特性を計測した後、アプタマー分解酵素を導入することによって、本来、分泌されるはずであった伝達物質３０５がリリースされ、その応答を計測することができる。 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.
　以上、本発明の例示的実施形態を説明したが、種々の変更、改変、および改良が当業者に容易に想起される。上記は、本発明の原理を例示的に示したものに過ぎず、当業者であれば、本発明の範囲および精神から逸脱することなく、様々な改変を為し得る。 While exemplary embodiments of the present invention have been described above, various changes, modifications, and improvements will readily occur to those skilled in the art. The foregoing is merely illustrative of the principles of this invention and various modifications can be made by those skilled in the art without departing from the scope and spirit of the invention.
　本発明によれば細胞表面の標的分子に特異的に結合するＤＮＡ鎖等の核酸鎖を修飾する事で容易に異種細胞を得ることができるので、創薬、医療分野の研究発展に貢献することができる。 According to the present invention, 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.
細胞表面のさまざまな標的分子に特異的に結合するＤＮＡ鎖等の核酸鎖を用いて機能的に異種細胞を製作し、これを空間配置する手法の手順を示した模式図である。It is the schematic diagram which showed the procedure of the method of producing a heterogeneous cell functionally using nucleic acid chains, such as a DNA chain | strand which specifically couple | bonds with various target molecules on a cell surface, and arranging this spatially. アプタマーによるイオンチャンネルブロックによって異種心筋細胞を空間配置した例を示した図である。It is the figure which showed the example which arranged the heterogeneous cardiomyocyte spatially by the ion channel block by an aptamer. 神経細胞ネットワークを用いた例を示した模式図である。It is the schematic diagram which showed the example using a nerve cell network.
　１０１、１０１１、１０１２、１０１３…細胞
　１０２１、１０２２、１０２３…細胞表面の標的分子
　１０３１，１０３２，１０３３…標的分子に結合するＤＮＡ鎖等の核酸鎖
　２０１１、２０１２…心筋細胞
　３０１、３０２…神経細胞
　３０３…シナプス結合
　３０４、３０５…神経伝達物質 101, 1011, 1012, 1013 ... cells 1021, 1022, 1023 ... target molecules on the cell surface 1031, 1032, 1033 ... 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

Claims (6)

  1. 　（ｉ）標的細胞または標的細胞の集団を準備し、前記標的細胞の細胞表面に発現している標的分子に特異的に結合する核酸または核酸誘導体もしくは核酸修飾体を前記標的細胞の表面に発現している前記標的分子に特異的に結合させて、核酸修飾細胞を作製する工程、
    　（ｉｉ）前記標的細胞の表面に発現している前記標的分子に特異的に結合しなかった核酸を除去する工程、
    　（ｉｉｉ）前記工程（ｉｉ）からの核酸修飾細胞に対して、前記標的細胞の細胞表面に発現している、前記標的分子とは異なる標的分子に特異的に結合する核酸または核酸誘導体あるいは核酸修飾体を用いて、前記工程（ｉ）および（ｉｉ）を行う工程、
    　（ｉｖ）前記工程（ｉ）および（ｉｉ）の操作、または前記工程（ｉｉｉ）の操作を繰り返すことによって作製した２種類以上の異なる核酸修飾細胞を、共通の細胞培養環境中の培養基板上で、前記各核酸修飾細胞の空間配置を制御して配列して細胞ネットワークを構築する工程、
    を含む、細胞機能制御方法。     (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 expressed on the surface of the target cell. Producing a nucleic acid-modified cell by specifically binding to the target molecule
    (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 steps (i) and (ii) using a body,
    (Iv) Two or more kinds of different nucleic acid-modified cells prepared by repeating the operations of the steps (i) and (ii) or the step (iii) on a culture substrate in a common cell culture environment The step of constructing a cell network by controlling and arranging the spatial arrangement of each of the nucleic acid-modified cells,
    A cell function control method comprising:
  2. 　さらに、
    　（ｖ）前記工程（ｉｖ）で培養している前記核酸修飾細胞の前記標的分子に特異的に結合する前記核酸または前記核酸誘導体もしくは前記核酸修飾体を分解させる核酸分解酵素を前記細胞培養環境中に導入する工程、を含む、請求項１に記載の方法。     further,
    (V) in the cell culture environment, the nucleic acid that specifically binds to the target molecule of the nucleic acid-modified cell cultured in the step (iv) or the nucleic acid derivative that degrades the nucleic acid derivative or the modified nucleic acid in the cell culture environment The method according to claim 1, comprising the step of:
  3. 　前記核酸が一本鎖核酸またはアプタマーである、請求項１または２に記載の細胞機能制御方法。 The cell function control method according to claim 1 or 2, wherein the nucleic acid is a single-stranded nucleic acid or an aptamer.
  4. 　前記標的細胞が、心筋細胞、神経細胞、線維芽細胞、グリア細胞、肝臓細胞、膵臓細胞、または幹細胞である、請求項１～３のいずれかに記載の細胞機能制御方法。 The cell function control method according to any one of claims 1 to 3, wherein the target cells are cardiomyocytes, nerve cells, fibroblasts, glial cells, liver cells, pancreatic cells, or stem cells.
  5. 　請求項１～４のいずれか一項に記載の細胞機能制御方法により作製された核酸修飾細胞を含む細胞ネットワーク。 A cell network comprising nucleic acid-modified cells produced by the cell function control method according to any one of claims 1 to 4.
  6. 　請求項５に記載の細胞ネットワークを使用して細胞機能を検査する方法。 A method for examining cell function using the cell network according to claim 5.
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