WO2007043523A1 - Transfection device - Google Patents

Transfection device Download PDF

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Publication number
WO2007043523A1
WO2007043523A1 PCT/JP2006/320208 JP2006320208W WO2007043523A1 WO 2007043523 A1 WO2007043523 A1 WO 2007043523A1 JP 2006320208 W JP2006320208 W JP 2006320208W WO 2007043523 A1 WO2007043523 A1 WO 2007043523A1
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Prior art keywords
gene
solid phase
transfection
sample
sirna
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PCT/JP2006/320208
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French (fr)
Japanese (ja)
Inventor
Tomohiro Yoshikawa
Naoto Kagiyama
Yoshiji Fujita
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Cytopathfinder, Inc.
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Publication of WO2007043523A1 publication Critical patent/WO2007043523A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture

Definitions

  • the present invention provides a gene sample (eg, single-stranded or double-stranded doxyribonucleic acid (DNA), ribonucleic acid (RNA), aptamer, or chemically modified derivatives thereof) from the outside to animal cells. It is related with the transformation device which introduces.
  • a gene sample eg, single-stranded or double-stranded doxyribonucleic acid (DNA), ribonucleic acid (RNA), aptamer, or chemically modified derivatives thereof
  • a device in which a gene sample is fixed to a solid phase substrate for the purpose of transfer can be used for gene therapy or gene analysis.
  • Transfusion device technologies that have been reported so far include gene samples fixed together with intercellular matrix proteins such as gelatin and fibronectin (see Patent Document 2 or Non-Patent Document 1), Some of them are precipitated together with calcium phosphate salt on the solid surface (see Non-Patent Documents 2 to 6).
  • Patent Document 1 US Pat. No. 664297
  • Patent Document 2 US Pat. No. 324780
  • Non-patent literature 1 J. Ziauddin and D. M. Sabatini. Microarrays of cells expressed defined cDNAs. Nature 411, 107-110 (2001).
  • Patent Document 2 R. Z. Wu et al. Cell-biological applications of transfected— cell microar rays. Trends in Cell Biology 12, 485-488 (2002).
  • Non-Patent Document 3 T. Ochiya et al. New delivery system for plasmid DNA in vivousing at elocollagenas a carrier material: the Minipellet.Nature Med. 5, 707-710 (1999)
  • Non-Patent Document 4 K. Honma et al. Atelocollagen- based gene transfer in cells allows high -throughput screening of gene functions. Biochem. Biophys. Res. Commu. 289, 1075 -1081 (2001)
  • Non-Patent Document 5 T. Yoshikawa et al. Transfection microarray of human mesenchymalst em cells and on-chip siRNA gene knockdown. J. Controlled Release 96, 227-232 (2 004).
  • Non-Patent Document 6 E. Uchimura et al. On-chip transfection of PC12 cells based on the ra tional understanding of the role of ECM molecules: efficient, non—viraltransfectionof PC12 cells using collagenl V. Neuroscience Lett. 378, 40—43 ( 2005).
  • the present invention provides a novel transfection device capable of realizing high gene transfer efficiency and reproducibility.
  • the first characteristic configuration of the transfer device of the present invention includes, in addition to a gene sample, a gene delivery material, and a cell adhesion factor, glucose, sucrose, glycogen, glutamine, anoleginin, histidine, lysine , Threonine, tryptophan, parin, alanine, glycine, proline, serine, which contains at least one kind of selected compound, is fixed to a solid phase substrate.
  • the transfection device of the present invention in addition to the gene sample, the gene delivery material, and the cell adhesion factor, at least one compound selected from the above-mentioned various saccharides and various amino acid group forces is fixed to the solid phase substrate. Then, the knowledge that gene transfer efficiency can be improved was found for the first time by the inventors' diligent research. With this configuration, although there are differences depending on the cells, there are differences depending on the cell, compared to conventional transfection devices that adhere a gene sample, a gene delivery material, and a substance containing a cell adhesion factor to a solid substrate and do not adhere the above compound. It is possible to improve gene transfer efficiency by about 2 to 10 times (see Examples described later). As a result, high reproducibility of the introduced gene Can be secured. This can be expected to improve the practicality of the transfer device.
  • the gene sample is at least one kind selected from the group consisting of single-stranded or double-stranded deoxyribonucleic acid, ribonucleic acid, abutama, or a chemically modified derivative thereof. At the point to be selected.
  • the gene sample is a nucleic acid
  • gene overexpression, antisense effect and RNA interference are expected. This makes it possible to analyze the function of genes in a transfection device. Chemical modification of these nucleic acids can be expected to stabilize the nucleic acids or prevent non-specific reactions. Therefore, when the gene sample is a chemically modified derivative of the nucleic acid, the function of the nucleic acid can be analyzed more accurately.
  • Abutama it is possible to cause functional inhibition by firmly binding to the target protein. This enables functional analysis of the target protein in the transfer device.
  • By chemically modifying the abutama it is possible to stabilize the abutama, suppress non-specific reactions, or specifically label the target protein. For this reason, if the gene sample is a chemical modification derivative of the above-mentioned Abuta, the function analysis of the target protein can be performed more accurately.
  • the third characteristic configuration of the present invention is that the gene delivery material contains at least one selected from the group consisting of a cationic polymer, a force thionic lipid, and a mineral force.
  • the gene delivery material has a function of inducing endocytosis, pinocytosis, phagocytosis, fusion with a cell membrane, and the like when a gene sample is introduced into a cell. Therefore, when the above-mentioned material is used as a gene delivery material, the gene delivery material and the gene sample form a complex and pass through the cell membrane. This makes it possible to efficiently send the gene sample into the cell.
  • a fourth characteristic configuration of the present invention is that the cell adhesion factor is gelatin, fibronectin, collagen, laminin, vitronectin, anti-integrin antibody force group force at least one kind Is at the point where is selected.
  • Cell adhesion factors promote the attachment of cells to the solid phase. In addition, it induces reorganization of the intracellular skeleton by acting on integrin proteins. This promotes flattening of cell morphology and flattening of cell nuclei. Therefore, in particular, when the above material is used as a cell adhesion factor, the probability that a gene sample is introduced into a cell is improved by the above-described effect.
  • the material constituting the solid phase substrate is glass or a synthetic polymer.
  • the material that constitutes the solid phase substrate is glass or synthetic polymer (plastic) 'metals, and these materials constitute a hard plate, it is possible to provide an environment suitable as a substrate such as a cell array. it can.
  • a glass' synthetic polymer is used as the material to form a highly permeable substrate, microscopic observation and the like can be easily performed.
  • a conductive base material such as a metal is used as the material, a change in potential can be measured.
  • the material constituting the solid phase base material is a natural polymer and the material constitutes a flexible plate such as a polymer film, it is possible to provide a sheet-like transfer device, patch base material, etc. Applicable.
  • the DDS technique can be used for gene sample delivery in vivo and for use as a sustained-release substrate.
  • the solid phase system is a technique for introducing a gene sample into a cell such as an animal cell by fixing the gene sample to a solid phase substrate.
  • a device in which a gene sample or the like is fixed to a solid phase base material for use in solid phase transformation is referred to as a transfusion device.
  • FIG. 1 shows a manufacturing method of a glass array which is an example of the transformation device of the present invention. 1 shows an embodiment.
  • a desired gene sample is dissolved in a buffer containing various sugars and amino acids, and then an appropriate gene delivery material is added. And react under the prescribed conditions. After a predetermined reaction time has elapsed, a cell adhesion factor is added to the reaction solution and mixed.
  • the mixed solution 1 is printed by spot formation on a glass slide 3 (an example of a solid phase substrate) with an appropriate printing apparatus 2. Allow to dry completely.
  • the spotted glass slide 3 is housed in a petri dish 5 and contains appropriate cells.
  • the suspension 4 is poured into the petri dish 5 and the cells are cultured on the glass slide 3 to introduce the gene into the cells.
  • the cells into which the gene has been introduced are observed only in the print region (spot formation region) of the mixed solution 1.
  • the buffer applicable to the present invention contains at least one compound of glucose, sucrose, glycogen, gnoretamine, anoleginine, histidine, lysine, threonine, tryptophan, norin, alanine, glycine, proline and serine.
  • Examples include aqueous solutions.
  • gene samples applicable to the present invention include single-stranded or double-stranded deoxyribonucleic acid (DNA), ribonucleic acid (RNA), abutama, or chemically modified derivatives thereof.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • abutama or chemically modified derivatives thereof.
  • RNA extracted from a cell or tissue using a known method can be used.
  • DNA or RNA extracted from a cell or tissue using a known method can be used.
  • linear or circular genomic DNA or plasmid DNA can be used as the DNA.
  • these can be used with restriction enzymes, or chemically cleaved DNA fragments, DNA synthesized with enzymes in a test tube, or chemically synthesized oligonucleotides.
  • Abutama is, for example, a nucleic acid (DNA, RNA) having an ability to specifically bind to a physiologically active site of a protein target substance.
  • Abutama which specifically acts on a protein that causes pathogenicity, can inhibit the function of the protein in the cell. This makes it possible to analyze functions such as physiological activity of the target protein.
  • gene delivery materials include cationic polymers, cationic lipids, minerals and the like, and these can be used alone or in any combination.
  • Cationic polymers and cationic lipids are cationic substances that aggregate nucleic acids.
  • the cationic substance can be introduced into the target cell by moving into the target cell in a state where it is electrostatically bound to the nucleic acid to form a complex. That is, the cationic substance becomes the gene carrier.
  • the gene delivery material applied to the present invention is not particularly limited as long as it is a cationic substance having such properties.
  • Cell adhesion factors promote the attachment of cells to the solid phase. In addition, it induces reorganization of the intracellular skeleton by acting on integrin proteins. This promotes flattening of cell morphology and flattening of cell nuclei. This effect increases the probability that the gene sample is introduced into the cell.
  • the cell adhesion factor applied to the present invention is not particularly limited as long as it is a substance having such an action.
  • cell adhesion factors include gelatin, fibronectin, collagen, laminin, vitronectin, anti-integrin antibody, and the like, and these may be used alone or in any combination. it can.
  • the printing apparatus applicable to the present invention is not limited to an apparatus capable of forming a spot on a solid phase substrate, such as an inkjet printer.
  • Examples of the material constituting the solid phase substrate applicable to the present invention include glass, synthetic high molecular weight, and the like. Examples thereof include, but are not limited to, children, metals, and natural polymers. Examples of the shape of the solid phase substrate applicable to the present invention include, but are not limited to, a plate-like body, a woven fabric, a needle, a porous fiber, and a bead.
  • the solid phase substrate may be an aggregate in which a large number of particles of the material are aggregated! ⁇ .
  • the inside can have a porous structure, and the gene sample can be carried in the porous structure. Therefore, in the case of the solid phase substrate of such an embodiment, it can be expected as a DDS technique to carry a gene sample to a desired site in vivo.
  • the solid phase substrate may be a polymer matrix such as a molecular complex as long as it can carry a gene sample.
  • cubic crystals can be used as the molecular aggregate.
  • Cubic crystals have a regular three-dimensional structure in which various types of molecular aggregates (spherical, rod-shaped, or bimolecular membranes) formed by amphiphilic lipids are structural units.
  • a gene sample can be carried in the internal space of the cubic crystal.
  • Solid phase substrate S In such an embodiment, DDS technology can be used for sustained release substrates that can control the release of gene samples in vivo.
  • any cell that can be cultured can be used. Examples include, but are not limited to, HeLa cells (human cervical cancer cells).
  • the cell can be a cell constituting a living body.
  • aqueous solution of various saccharides such as monosaccharide (glucose), disaccharide (sucrose, maltose, latatose), trisaccharide (maltotriose) and polysaccharide (glycogen) described in Table 1 below is given.
  • pEGFP-Nl plasmid DNA expressing green fluorescent protein EGFP
  • pEGFP—Nl plasmid DNA expressing green fluorescent protein EGFP
  • DNase NA-degrading enzyme
  • RNase RNase
  • LipofectAMINE200 a cationic lipid, as a gene delivery material
  • a 4 mg / mL fibronectin solution was prepared with sterile distilled water.
  • Each of the above reagents was mixed at the blending ratio shown in Table 2 below.
  • each buffer (17 ⁇ L) was mixed with plasmid DNA (pEGFP-N1) solution (1 ⁇ L), and then LipofectAMINE2000 (2 L) was added. Mix and mix for 20 minutes at room temperature. Next, a fibronectin solution (5 was added to the reaction solution and mixed to obtain 21 types of mixed solution.
  • the mixed solution is hereinafter referred to as a sample.
  • each slide was seeded with 10 mL of HeLa cell suspension (2 X 10 5 cells / mL), cultured for 24 hours (5% CO, 37 ° C), and solid phase transfection was performed. did. After 24 hours of cell culture, fluorescence images of each spot were obtained using an image scanner, and the effects of various saccharides on the solid phase system were evaluated.
  • the fluorescence image is a green image emitted from HeLa cells into which plasmid DNA (pEGFP-Nl) has been introduced.
  • FIG. 3 is an evaluation of the influence of glucose, and shows fluorescence images of spots (FIGS. 3 (a) and (b) each of 8 spots).
  • Fig. 3 (a) shows the control using sterilized distilled water as the buffer
  • Fig. 3 (b) shows the glucose aqueous solution (4.5 g / L) as the buffer.
  • glucose with higher fluorescence intensity in the sample using the aqueous glucose solution (Fig. 3 (b)) than in the control (Fig. 3 (a)) can increase the gene transfer efficiency. Power.
  • FIGS. 4 to 8 show evaluations of the effects of sucrose, maltose, ratatoose, maltotriose and glycogen, respectively. Each figure shows the fluorescence image of the spot on the above three slides (bottom), and the fluorescence image quantified by the image analysis software and graphed (top). .
  • a bar graph is prepared for each of the sterilized distilled water (control) and each sample (for each concentration of saccharide used), and the vertical axis of the graph indicates the amount of fluorescence (fluorescence intensity). Yes.
  • each bar graph corresponds to the fluorescent image (three slides) immediately below it.
  • a total of 12 spot fluorescence images are obtained for each sample (or control) and a bar graph is created. Note that the error bars in the graph indicate the variation from spot to spot.
  • the evaluation index of the gene transfer state was efficiency (gene transfer efficiency), localization, and uniformity using the fluorescence intensity as an index.
  • efficiency gene transfer efficiency
  • localization localization
  • uniformity using the fluorescence intensity as an index.
  • green fluorescence emitted from HeLa cells into which plasmid DNA (pEGFP-Nl) was introduced was quantified by image processing and evaluated as the amount of gene transfer.
  • the quantification was performed using an image scanner and image analysis software for the integrated luminance value of green fluorescent protein EGFP.
  • each sample and control (sterile distilled water) were prepared, three slides were prepared, and solid phase transformation was performed.
  • L-arginine a gene delivery material (LipofectAMINE2000 (Invitrogen)) was not included! /, And a sample (L-arginine (without LF2000)) was also prepared.
  • FIGS. 9 to 28 show glutamine, L-arginine, L-arginine (without LF2000), L-cystine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine and DL, respectively.
  • glucose (.25 g / L), which is a sugar species with good results
  • histidine (2.111 ⁇ / 1 ⁇ (1), 5.2511 ⁇ /] ⁇ (2.5) among amino acids.
  • threonine (0.95mg / L (Xl), 2.125mg / L (X2.5), 4.25mg / L (X5)
  • alanine (0.89mg / L (X1)
  • serine (1.05mg / L (Xl), 2.625mg / L (X 2.5), 5.25mg / L (X 5)
  • a mixture of equal amounts was used as a buffer and tested in the same manner as in Examples 1 and 2 above.
  • D-form and L-form were prepared for each of the above amino acids, and compared with the control (sterile distilled water) in terms of gene introduction efficiency, localization, and uniformity.
  • FIGS. 29 to 32 show the effects of glucose and D, L-histidine, glucose and D, L-threonine, glucose and D, L-alanine, glucose and D, L-serine, respectively. is there.
  • the fluorescence images of the spots on the two slides (bottom) and their fluorescence image power are quantified using the image analysis software, and the values for each spot are averaged and graphed (top).
  • a bar graph is created for each slide for each of the sterilized distilled water (control) and each sample (concentration of D-form and L-form amino acids used), and the vertical axis of the graph represents the fluorescence. Show the amount (fluorescence intensity)!
  • SiRNA short interference RNA
  • siRNA siRNA that exhibits RNA interference was used as the nucleic acid (gene sample) used in the manufacture of the transfer device.
  • HiPerFect Transfection Reagent Qiagen
  • siRNA anti-EGFP siRNA that can suppress the expression of green fluorescent protein (EGFP) and negative control siRNA that does not suppress the expression of green fluorescent protein (EGFP) were used.
  • EGFP green fluorescent protein
  • EGFP negative control siRNA that does not suppress the expression of green fluorescent protein
  • HiPerFect reagent was added and incubated at room temperature for 10 minutes. Next, a fibronectin solution was added and printed on a slide to produce a transfection device.
  • FIG. 34 shows the results of the solid phase transfection carried out in this way.
  • the transfection device of the present invention can perform solid-phase transfection in a state where the function of siRNA (suppression of gene expression) is maintained.
  • siRNA exhibiting RNA interference is used as a gene sample, even if siRNA having an effect of suppressing gene expression is used due to the unstable nature of siRNA molecules, The effect may not be shown. Especially this present The elephant is prominent when gene transfer is performed in vivo as a target.
  • the stability of siRNA molecules in vivo is an important factor when manufacturing a transfection device for gene transfer in vivo.
  • a method for evaluating the stability of siRNA molecules in vivo a method using the degradation rate of siRNA molecules in serum as an index is common.
  • Fig. 35 shows a method for adjusting the reagent used in the present embodiment.
  • Various samples (glucose / alanine solution, siRNA, plasmid DNA, LipofectAMINE2000, fibronectin solution) shown in Fig. 35 (a) were prepared.
  • the negative control siRNA described above was used as siRNA, and pUC 19 was used as plasmid DNA.
  • the various samples were mixed as appropriate to produce two types of samples (TMA mix, siRNA only).
  • the stability evaluation solution is prepared by using siRNA, plasmid DN in a glucose / alanin-containing buffer.
  • a and LipofectAMINE2000 were dissolved and incubated at room temperature for 20 minutes, and a fibronectin solution was added (FIG. 35 (b)).
  • RiboGreen solution Diluted 00 times (hereinafter referred to as RiboGreen solution) and used for the following experiments.
  • the siRNA contained in the sample (TMA mix) used to manufacture the transfection device under 50% serum conditions is higher than the sample (siRNA only) in which only the siRNA is dissolved in the buffer. ! It has been confirmed that it has stability. Similar results were obtained in the two experiments (Runl, Run2).
  • the present invention provides an external force gene sample (eg, single-stranded or double-stranded deoxyribonucleic acid (DNA), ribonucleic acid (RNA), aptamer, or the like) to animal cells that are eukaryotic cells. It can be used for a transfer device for introducing a chemically modified derivative of Brief Description of Drawings
  • FIG. 1 is a diagram showing an example of a method for manufacturing a glass array, which is an example of a transformation device of the present invention.
  • FIG. 2 is a diagram showing an example of evaluation of the state of gene introduction at a spot when solid phase transfection is performed using the transfection device of the present invention.
  • FIG. 35 Diagram showing the method of preparing the reagents used to evaluate the stability of siRNA
  • FIG. 36 shows the evaluation results of siRNA measurement sensitivity.
  • FIG.37 Diagram showing the results of time-dependent changes in siRNA concentration in the presence of 50% urine fetal serum.

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Abstract

A novel transfection device capable of realizing high gene introduction efficiency and reproduction. There is provided a transfection device comprising a solid-phase base material and, fixed thereonto, a composition containing not only a gene sample, a gene delivery material and a cell adhesion factor but also at least one compound selected from the group consisting of glucose, sucrose, glycogen, glutamine, arginine, histidine, lysine, threonine, tryptophan, valine, alanine, glycine, proline and serine.

Description

明 細 書  Specification
トランスフエクシヨンデバイス 技術分野  Transfusion device technology
[0001] 本発明は、動物細胞に外部から遺伝子試料 (例えば、一本鎖又は二本鎖のデォキ シリボ核酸 (DNA)、リボ核酸 (RNA)、ァプタマ一、あるいはこれらの化学修飾誘導 体等)を導入するトランスフエクシヨンデバイスに関する。  [0001] The present invention provides a gene sample (eg, single-stranded or double-stranded doxyribonucleic acid (DNA), ribonucleic acid (RNA), aptamer, or chemically modified derivatives thereof) from the outside to animal cells. It is related with the transformation device which introduces.
背景技術  Background art
[0002] トランスフエクシヨン (遺伝子導入)を目的として遺伝子試料を固相基板に固着させ たデバイスは、遺伝子治療用途や遺伝子解析用途に用いることが可能である。これ まで報告されてきたトランスフエクシヨンデバイス技術としては、遺伝子試料をゼラチン ゃフイブロネクチン等の細胞間マトリックスタンパク質と一緒に固着したものや (特許 文献 特許文献 2又は非特許文献 1参照）、遺伝子試料をリン酸カルシウム塩と一 緒に固相表面に析出させたものなどがある (非特許文献 2〜6参照)。  [0002] A device in which a gene sample is fixed to a solid phase substrate for the purpose of transfer (gene transfer) can be used for gene therapy or gene analysis. Transfusion device technologies that have been reported so far include gene samples fixed together with intercellular matrix proteins such as gelatin and fibronectin (see Patent Document 2 or Non-Patent Document 1), Some of them are precipitated together with calcium phosphate salt on the solid surface (see Non-Patent Documents 2 to 6).
特許文献 1：米国特許第 664297号公報  Patent Document 1: US Pat. No. 664297
特許文献 2：米国特許第 324780号公報  Patent Document 2: US Pat. No. 324780
非特干文献 1： J. Ziauddin and D. M. Sabatini. Microarrays of cells expressed defined cDNAs. Nature 411, 107-110 (2001).  Non-patent literature 1: J. Ziauddin and D. M. Sabatini. Microarrays of cells expressed defined cDNAs. Nature 411, 107-110 (2001).
特許文献 2： R. Z. Wu et al. Cell-biological applications of transfected— cell microar rays. Trends in Cell Biology 12, 485—488 (2002).  Patent Document 2: R. Z. Wu et al. Cell-biological applications of transfected— cell microar rays. Trends in Cell Biology 12, 485-488 (2002).
非特許文献 3 : T. Ochiya et al. New delivery system for plasmid DNA in vivousing at elocollagenas a carrier material: the Minipellet. Nature Med. 5, 707-710 (1999) 非特許文献 4 : K. Honma et al. Atelocollagen- based gene transfer in cells allows high -throughput screening of gene functions. Biochem. Biophys. Res. Commu.289, 1075 -1081 (2001)  Non-Patent Document 3: T. Ochiya et al. New delivery system for plasmid DNA in vivousing at elocollagenas a carrier material: the Minipellet.Nature Med. 5, 707-710 (1999) Non-Patent Document 4: K. Honma et al. Atelocollagen- based gene transfer in cells allows high -throughput screening of gene functions. Biochem. Biophys. Res. Commu. 289, 1075 -1081 (2001)
非特許文献 5 : T. Yoshikawa et al. Transfection microarray of human mesenchymalst em cells and on-chip siRNA gene knockdown. J. Controlled Release 96, 227-232 (2 004). 非特許文献 6 : E. Uchimura et al. On-chip transfection of PC12 cells based on the ra tional understanding of the role of ECM molecules: efficient, non— viraltransfectionof PC12 cells using collagenlV. Neuroscience Lett. 378, 40—43 (2005). Non-Patent Document 5: T. Yoshikawa et al. Transfection microarray of human mesenchymalst em cells and on-chip siRNA gene knockdown. J. Controlled Release 96, 227-232 (2 004). Non-Patent Document 6: E. Uchimura et al. On-chip transfection of PC12 cells based on the ra tional understanding of the role of ECM molecules: efficient, non—viraltransfectionof PC12 cells using collagenl V. Neuroscience Lett. 378, 40—43 ( 2005).
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0003] し力しながら、上記文献に記載される従来のトランスフエクシヨンデバイス技術にお いては、特に遺伝子導入効率について改良の余地が大いに残されており、再現性が 確保し難ぐ実用化のための大きな障壁になっていた。すなわち、遺伝子試料、遺伝 子デリバリー材料、細胞接着因子を固相基材に固着してあるトランスフエクシヨンデバ イスにおいて、そのトランスフエクシヨンデバイスが実用的かどうかは、導入効率の高さ と再現性にかかって ヽると ヽつても過言ではな ヽ。 [0003] However, in the conventional transfection device technology described in the above-mentioned literature, there is still much room for improvement in terms of gene transfer efficiency, and it is difficult to ensure reproducibility. Had become a big barrier for. In other words, in a transfer device in which a gene sample, gene delivery material, and cell adhesion factor are fixed to a solid phase substrate, whether the transfer device is practical or not is highly efficient and reproducible. It's not an exaggeration to say that it depends on you.
[0004] 本発明は、高い遺伝子導入効率と再現性とを実現し得る新規なトランスフエクシヨン デバイスを提供するものである。 [0004] The present invention provides a novel transfection device capable of realizing high gene transfer efficiency and reproducibility.
課題を解決するための手段  Means for solving the problem
[0005] 上記目的を達成するための本発明のトランスフエクシヨンデバイス第一特徴構成は 、遺伝子試料、遺伝子デリバリー材料、細胞接着因子に加え、グルコース、スクロー ス、グリコーゲン、グルタミン、ァノレギニン、ヒスチジン、リシン、トレオニン、トリプトファ ン、パリン、ァラニン、グリシン、プロリン、セリンカもなる群力も選択される少なくとも一 種の化合物を含むものを固相基材に固着した点にある。 [0005] In order to achieve the above object, the first characteristic configuration of the transfer device of the present invention includes, in addition to a gene sample, a gene delivery material, and a cell adhesion factor, glucose, sucrose, glycogen, glutamine, anoleginin, histidine, lysine , Threonine, tryptophan, parin, alanine, glycine, proline, serine, which contains at least one kind of selected compound, is fixed to a solid phase substrate.
[0006] 〔作用及び効果〕 [0006] [Action and effect]
本発明のトランスフエクシヨンデバイスにおいて、遺伝子試料、遺伝子デリバリー材 料、細胞接着因子に加えて、上述した各種糖類および各種アミノ酸力もなる群力も選 択される少なくとも一種の化合物を固相基材に固着すると、遺伝子導入効率を向上 させることができるという知見は、本発明者らの鋭意研究によって初めて見出された。 本構成により、遺伝子試料、遺伝子デリバリー材料、細胞接着因子を含むものを固 相基材に固着して上記化合物を固着しない従来のトランスフエクシヨンデバイスに比 ベて、細胞によって差はあるが、およそ 2〜10倍程度、遺伝子導入効率を向上させる ことが可能である（後述の実施例参照)。その結果、導入した遺伝子の高い再現性を 確保することができる。これにより、トランスフエクシヨンデバイスの実用性の向上が期 待できる。 In the transfection device of the present invention, in addition to the gene sample, the gene delivery material, and the cell adhesion factor, at least one compound selected from the above-mentioned various saccharides and various amino acid group forces is fixed to the solid phase substrate. Then, the knowledge that gene transfer efficiency can be improved was found for the first time by the inventors' diligent research. With this configuration, although there are differences depending on the cells, there are differences depending on the cell, compared to conventional transfection devices that adhere a gene sample, a gene delivery material, and a substance containing a cell adhesion factor to a solid substrate and do not adhere the above compound. It is possible to improve gene transfer efficiency by about 2 to 10 times (see Examples described later). As a result, high reproducibility of the introduced gene Can be secured. This can be expected to improve the practicality of the transfer device.
[0007] 本発明の第二特徴構成は、前記遺伝子試料が、一本鎖又は二本鎖のデォキシリ ボ核酸、リボ核酸、アブタマ一、あるいはこれらの化学修飾誘導体からなる群から少 なくとも一種が選択される点にある。  [0007] In the second characteristic configuration of the present invention, the gene sample is at least one kind selected from the group consisting of single-stranded or double-stranded deoxyribonucleic acid, ribonucleic acid, abutama, or a chemically modified derivative thereof. At the point to be selected.
[0008] 〔作用及び効果〕  [0008] [Action and effect]
遺伝子試料が核酸である場合、遺伝子の過剰発現、アンチセンス効果及び、 RNA 干渉などが期待される。これによつて、トランスフエクシヨンデバイスにおいて遺伝子の 機能解析が可能となる。これら核酸を化学修飾することで当該核酸を安定化させる、 又は非特異的反応を防止することが期待できる。そのため、遺伝子試料を当該核酸 の化学修飾誘導体とすると、より正確に核酸の機能解析を行うことができる。  When the gene sample is a nucleic acid, gene overexpression, antisense effect and RNA interference are expected. This makes it possible to analyze the function of genes in a transfection device. Chemical modification of these nucleic acids can be expected to stabilize the nucleic acids or prevent non-specific reactions. Therefore, when the gene sample is a chemically modified derivative of the nucleic acid, the function of the nucleic acid can be analyzed more accurately.
アブタマ一の場合、標的とするタンパク質と強固に結合させることで、機能阻害を生 じさせることが可能である。これによりトランスフエクシヨンデバイスにおいて標的タンパ ク質の機能解析が可能となる。当該アブタマ一をィ匕学修飾することで、当該アブタマ 一を安定化させる、非特異的反応を抑制する、或いは標的タンパク質を特異的にラ ベルイ匕することができる。そのため、遺伝子試料を当該アブタマ一の化学修飾誘導 体とすると、より正確に標的タンパク質の機能解析を行うことができる。  In the case of Abutama, it is possible to cause functional inhibition by firmly binding to the target protein. This enables functional analysis of the target protein in the transfer device. By chemically modifying the abutama, it is possible to stabilize the abutama, suppress non-specific reactions, or specifically label the target protein. For this reason, if the gene sample is a chemical modification derivative of the above-mentioned Abuta, the function analysis of the target protein can be performed more accurately.
[0009] 本発明の第三特徴構成は、前記遺伝子デリバリー材料が、カチオン性ポリマー、力 チオン性脂質、ミネラル力 なる群力 選択される少なくとも一種を含有する点にある  [0009] The third characteristic configuration of the present invention is that the gene delivery material contains at least one selected from the group consisting of a cationic polymer, a force thionic lipid, and a mineral force.
[0010] 〔作用及び効果〕 [0010] [Action and effect]
遺伝子デリバリー材料は、遺伝子試料を細胞に導入する際、エンドサイト一シス、ピ ノサイト一シス、ファゴサイト一シス、細胞膜との融合などを誘導する働きを有する。そ のため、遺伝子デリバリー材料として上記材料を使用すれば、当該遺伝子デリバリー 材料と遺伝子試料とが複合体を形成して細胞膜を通過する。これにより、効率的に遺 伝子試料を細胞内に送り込むことが可能となる。  The gene delivery material has a function of inducing endocytosis, pinocytosis, phagocytosis, fusion with a cell membrane, and the like when a gene sample is introduced into a cell. Therefore, when the above-mentioned material is used as a gene delivery material, the gene delivery material and the gene sample form a complex and pass through the cell membrane. This makes it possible to efficiently send the gene sample into the cell.
[0011] 本発明の第四特徴構成は、前記細胞接着因子が、ゼラチン、フイブロネクチン、コ ラーゲン、ラミニン、ビトロネクチン、抗インテグリン抗体力 なる群力 少なくとも一種 が選択される点にある。 [0011] A fourth characteristic configuration of the present invention is that the cell adhesion factor is gelatin, fibronectin, collagen, laminin, vitronectin, anti-integrin antibody force group force at least one kind Is at the point where is selected.
[0012] 〔作用及び効果〕  [0012] [Action and effect]
細胞接着因子は、細胞が固相に付着することを促進する。その他に、インテグリンタ ンパク質などへ働きかけることにより、細胞内骨格の再編成を誘導する。これにより、 細胞形態の扁平化及び、細胞核の扁平化を促進する。そのため、特に、細胞接着因 子として上記材料を使用すれば、上述の効果により、遺伝子試料が細胞へ導入され る確率が向上する。  Cell adhesion factors promote the attachment of cells to the solid phase. In addition, it induces reorganization of the intracellular skeleton by acting on integrin proteins. This promotes flattening of cell morphology and flattening of cell nuclei. Therefore, in particular, when the above material is used as a cell adhesion factor, the probability that a gene sample is introduced into a cell is improved by the above-described effect.
[0013] 本発明の第五特徴構成は、前記固相基材を構成する材料が、ガラス、合成高分子 [0013] According to a fifth characteristic configuration of the present invention, the material constituting the solid phase substrate is glass or a synthetic polymer.
、金属類、天然高分子からなる群から選択される点にある。 , Metals and natural polymers.
[0014] 〔作用及び効果〕 [0014] [Action and effect]
固相基材を構成する材料がガラス ·合成高分子 (プラスチック） '金属類であり、これ ら材料が硬質板を構成する場合は、細胞アレイなどの基材として適した環境を提供 することができる。特にガラス'合成高分子を当該材料に利用し、透過性の高い基材 とした場合、顕微観察などが容易に可能となる。また、当該材料として金属などの導 電基材とした場合では、電位変化の測定などが可能となる。  If the material that constitutes the solid phase substrate is glass or synthetic polymer (plastic) 'metals, and these materials constitute a hard plate, it is possible to provide an environment suitable as a substrate such as a cell array. it can. In particular, when a glass' synthetic polymer is used as the material to form a highly permeable substrate, microscopic observation and the like can be easily performed. Further, when a conductive base material such as a metal is used as the material, a change in potential can be measured.
また、固相基材を構成する材料が天然高分子であり、当該材料が高分子フィルム などのように軟質板を構成する場合は、シート状トランスフエクシヨンデバイスの提供 や、パッチ基材などへ適用可能である。  In addition, when the material constituting the solid phase base material is a natural polymer and the material constitutes a flexible plate such as a polymer film, it is possible to provide a sheet-like transfer device, patch base material, etc. Applicable.
固相基材の態様が粒子及び高分子マトリクスの場合には、 DDS技術として、 in vi voにおける遺伝子試料のデリバリーや、徐放化基材への利用用途が考えられる。 発明を実施するための最良の形態  When the solid-phase substrate is in the form of particles and a polymer matrix, the DDS technique can be used for gene sample delivery in vivo and for use as a sustained-release substrate. BEST MODE FOR CARRYING OUT THE INVENTION
[0015] 以下に本発明の実施の形態を図面に基づいて説明する。 Hereinafter, embodiments of the present invention will be described with reference to the drawings.
固相系トランスフエクシヨンとは、遺伝子試料を固相基材に固着することにより、動物 細胞等の細胞へ遺伝子試料を導入する手法である。本明細書では、固相系トランス フエクシヨンに利用するため、遺伝子試料等を固相基材に固着したデバイスをトラン スフエクシヨンデバイスと 、う。  The solid phase system is a technique for introducing a gene sample into a cell such as an animal cell by fixing the gene sample to a solid phase substrate. In the present specification, a device in which a gene sample or the like is fixed to a solid phase base material for use in solid phase transformation is referred to as a transfusion device.
[0016] (実施形態） [0016] (Embodiment)
図 1は、本発明のトランスフエクシヨンデバイスの一例であるガラスアレイの製造方法 の一実施形態を示したものである。 FIG. 1 shows a manufacturing method of a glass array which is an example of the transformation device of the present invention. 1 shows an embodiment.
図 1 (a)に示すように、例えばエツペンドルフチューブなどの適当な容器において、 各種糖類やアミノ酸類を含有するバッファーに、所望の遺伝子試料を溶解させた後、 適当な遺伝子デリバリー材料を加えて混合し、所定条件下にて反応させる。所定の 反応時間が経過した後、その反応液に細胞接着因子を加えて混合する。  As shown in Fig. 1 (a), in a suitable container such as an Eppendorf tube, a desired gene sample is dissolved in a buffer containing various sugars and amino acids, and then an appropriate gene delivery material is added. And react under the prescribed conditions. After a predetermined reaction time has elapsed, a cell adhesion factor is added to the reaction solution and mixed.
[0017] 次いで、図 1 (b)に示されるように、その混合液 1を、適当なプリント装置 2により、ガ ラススライド 3 (固相基材の一例）上にスポット形成することでプリントし、完全に自然乾 燥させる。 Next, as shown in FIG. 1 (b), the mixed solution 1 is printed by spot formation on a glass slide 3 (an example of a solid phase substrate) with an appropriate printing apparatus 2. Allow to dry completely.
また、固相系トランスフ クシヨン (遺伝子導入)を実施する際は、図 1 (b)に示される ように、スポット形成されたガラススライド 3をシャーレ 5内に収容し、適当な細胞を含 有する細胞懸濁液 4をシャーレ 5内に注ぎ込み、ガラススライド 3上で細胞培養を行う ことで当該細胞内への遺伝子導入を行う。  When performing solid-phase transformation (gene transfer), as shown in FIG. 1 (b), the spotted glass slide 3 is housed in a petri dish 5 and contains appropriate cells. The suspension 4 is poured into the petri dish 5 and the cells are cultured on the glass slide 3 to introduce the gene into the cells.
尚、遺伝子導入された細胞は、混合液 1のプリント領域 (スポット形成領域)のみで 観察される。  The cells into which the gene has been introduced are observed only in the print region (spot formation region) of the mixed solution 1.
[0018] (バッファー） [0018] (buffer)
本発明に適用可能なバッファ一としては、グルコース、スクロース、グリコーゲン、グ ノレタミン、ァノレギニン、ヒスチジン、リシン、トレオニン、トリプトファン、ノ リン、ァラニン、 グリシン、プロリン、セリンのうち少なくとも 1種の化合物を含有する水溶液等が挙げら れる。  The buffer applicable to the present invention contains at least one compound of glucose, sucrose, glycogen, gnoretamine, anoleginine, histidine, lysine, threonine, tryptophan, norin, alanine, glycine, proline and serine. Examples include aqueous solutions.
[0019] (遺伝子試料）  [0019] (gene sample)
本発明に適用可能な遺伝子試料としては、一本鎖又は二本鎖のデォキシリボ核酸 (DNA)、リボ核酸 (RNA)、アブタマ一、あるいはこれらの化学修飾誘導体等が挙げ られる。  Examples of gene samples applicable to the present invention include single-stranded or double-stranded deoxyribonucleic acid (DNA), ribonucleic acid (RNA), abutama, or chemically modified derivatives thereof.
[0020] 核酸を用いる場合には、公知の方法を用いて細胞もしくは組織より抽出された DN A或いは RNAを利用することができる。例えば DNAとしては、鎖状若しくは環状の ゲノミック DNAやプラスミド DNAが利用できる。また、これらを制限酵素により、ある いは、化学的に切断した DNA断片、試験管内で酵素等により合成された DNA、又 は化学合成したオリゴヌクレオチド等を用いることもできる。 [0021] アブタマ一とは、例えばタンパク質の標的物質の生理活性部位等に特異的に結合 する能力を有する核酸 (DNA, RNA)である。例えば病原性の原因となるタンパク質 に特異的に作用するアブタマ一は、そのタンパク質の機能を細胞内で阻害できる。こ れにより当該標的タンパク質の生理活性などの機能解析が可能となる。 [0020] When a nucleic acid is used, DNA or RNA extracted from a cell or tissue using a known method can be used. For example, linear or circular genomic DNA or plasmid DNA can be used as the DNA. In addition, these can be used with restriction enzymes, or chemically cleaved DNA fragments, DNA synthesized with enzymes in a test tube, or chemically synthesized oligonucleotides. [0021] Abutama is, for example, a nucleic acid (DNA, RNA) having an ability to specifically bind to a physiologically active site of a protein target substance. For example, Abutama, which specifically acts on a protein that causes pathogenicity, can inhibit the function of the protein in the cell. This makes it possible to analyze functions such as physiological activity of the target protein.
[0022] (遺伝子デリバリー材料）  [0022] (Gene delivery material)
本発明に適用可能な遺伝子デリバリー材料としては、カチオン性ポリマー、カチォ ン性脂質、ミネラル等が挙げられ、これらを単独で、あるいは任意に組み合わせて使 用することができる。  Examples of gene delivery materials applicable to the present invention include cationic polymers, cationic lipids, minerals and the like, and these can be used alone or in any combination.
[0023] カチオン性ポリマーおよびカチオン性脂質は、核酸を凝集化するカチオン性物質 である。当該カチオン性物質は、核酸と静電的に結合して複合体を形成した状態で 標的細胞内に移行することにより、核酸を標的細胞内に導入することができる。即ち 、当該カチオン性物質は遺伝子キャリア一となる。本発明に適用される遺伝子デリバ リー材料は、このような性質を有するカチオン性物質であれば、特に限定されるもの ではない。  [0023] Cationic polymers and cationic lipids are cationic substances that aggregate nucleic acids. The cationic substance can be introduced into the target cell by moving into the target cell in a state where it is electrostatically bound to the nucleic acid to form a complex. That is, the cationic substance becomes the gene carrier. The gene delivery material applied to the present invention is not particularly limited as long as it is a cationic substance having such properties.
[0024] (細胞接着因子）  [0024] (Cell adhesion factor)
細胞接着因子は、細胞が固相に付着することを促進する。その他に、インテグリンタ ンパク質などへ働きかけることにより、細胞内骨格の再編成を誘導する。これにより、 細胞形態の扁平化及び、細胞核の扁平化を促進する。この効果により、遺伝子試料 が細胞へ導入される確率が向上する。本発明に適用される細胞接着因子は、このよ うな作用を有する物質であれば、特に限定されるものではな 、。  Cell adhesion factors promote the attachment of cells to the solid phase. In addition, it induces reorganization of the intracellular skeleton by acting on integrin proteins. This promotes flattening of cell morphology and flattening of cell nuclei. This effect increases the probability that the gene sample is introduced into the cell. The cell adhesion factor applied to the present invention is not particularly limited as long as it is a substance having such an action.
[0025] 本発明に適用可能な細胞接着因子としては、ゼラチン、フイブロネクチン、コラーゲ ン、ラミニン、ビトロネクチン、抗インテグリン抗体等が挙げられ、これらを単独で、ある いは任意に組み合わせて使用することができる。  [0025] Examples of cell adhesion factors applicable to the present invention include gelatin, fibronectin, collagen, laminin, vitronectin, anti-integrin antibody, and the like, and these may be used alone or in any combination. it can.
[0026] (プリント装置）  [0026] (Printing device)
本発明に適用可能なプリント装置としては、固相基材上にスポットを形成できる装置 、例えばインクジェットプリンタ一等が挙げられる力 これに限定されるものではない。  The printing apparatus applicable to the present invention is not limited to an apparatus capable of forming a spot on a solid phase substrate, such as an inkjet printer.
[0027] (固相基材）  [0027] (Solid phase substrate)
本発明に適用可能な固相基材を構成する材料としては、例えば、ガラス、合成高分 子、金属類、天然高分子等が挙げられるがこれらに限定されるものではない。また、 本発明に適用可能な固相基材の形状としては、例えば、板状体、織布、ニードル、 多孔性繊維、ビーズ等が挙げられるが、これらに限定されるものではない。 Examples of the material constituting the solid phase substrate applicable to the present invention include glass, synthetic high molecular weight, and the like. Examples thereof include, but are not limited to, children, metals, and natural polymers. Examples of the shape of the solid phase substrate applicable to the present invention include, but are not limited to, a plate-like body, a woven fabric, a needle, a porous fiber, and a bead.
[0028] また、固相基材の態様を、前記材料の粒子を多数凝集させた集合体としてもよ！ヽ。 [0028] The solid phase substrate may be an aggregate in which a large number of particles of the material are aggregated!ヽ.
このような基材であれば内部をポーラス構造とすることができ、遺伝子試料を当該ポ 一ラス構造内で担持することができる。そのため、このような態様の固相基材であれ ば、 DDS技術として、 in vivoにおいて遺伝子試料を所望の部位に運ぶことが期待 できる。  With such a base material, the inside can have a porous structure, and the gene sample can be carried in the porous structure. Therefore, in the case of the solid phase substrate of such an embodiment, it can be expected as a DDS technique to carry a gene sample to a desired site in vivo.
[0029] さらに、固相基材としては、遺伝子試料を担持できる態様であれば、例えば分子会 合体のような高分子マトリクスであってもよい。分子会合体としては、例えばキュービッ ク結晶等が適用可能である。キュービック結晶は、両親媒性脂質が形成する様々な 形態の分子会合体 (球状、ロッド状、あるいは二分子膜など)が構造単位となり、規則 的な三次元構造をとつて 、る。当該キュービック結晶の内部空間に遺伝子試料を担 持することができる。固相基材カ Sこのような態様であれば、 DDS技術として、 in vivo において遺伝子試料の放出性を制御できる徐放化基材への利用用途が考えられる  [0029] Furthermore, the solid phase substrate may be a polymer matrix such as a molecular complex as long as it can carry a gene sample. For example, cubic crystals can be used as the molecular aggregate. Cubic crystals have a regular three-dimensional structure in which various types of molecular aggregates (spherical, rod-shaped, or bimolecular membranes) formed by amphiphilic lipids are structural units. A gene sample can be carried in the internal space of the cubic crystal. Solid phase substrate S In such an embodiment, DDS technology can be used for sustained release substrates that can control the release of gene samples in vivo.
[0030] (細胞） [0030] (Cell)
本発明のトランスフエクシヨンデバイスを使用して遺伝子試料を導入し得る細胞とし ては、培養可能な真核細胞であれば任意の細胞が適用可能である。例えば、 HeLa 細胞 (ヒト子宮頸がん細胞)等が挙げられるがこれに限定されるものではない。  As a cell into which a gene sample can be introduced using the transfection device of the present invention, any cell that can be cultured can be used. Examples include, but are not limited to, HeLa cells (human cervical cancer cells).
さらに、本発明のトランスフエクシヨンデバイスを DDS技術に適用する場合は、当該 細胞は、生体を構成する細胞とすることが可能である。  Furthermore, when the transformation device of the present invention is applied to the DDS technology, the cell can be a cell constituting a living body.
実施例  Example
[0031] (実施例 1) [Example 1]
ノ ッファーとして、以下の表 1に記載される単糖 (グルコース）、二糖 (スクロース、マ ルトース、ラタトース）、三糖 (マルトトリオース）、多糖 (グリコーゲン)の各種糖類の水 溶液を所定の濃度（グルコース（4. 5gZL)、スクロース（lmgZL、 2mg/L, 3mg ZLゝ 4. 5mgZU、マルトース（lmgZLゝ 2mg/L, 3mg/L, 4. 5mgZU、ラタト ース（lmgZL、 2mgZL、 3mg/L, 4. 5mgZU、マルトトリオース（lmgZL、 2m gZL、 3mgZL、 4. 5mgZU、グリコーゲン（lmgZL、 2mg/L, 3mg/L, 4. 5 mgZU )にて調製し、合計で 21種類のバッファーを得た。 As a noffer, an aqueous solution of various saccharides such as monosaccharide (glucose), disaccharide (sucrose, maltose, latatose), trisaccharide (maltotriose) and polysaccharide (glycogen) described in Table 1 below is given. Concentrations (glucose (4.5 gZL), sucrose (lmgZL, 2 mg / L, 3 mg ZL ゝ 4.5 mgZU, maltose (lmgZL ゝ 2 mg / L, 3 mg / L, 4.5 mgZU, ratato Source (lmgZL, 2mgZL, 3mg / L, 4.5mgZU, maltotriose (lmgZL, 2mgZL, 3mgZL, 4.5mgZU, glycogen (lmgZL, 2mg / L, 3mg / L, 4.5mgZU)) In total, 21 types of buffers were obtained.
[表 1] [table 1]
バッファ—に用いた試薬類-—覧  Reagents used in the buffer
Figure imgf000010_0001
Figure imgf000010_0001
また、遺伝子試料として、 1 μ g/ Lの pEGFP- Nl (緑色蛍光タンパク質 EGFPを 発現するプラスミド DNA)溶液を滅菌蒸留水で調製した。尚、実験に使用したプラス ミド DNA(pEGFP— Nl)は、エンドトキシン除去工程を経て生成したものであり、 D NA分解酵素（DNase)及び RNA分解酵素 (RNase)を含まな ヽ滅菌蒸留水に溶解 させたものを使用した。 As a gene sample, a 1 μg / L pEGFP-Nl (plasmid DNA expressing green fluorescent protein EGFP) solution was prepared with sterile distilled water. The plasmid DNA (pEGFP—Nl) used in the experiment was produced through the endotoxin removal step. A solution dissolved in sterile distilled water containing NA-degrading enzyme (DNase) and RNase (RNase) was used.
遺伝子デリバリー材料として、カチオン性脂質である市販の LipofectAMINE200 Commercially available LipofectAMINE200, a cationic lipid, as a gene delivery material
0 (インビトロジェン社製)を使用した。 0 (Invitrogen) was used.
細胞接着因子として、 4mg/mLのフイブロネクチン溶液を滅菌蒸留水で調製した。 上記各試薬にっ ヽて、以下の表 2に記載される配合割合で混合した。  As a cell adhesion factor, a 4 mg / mL fibronectin solution was prepared with sterile distilled water. Each of the above reagents was mixed at the blending ratio shown in Table 2 below.
[0034] [表 2] [0034] [Table 2]
Figure imgf000011_0001
Figure imgf000011_0001
[0035] 詳細には、エツペンドルフチューブにおいて、各バッファー（17 μ L)に、プラスミド DNA(pEGFP- N1)溶液（1 μ L)をカ卩えて混合し、さらに、 LipofectAMINE2000 (2 L)をカ卩えて混合して、 20分間室温にて反応させた。次いで、その反応液にフィ ブロネクチン溶液（5 を加えて混合し、 21種類の混合液を得た。当該混合液は、 以下、サンプルと称する。  [0035] Specifically, in an Eppendorf tube, each buffer (17 μL) was mixed with plasmid DNA (pEGFP-N1) solution (1 μL), and then LipofectAMINE2000 (2 L) was added. Mix and mix for 20 minutes at room temperature. Next, a fibronectin solution (5 was added to the reaction solution and mixed to obtain 21 types of mixed solution. The mixed solution is hereinafter referred to as a sample.
[0036] グルコース含有バッファーを使用したサンプル以外の各サンプルについて、インク ジェットプリンターによって 1枚の PLLコート DNAマイクロアレイスライド（以下、スライ ドと称する）上に 4スポットずつプリント (スポット形成）し、乾燥させた。この操作を繰り 返し、 3枚のスライドを作製した。  [0036] For each sample other than the sample using a glucose-containing buffer, four spots are printed (spot formation) on a single PLL-coated DNA microarray slide (hereinafter referred to as a slide) by an ink jet printer and dried. It was. This operation was repeated to produce three slides.
[0037] また、ノ ッファーとして滅菌蒸留水 (DDW)を使用して、上述と同様に混合液を調 製し、 3枚の各スライドに 4スポットずつプリントしたものをコントロールとした。  [0037] In addition, using distilled distilled water (DDW) as a noffer, a mixture was prepared in the same manner as described above, and four spots printed on each of three slides were used as controls.
[0038] 尚、グルコース含有バッファーを使用したサンプルについては、 1枚のスライドにそ れぞれ 8個のサンプルスポットとコントロールスポットをプリントしたものを作製した。 次いで、各スライドについて、 HeLa細胞の細胞懸濁液（2 X 105cells/mL) lOmLを 播種し、 24時間培養（5%CO ,37°C)して、固相系トランスフエクシヨンを実施した。 24時間の細胞培養を行った後、イメージスキャナによって、各スポットの蛍光画像 を取得し、各種糖類の固相系トランスフエクシヨンへの影響を評価した。蛍光画像は、 プラスミド DNA (pEGFP-Nl)が導入された HeLa細胞が発する緑色の画像である。 [0038] As for samples using the glucose-containing buffer, 8 samples spots and control spots were printed on each slide. Next, each slide was seeded with 10 mL of HeLa cell suspension (2 X 10 5 cells / mL), cultured for 24 hours (5% CO, 37 ° C), and solid phase transfection was performed. did. After 24 hours of cell culture, fluorescence images of each spot were obtained using an image scanner, and the effects of various saccharides on the solid phase system were evaluated. The fluorescence image is a green image emitted from HeLa cells into which plasmid DNA (pEGFP-Nl) has been introduced.
[0039] 図 3は、グルコースの影響を評価したものであり、スポット（図 3 (a)及び (b)各々 8ス ポット）の蛍光画像を示している。図 3 (a)はバッファ一として滅菌蒸留水を使用したも の（コントロール）であり、図 3 (b)はバッファ一としてグルコース水溶液（4. 5g/L)を使 用したものである。図 3から明らかなように、コントロール（図 3 (a) )よりも、グルコース 水溶液を使用したサンプル（図 3 (b) )の蛍光強度が高ぐグルコースは遺伝子導入 効率を上昇させ得ることが分力る。  FIG. 3 is an evaluation of the influence of glucose, and shows fluorescence images of spots (FIGS. 3 (a) and (b) each of 8 spots). Fig. 3 (a) shows the control using sterilized distilled water as the buffer, and Fig. 3 (b) shows the glucose aqueous solution (4.5 g / L) as the buffer. As can be seen from Fig. 3, glucose with higher fluorescence intensity in the sample using the aqueous glucose solution (Fig. 3 (b)) than in the control (Fig. 3 (a)) can increase the gene transfer efficiency. Power.
[0040] 図 4〜図 8は、それぞれスクロース、マルトース、ラタトース、マルトトリオース、グリコ 一ゲンの影響を評価したものを示す。各図には、 3枚の上記スライドにおけるスポット の蛍光画像 (下段）と、その蛍光画像カゝら画像解析ソフトにより蛍光量を定量ィ匕してグ ラフ化 (上段)したものを示してある。  [0040] FIGS. 4 to 8 show evaluations of the effects of sucrose, maltose, ratatoose, maltotriose and glycogen, respectively. Each figure shows the fluorescence image of the spot on the above three slides (bottom), and the fluorescence image quantified by the image analysis software and graphed (top). .
[0041] グラフは、滅菌蒸留水（コントロール）と、各サンプル (使用した糖類の濃度毎）につ いて、それぞれ棒グラフが作成されており、グラフの縦軸は蛍光量 (蛍光強度)を示し ている。  [0041] A bar graph is prepared for each of the sterilized distilled water (control) and each sample (for each concentration of saccharide used), and the vertical axis of the graph indicates the amount of fluorescence (fluorescence intensity). Yes.
[0042] すなわち、各棒グラフと、そのすぐ下の蛍光画像（3枚のスライド）とが対応している。  That is, each bar graph corresponds to the fluorescent image (three slides) immediately below it.
各サンプル (又はコントロール）にっき合計 12個のスポット蛍光画像が得られ、棒ダラ フが作成される。尚、グラフ中のエラーバーは、スポット毎のばらつきを示す。  A total of 12 spot fluorescence images are obtained for each sample (or control) and a bar graph is created. Note that the error bars in the graph indicate the variation from spot to spot.
[0043] 尚、遺伝子導入状態の評価指標は、蛍光強度を指標とした効率 (遺伝子導入効率 )、局在性、及び均一性とした。例えば、図 2において、試料 1〜3に示されるようなス ポット蛍光画像を取得した場合、各スポットにおける、効率、局在性、均一性は、図 2 に示されるように評価される（〇：良、 X：不良)。  [0043] It should be noted that the evaluation index of the gene transfer state was efficiency (gene transfer efficiency), localization, and uniformity using the fluorescence intensity as an index. For example, in FIG. 2, when spot fluorescent images as shown in samples 1 to 3 are acquired, efficiency, localization, and uniformity at each spot are evaluated as shown in FIG. : Good, X: bad).
効率 (遺伝子導入効率）については、プラスミド DNA (pEGFP-Nl)が導入された HeLa細胞が発する緑色蛍光を画像処理によって定量化し、遺伝子導入量として評 価した。尚、定量化は、緑色蛍光タンパク質 EGFPの積分輝度値を、イメージスキャナ 及び画像解析ソフトを用いて行った。  For efficiency (gene transfer efficiency), green fluorescence emitted from HeLa cells into which plasmid DNA (pEGFP-Nl) was introduced was quantified by image processing and evaluated as the amount of gene transfer. The quantification was performed using an image scanner and image analysis software for the integrated luminance value of green fluorescent protein EGFP.
[0044] 図 3〜図 8に示される結果から、各サンプル及びコントロール (滅菌蒸留水）におけ る遺伝子導入効率、局在性、均一性について比較検討した。尚、同じ糖類を使用し ても、その濃度 (サンプル）によって蛍光強度に差異が生じるため、各種糖類毎に総 合的に判断評価してまとめたものを以下の表 3に示した（+ + :滅菌蒸留水 (コント口 ール）に比べ良好、 + :コントロールと同等、一：コントロールに比べ不良）。 [0044] From the results shown in Fig. 3 to Fig. 8, in each sample and control (sterile distilled water), We compared gene transfer efficiency, localization, and homogeneity. Even if the same saccharide is used, the fluorescence intensity varies depending on its concentration (sample). Therefore, the results of comprehensive judgment and evaluation for each saccharide are shown in Table 3 below (++ : Good compared to sterilized distilled water (conte), +: Equivalent to control, 1: Bad compared to control).
その結果、グルコース、スクロース、グリコーゲンをバッファー溶液として用いた場合 に、著しく遺伝子導入効率が上昇すると認められた。  As a result, it was confirmed that gene transfer efficiency was remarkably increased when glucose, sucrose, or glycogen was used as a buffer solution.
[0045] [表 3] [0045] [Table 3]
Figure imgf000013_0001
Figure imgf000013_0001
[0046] (実施例 2)  [Example 2]
ノ ッファーとして、上記表 1に記載される必須アミノ酸 (アルギニン、システィン、ヒス チジン、イソロイシン、ロイシン、リシン、メチォニン、フエ二ルァラニン、トレオニン、トリ プトファン、チロシン、ノ リン、グルタミン、グルタミン酸）、非必須アミノ酸 (ァラニン、ァ スパラギン酸、グリシン、プロリン、セリン）の各種アミノ酸類の水溶液を以下の表 4に 記載される濃度にて調製し、合計 57種類のバッファーを得た。  The essential amino acids listed in Table 1 above (arginine, cystine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, norin, glutamine, glutamic acid), non-essential Aqueous solutions of various amino acids of amino acids (alanine, aspartic acid, glycine, proline, serine) were prepared at the concentrations shown in Table 4 below to obtain a total of 57 types of buffers.
[0047] 次いで、上記実施例 1と同様にして、各サンプルとコントロール (滅菌蒸留水）とを調 製し、 3枚のスライドを作成して、固相系トランスフ クシヨンを実施した。尚、 L-アルギ ニンについては、遺伝子デリバリー材料（LipofectAMINE2000 (インビトロジェン 社製) )を含まな!/、サンプル (L-アルギニン (LF2000無し) )も調製した。  [0047] Next, in the same manner as in Example 1, each sample and control (sterile distilled water) were prepared, three slides were prepared, and solid phase transformation was performed. For L-arginine, a gene delivery material (LipofectAMINE2000 (Invitrogen)) was not included! /, And a sample (L-arginine (without LF2000)) was also prepared.
コントロールにつ 、ては、 3枚の各スライドに 8スポットずつ（合計 24スポット）プリント した。  For the controls, 8 spots were printed on each of the 3 slides (24 spots in total).
[0048] 図 9〜図 28は、それぞれグルタミン、 L-アルギニン、 L-アルギニン（LF2000無し） 、 L-システィン、 L-ヒスチジン、 L-イソロイシン、 L-ロイシン、 L-リシン、 L-メチォニン 、 DL-フエ二ルァラニン、 L-トレオニン、 L-トリプトファン、 L-チロシン、 L -パリン、 L- グルタミン酸、 L-ァラニン、 L-ァスパラギン酸、 L-グリシン、 L-プロリン、 L-セリンの影 響を評価したものである。 [0048] FIGS. 9 to 28 show glutamine, L-arginine, L-arginine (without LF2000), L-cystine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine and DL, respectively. -Shade of phenylalanine, L-threonine, L-tryptophan, L-tyrosine, L-parin, L-glutamic acid, L-alanine, L-aspartic acid, L-glycine, L-proline, L-serine This is an evaluation of the sound.
[0049] 図 9〜図 28 (図 11 (LF2000無し）を除く）に示される結果から、各サンプル及びコ ントロール (滅菌蒸留水）における遺伝子導入効率、局在性、均一性について比較し 、評価してまとめたものを以下の表 4に示した（+ +：滅菌蒸留水（コントロール）に比 ベ良好、 + :コントロールと同等、一 ：コントロールに比べ不良）。  [0049] From the results shown in Fig. 9 to Fig. 28 (excluding Fig. 11 (without LF2000)), the gene transfer efficiency, localization, and uniformity of each sample and control (sterile distilled water) were compared and evaluated. The results are summarized in Table 4 below (++: better than sterile distilled water (control), +: equivalent to control, 1: poor compared to control).
[0050] [表 4]  [0050] [Table 4]
Figure imgf000014_0001
[0051] 遺伝子導入効率では、グルタミン、アルギニン、ヒスチジン、リシン、トレオニン、トリ プトファン、ノ リン、ァラニン、グリシン、プロリン、セリンにおいて、コントロール（滅菌 蒸留水）と比べ、高い遺伝子導入効率を確認した。一方、システィン、イソロイシン、口 イシン、フエ-ルァラニン、グルタミン酸、ァスパラギン酸では、コントロール (滅菌蒸留 水）と比べ、低い遺伝子導入効率を確認した。
Figure imgf000014_0001
[0051] In terms of gene transfer efficiency, higher gene transfer efficiencies were confirmed for glutamine, arginine, histidine, lysine, threonine, tryptophan, norin, alanine, glycine, proline, and serine compared to the control (sterile distilled water). On the other hand, cystine, isoleucine, oral isine, ferulalanin, glutamic acid, and aspartic acid confirmed low gene transfer efficiency compared to control (sterile distilled water).
[0052] 遺伝子導入の局在性では、グルタミン、システィン、ヒスチジン、リシン、トレオニン、 チロシン、ァラニン、プロリン、セリンにおいて、コントロールと比べ、高い遺伝子導入 の局在性を確認した。一方、アルギニン、ロイシン、フエ二ルァラニンでは、コントロー ルと比べ、低い局在性を示した。  [0052] Regarding the localization of gene introduction, higher localization of gene introduction was confirmed in glutamine, cysteine, histidine, lysine, threonine, tyrosine, alanine, proline, and serine compared to the control. On the other hand, arginine, leucine, and phenylalanine showed low localization compared to control.
[0053] 遺伝子導入の均一性では、ヒスチジン、リシン、トレオニン、チロシン、ァラニン、プロ リン、セリンにおいて、コントロールと比べ、高い遺伝子導入の均一性を確認した。一 方、フエ-ルァラニンでは、コントロールと比べ、遺伝子導入が不均一に生じることが 確認された。  [0053] Regarding the homogeneity of gene introduction, higher homogeneity of gene introduction was confirmed in histidine, lysine, threonine, tyrosine, alanine, proline, and serine compared to the control. On the other hand, it was confirmed that the transfer of feralanine was heterogeneous compared to the control.
[0054] (実施例 3)  [Example 3]
実施例 1、 2において、良好な結果を得た糖種であるグルコース (2.25g/L)と、ァミノ 酸のうち、ヒスチジン（2.111^/1^( 1)、5.2511^/]^( 2.5)、 10.5mg/L(X5))、トレオ ニン（0.95mg/L(Xl)、 2.125mg/L(X2.5)、 4.25mg/L(X5))、ァラニン（0.89mg/ L(X1)、 2.225mg/L(X2.5)、 4.45mg/L(X5))、セリン（1.05mg/L(Xl)、 2.625m g/L ( X 2.5)、 5.25mg/L ( X 5) )とをそれぞれ等量混合したものをバッファ一として使 用し、上記実施例 1及び 2と同様に試験した。  In Examples 1 and 2, glucose (2.25 g / L), which is a sugar species with good results, and histidine (2.111 ^ / 1 ^ (1), 5.2511 ^ /] ^ (2.5) among amino acids. , 10.5mg / L (X5)), threonine (0.95mg / L (Xl), 2.125mg / L (X2.5), 4.25mg / L (X5)), alanine (0.89mg / L (X1)), 2.225mg / L (X2.5), 4.45mg / L (X5)), serine (1.05mg / L (Xl), 2.625mg / L (X 2.5), 5.25mg / L (X 5)) A mixture of equal amounts was used as a buffer and tested in the same manner as in Examples 1 and 2 above.
尚、スライドは 2枚使用し、各サンプル及びコントロールを各スライドに 6スポットずつ プリントした。その際、上記各アミノ酸について D体及び L体をそれぞれ調製し、遺伝 子導入効率、局在性、均一性についてコントロール (滅菌蒸留水）と比較した。  Two slides were used, and 6 samples of each sample and control were printed on each slide. At that time, D-form and L-form were prepared for each of the above amino acids, and compared with the control (sterile distilled water) in terms of gene introduction efficiency, localization, and uniformity.
[0055] 図 29〜図 32は、それぞれグルコース及び D, L-ヒスチジン、グルコース及び D, L- トレオニン、グルコース及び D, L-ァラニン、グルコース及び D, L-セリンの影響を評 価したものである。各図において、 2枚のスライドにおけるスポットの蛍光画像（下段） と、その蛍光画像力 画像解析ソフトにより蛍光量を定量ィ匕し、スポット毎の値を平均 化してグラフ化 (上段)したものを示す。 [0056] グラフは、滅菌蒸留水（コントロール）と、各サンプル (使用した D体及び L体のアミノ 酸類の濃度毎）について、それぞれスライド毎に棒グラフが作成されており、グラフの 縦軸は蛍光量 (蛍光強度)を示して!/ヽる。 [0055] FIGS. 29 to 32 show the effects of glucose and D, L-histidine, glucose and D, L-threonine, glucose and D, L-alanine, glucose and D, L-serine, respectively. is there. In each figure, the fluorescence images of the spots on the two slides (bottom) and their fluorescence image power are quantified using the image analysis software, and the values for each spot are averaged and graphed (top). Show. [0056] A bar graph is created for each slide for each of the sterilized distilled water (control) and each sample (concentration of D-form and L-form amino acids used), and the vertical axis of the graph represents the fluorescence. Show the amount (fluorescence intensity)!
[0057] 図 29〜図 32に示される結果から、各サンプル及びコントロール (滅菌蒸留水）にお ける遺伝子導入効率、局在性、均一性について比較し、評価してまとめたものを以下 の表 5に示した（+ +：滅菌蒸留水（コントロール）に比べ良好、 +：コントロールと同 等、一：コントロールに比べ不良）。  [0057] From the results shown in Fig. 29 to Fig. 32, the following table summarizes the results of comparing, evaluating, and summarizing the gene transfer efficiency, localization, and homogeneity in each sample and control (sterile distilled water). Shown in 5 (++: good compared to sterile distilled water (control), +: equivalent to control, 1: poor compared to control).
[0058] 表 5に示すように、全てのサンプルにおいて、コントロールと比べて遺伝子導入効率 、局在性、均一性は良好であることが確認された。特に、アミノ酸を糖溶液に混合す ることで、著しい局在性、均一性の向上が確認された。例えば、グルコース及び D, L -トレオニン（図 30)、および、グルコース及び D, L-セリン（図 32)をバッファーに添カロ した場合、積分輝度値の比較に基づくと、遺伝子導入効率はコントロールと比べて 1 0倍程度も向上して ヽると認められた。  [0058] As shown in Table 5, it was confirmed that the gene transfer efficiency, localization, and uniformity were better in all samples than in the control. In particular, it was confirmed that the localization and uniformity were significantly improved by mixing the amino acid into the sugar solution. For example, when glucose and D, L-threonine (Fig. 30) and glucose and D, L-serine (Fig. 32) are added to the buffer, the efficiency of gene transfer is controlled and controlled based on the comparison of integrated luminance values. It was recognized that it was improved by about 10 times.
また、 D体を用いても、固相系トランスフエクシヨン結果に、大きな影響を与えるもの ではないことも確認した。  It was also confirmed that the use of Form D does not significantly affect the solid-phase transfection results.
[0059] [表 5]  [0059] [Table 5]
Figure imgf000016_0001
Figure imgf000016_0001
[0060] (実施例 4)  [Example 4]
トランスフエクシヨンデバイス製造時に使用する核酸 (遺伝子試料)として、 RNA干 渉作用を示す siRNA (short interference RNA)を用いた。 siRNAをトランスフ ェクシヨンデバイスに用いる場合には、遺伝子デリバリー材料として、 HiPerFect Tr ansfection Reagent (キアゲン社製）を使用することが望ましい。  SiRNA (short interference RNA) that exhibits RNA interference was used as the nucleic acid (gene sample) used in the manufacture of the transfer device. When siRNA is used in a transfection device, it is desirable to use HiPerFect Transfection Reagent (Qiagen) as a gene delivery material.
[0061] 図 33 (b)に示される各種試料（グルコース/ァラニン溶液、 siRNA、 HiPerFect試 薬、フイブロネクチン溶液)を調製し、図 33 (a)に示す方法に従いトランスフエクシヨン デバイスを製造した。 [0061] Various samples shown in Fig. 33 (b) (glucose / alanine solution, siRNA, HiPerFect test) The drug, fibronectin solution) was prepared, and a transfection device was manufactured according to the method shown in FIG.
図 33 (a)に記載の方法を以下に詳述する。  The method described in Figure 33 (a) is described in detail below.
siRNAとして、緑色蛍光タンパク質（EGFP)の発現を抑制し得る anti-EGFPsiR NAと、緑色蛍光タンパク質（EGFP)の発現を抑制しな!、ネガティブコントロール siR NAとを用いた。  As siRNA, anti-EGFP siRNA that can suppress the expression of green fluorescent protein (EGFP) and negative control siRNA that does not suppress the expression of green fluorescent protein (EGFP) were used.
グルコース/ァラニン含有バッファーに、 siRNAを溶解させた後、 HiPerFect試薬 を添カ卩し、室温で 10分間インキュベートした。次に、フイブロネクチン溶液を添カ卩して スライド上にプリントすることにより、トランスフエクシヨンデバイスを製造した。  After dissolving siRNA in a glucose / alanine-containing buffer, HiPerFect reagent was added and incubated at room temperature for 10 minutes. Next, a fibronectin solution was added and printed on a slide to produce a transfection device.
このスライドを細胞培養用ディッシュに移して HeLa細胞を播種し、 3〜6時間後に、 緑色蛍光タンパク質プラスミド DNA (pEGFP- N 1 )を Ef f ecten試薬によりトランスフ ェクシヨンデバイス上の HeLa細胞へトランスフエクシヨンした。  Transfer this slide to a cell culture dish and inoculate HeLa cells. After 3-6 hours, transfer green fluorescent protein plasmid DNA (pEGFP-N1) to HeLa cells on the transfection device using Effecten reagent. Excuse me.
このように実施された固相系トランスフエクシヨンの結果を、図 34に示す。  FIG. 34 shows the results of the solid phase transfection carried out in this way.
[0062] この結果、ネガティブコントロール siRNAを導入した細胞に比べ、緑色蛍光タンパ ク質発現を抑制し得る anti-EGFPsiRNAを導入した細胞では、有意に緑色蛍光タ ンパク質遺伝子の発現を抑制していることが確認された。 [0062] As a result, the expression of the green fluorescent protein gene was significantly suppressed in the cells introduced with anti-EGFP siRNA that can suppress the expression of the green fluorescent protein compared to the cells introduced with the negative control siRNA. It was confirmed.
[0063] すなわち、本発明のトランスフエクシヨンデバイスは、 siRNAの機能（遺伝子発現の 抑制）を保持した状態で固相系トランスフエクシヨンを実施可能であることが示唆され た。 [0063] That is, it was suggested that the transfection device of the present invention can perform solid-phase transfection in a state where the function of siRNA (suppression of gene expression) is maintained.
仮に siRNAの機能が損なわれた状態で上記固相系トランスフエクシヨンが実施され たとした場合、上記 anti-EGFPsiRNAは、緑色蛍光タンパク質（EGFP)の発現を 抑制することができない。その結果、 anti-EGFPsiRNAを導入した細胞と、ネガティ ブコントロール siRNAを導入した細胞とでは、緑色蛍光タンパク質（EGFP)の蛍光 輝度に図 34に示されるような有意差は生じ難いと考えられる。  If the solid-phase transfection is carried out with the siRNA function impaired, the anti-EGFP siRNA cannot suppress the expression of green fluorescent protein (EGFP). As a result, it is unlikely that a significant difference as shown in FIG. 34 occurs in the fluorescence intensity of green fluorescent protein (EGFP) between cells into which anti-EGFP siRNA has been introduced and cells into which negative control siRNA has been introduced.
[0064] (実施例 5) [0064] (Example 5)
RN A干渉作用を示す siRNAを遺伝子試料として用いた場合、 siRNA分子の不安 定性'分解され易さにより、遺伝子発現の抑制効果を有する siRNAを用いた場合で あっても、遺伝子導入される対象によってその効果を示さないことがある。特にこの現 象は、対象として in vivoへ遺伝子導入を行った際に顕著である。 When siRNA exhibiting RNA interference is used as a gene sample, even if siRNA having an effect of suppressing gene expression is used due to the unstable nature of siRNA molecules, The effect may not be shown. Especially this present The elephant is prominent when gene transfer is performed in vivo as a target.
in vivoへの遺伝子導入を目的としたトランスフエクシヨンデバイスの製造を行う際 には、 siRNA分子の in vivoにおける安定性が重要な因子となる。 in vivoにおける siRNA分子の安定性を評価する方法としては、血清中における siRNA分子の分解 速度を指標とする方法が一般的である。  The stability of siRNA molecules in vivo is an important factor when manufacturing a transfection device for gene transfer in vivo. As a method for evaluating the stability of siRNA molecules in vivo, a method using the degradation rate of siRNA molecules in serum as an index is common.
[0065] このような背景の下、トランスフエクシヨンデバイス製造に使用する試薬混合物の血 清中における安定性を調べ、その結果を未処理の siRNAと対比した。  [0065] Against this background, the stability of the reagent mixture used for the production of a transfection device during seralysis was examined, and the results were compared with untreated siRNA.
[0066] 本実施形態で使用する試薬の調整法を図 35に示した。  [0066] Fig. 35 shows a method for adjusting the reagent used in the present embodiment.
図 35 (a)に示される各種試料（グルコース/ァラニン溶液、 siRNA、プラスミド DNA 、 LipofectAMINE2000、フイブロネクチン溶液）をそれぞれ調製した。 siRNAとし て上述したネガティブコントロール siRNA、プラスミド DNAとして pUC 19を用いた。 当該各種試料を適宜混合し、二種類のサンプル (TMA mix, siRNA only)を作 製した。  Various samples (glucose / alanine solution, siRNA, plasmid DNA, LipofectAMINE2000, fibronectin solution) shown in Fig. 35 (a) were prepared. The negative control siRNA described above was used as siRNA, and pUC 19 was used as plasmid DNA. The various samples were mixed as appropriate to produce two types of samples (TMA mix, siRNA only).
安定性評価溶液は、グルコース/ァラニン含有バッファーに、 siRNA、プラスミド DN The stability evaluation solution is prepared by using siRNA, plasmid DN in a glucose / alanin-containing buffer.
A、 LipofectAMINE2000をそれぞれ溶解させた後、室温で 20分間インキュベート し、フイブロネクチン溶液を添加して調製した（図 35 (b) )。 A and LipofectAMINE2000 were dissolved and incubated at room temperature for 20 minutes, and a fibronectin solution was added (FIG. 35 (b)).
siRNA濃度の測定を行う際には、 RiboGreen RNA Quantitation Kit (R— 1 When measuring siRNA concentration, use RiboGreen RNA Quantitation Kit (R— 1
1490 :インビトロジェン社製）を使用した。この Kitの試薬を、 1 XTBEバッファーで 21490: manufactured by Invitrogen). Reagents from this kit with 1 XTBE buffer
00倍希釈（以下、 RiboGreen溶液と称する）して以下の実験に使用した。 Diluted 00 times (hereinafter referred to as RiboGreen solution) and used for the following experiments.
[0067] 図 36には、前記二種類のサンプルにおいて、 siRNA濃度を変化させた際の、 Rib oGreen RNA Quantitation Kitの測定感度を評価した。 In FIG. 36, the measurement sensitivity of the RiboGreen RNA Quantitation Kit when the siRNA concentration was changed in the two types of samples was evaluated.
[0068] siRNAのみがバッファ一中に溶解しているサンプル（siRNA only)では、 RiboGr een溶液の添カ卩量によらず、 siRNA量に依存的な蛍光量を示すことが分かった。 一方、トランスフエクシヨンデバイス製造に使用するサンプル (TMA mix)では、夾 雑物の影響で、 RiboGreen溶液の添カ卩量力 当該サンプル量の 8倍以上になった 際に、 siRNA量に依存的な蛍光量を示すことが分力つた。 [0068] It was found that the sample in which only siRNA was dissolved in the buffer (siRNA only) showed a fluorescence amount dependent on the amount of siRNA regardless of the amount of RiboGreen solution added. On the other hand, in the sample (TMA mix) used for the manufacture of a transfection device, the amount of addition of the RiboGreen solution depends on the amount of siRNA when the amount of the sample increases more than 8 times due to the influence of impurities. It was a component to show the amount of fluorescence.
[0069] この結果に基づき、 RiboGreen溶液の添力卩量をサンプル量の 10倍として、以下の 実験を行った。 [0070] 図 35 (a)に示した各種試料と同じ試料を適宜混合し、表 6に記載した二種類のサン プル（TMA mix, siRNA only)を作製した。これらサンプルのそれぞれに、終濃 度が 50%となるようにゥシ胎児血清を 25 μ L添カ卩した。 [0069] On the basis of this result, the following experiment was conducted with the amount of applied force of the RiboGreen solution being 10 times the amount of the sample. [0070] The same samples as the various samples shown in Fig. 35 (a) were appropriately mixed to prepare the two types of samples shown in Table 6 (TMA mix, siRNA only). Each of these samples was supplemented with 25 μL of rabbit fetal serum so that the final concentration was 50%.
[0071] [表 6]  [0071] [Table 6]
Figure imgf000019_0001
Figure imgf000019_0001
[0072] ゥシ胎児血清を添加した後、 0,10,20,30,60, 90, 120, 150, 180分間室温にて インキュベートし、各サンプル内の siRNA量を測定した結果を図 37に示した。同じ条 件で上記実験を 2回行った (Runl, Run2)。 [0072] After adding fetal bovine serum, incubation was performed at room temperature for 0, 10, 20, 30, 60, 90, 120, 150, 180 minutes, and the amount of siRNA in each sample was measured. Indicated. The above experiment was performed twice under the same conditions (Runl, Run2).
その結果、 50%血清条件において、トランスフエクシヨンデバイス製造に使用する サンプル（TMA mix)に含まれる siRNAが、 siRNAのみがバッファ一中に溶解して V、るサンプル (siRNA only)にくらべ、高!、安定性を有して 、ることが確認された。 なお、 2回行った実験 (Runl, Run2)では、同様の結果が得られている。  As a result, the siRNA contained in the sample (TMA mix) used to manufacture the transfection device under 50% serum conditions is higher than the sample (siRNA only) in which only the siRNA is dissolved in the buffer. ! It has been confirmed that it has stability. Similar results were obtained in the two experiments (Runl, Run2).
[0073] 以上より、トランスフエクシヨンデバイス製造に使用するサンプル (TMA mix)にお いて、 siRNA分子は in vivoにおいて安定性を有するものと認められた。 [0073] From the above, it was confirmed that the siRNA molecules were stable in vivo in the sample (TMA mix) used for the production of the transfection device.
産業上の利用可能性  Industrial applicability
[0074] 本発明は、真核細胞である動物細胞等に外部力 遺伝子試料 (例えば、一本鎖又 は二本鎖のデォキシリボ核酸（DNA)、リボ核酸 (RNA)、ァプタマ一、あるいはこれ らの化学修飾誘導体等)を導入するトランスフエクシヨンデバイスに利用できる。 図面の簡単な説明 [0074] The present invention provides an external force gene sample (eg, single-stranded or double-stranded deoxyribonucleic acid (DNA), ribonucleic acid (RNA), aptamer, or the like) to animal cells that are eukaryotic cells. It can be used for a transfer device for introducing a chemically modified derivative of Brief Description of Drawings
[0075] [図 1]本発明のトランスフエクシヨンデバイスの一例であるガラスアレイの製造方法の一 例を示した図  [0075] FIG. 1 is a diagram showing an example of a method for manufacturing a glass array, which is an example of a transformation device of the present invention.
[図 2]本発明のトランスフエクシヨンデバイスを用いて固相系トランスフエクシヨンを実施 した際のスポットにおける遺伝子導入状態の評価例を示した図  FIG. 2 is a diagram showing an example of evaluation of the state of gene introduction at a spot when solid phase transfection is performed using the transfection device of the present invention.
[図 3]固相系トランスフエクシヨンに際してグルコースの影響を評価した図 [図 4]固相系トランスフエクシヨンに際してスクロースの影響を評価した図 [Fig. 3] Evaluation of the effect of glucose on solid phase transfection [Fig.4] Evaluation of the effect of sucrose on solid phase transfection
[図 5]固相系トランスフエクシヨンに際してマルトースの影響を評価した図  [Fig.5] Evaluation of the effect of maltose on solid phase transformation
[図 6]固相系トランスフエクシヨンに際してラタトースの影響を評価した図  [Fig. 6] Evaluation of the effect of ratatoses on solid-phase transformation
[図 7]固相系トランスフエクシヨンに際してマルトトリオースの影響を評価した図  [Fig.7] Evaluation of the effect of maltotriose on solid phase transformation
[図 8]固相系トランスフエクシヨンに際してグリコーゲンの影響を評価した図  [Fig.8] Evaluation of the effect of glycogen on solid phase transfer
[図 9]固相系トランスフエクシヨンに際してグルタミンの影響を評価した図  [Figure 9] Evaluation of the effect of glutamine on solid-phase transfection
[図 10]固相系トランスフエクシヨンに際して L-アルギニンの影響を評価した図  [Figure 10] Evaluation of the effect of L-arginine during solid-phase transfection
[図 11]固相系トランスフエクシヨンに際して L-アルギニン (遺伝子デリバリー材料無し) の影響を評価した図  [Fig. 11] Evaluation of the effect of L-arginine (no gene delivery material) on solid phase transfection
[図 12]固相系トランスフエクシヨンに際して L-システィンの影響を評価した図  [Fig.12] Evaluation of the influence of L-cysteine during solid phase transfer
[図 13]固相系トランスフエクシヨンに際して L-ヒスチジンの影響を評価した図  [Figure 13] Evaluation of the effect of L-histidine during solid-phase transfection
[図 14]固相系トランスフエクシヨンに際して L-イソロイシンの影響を評価した図  [Fig.14] Evaluation of the effect of L-isoleucine on solid phase transfection
[図 15]固相系トランスフエクシヨンに際して L-ロイシンの影響を評価した図  [Fig.15] Evaluation of the effect of L-leucine on solid phase transfection
[図 16]固相系トランスフエクシヨンに際して L-リシンの影響を評価した図  [Fig.16] Evaluation of the effect of L-lysine on solid-phase transfection
[図 17]固相系トランスフエクシヨンに際して L-メチォニンの影響を評価した図  [Fig.17] Evaluation of the effect of L-methionine on solid phase transformation
[図 18]固相系トランスフエクシヨンに際して DL-フエ-ルァラニンの影響を評価した図 [Fig.18] Evaluation of the effect of DL-ferualanin on solid-phase transformation
[図 19]固相系トランスフエクシヨンに際して L-トレオニンの影響を評価した図 [Fig. 19] Evaluation of the effect of L-threonine on solid phase transfection
[図 20]固相系トランスフエクシヨンに際して L-トリブトファンの影響を評価した図  [Fig.20] Evaluation of the effect of L-tributophan on solid-phase transfection
[図 21]固相系トランスフエクシヨンに際して L-チロシンの影響を評価した図  [Fig.21] Evaluation of the effect of L-tyrosine during solid phase transfection
[図 22]固相系トランスフエクシヨンに際して L-パリンの影響を評価した図  [Fig.22] Evaluation of the effect of L-parin during solid-phase transfection
[図 23]固相系トランスフエクシヨンに際して L-グルタミン酸の影響を評価した図  [Fig.23] Evaluation of the effect of L-glutamic acid on solid phase transfection
[図 24]固相系トランスフエクシヨンに際して L-ァラニンの影響を評価した図  [Fig.24] Evaluation of the effect of L-alanine on solid phase transfection
[図 25]固相系トランスフエクシヨンに際して L-ァスパラギン酸の影響を評価した図 [Fig.25] Evaluation of the effect of L-aspartic acid on solid phase transfection
[図 26]固相系トランスフエクシヨンに際して L-グリシンの影響を評価した図 [Fig.26] Evaluation of the effect of L-glycine on solid-phase transfection
[図 27]固相系トランスフエクシヨンに際して L-プロリンの影響を評価した図  [Fig.27] Evaluation of the effect of L-proline on solid phase transfection
[図 28]固相系トランスフエクシヨンに際して L-セリンの影響を評価した図  [Fig.28] Evaluation of the effect of L-serine on solid phase transfection
[図 29]固相系トランスフエクシヨンに際してグルコース及び D, L-ヒスチジンの影響を 評価した図 [図 30]固相系トランスフエクシヨンに際してグルコース及び D, L-トレオニンの影響を 評価した図 [Fig.29] Evaluation of the effects of glucose and D, L-histidine during solid-phase transfection. [Fig. 30] Evaluation of the effects of glucose and D, L-threonine on solid phase transfection
[図 31]固相系トランスフエクシヨンに際してグルコース及び D, L-ァラニンの影響を評 価した図  [Fig.31] Evaluation of the effects of glucose and D, L-alanine on solid phase transfection
[図 32]固相系トランスフエクシヨンに際してグルコース及び D, L-セリンの影響を評価 した図  [Fig.32] Evaluation of the effects of glucose and D, L-serine during solid phase transfection
[図 33]siRNA単独での固相系トランスフエクシヨン及びその確認方法を示した図 [図 34]siRNA単独での固相系トランスフエクシヨンの結果を示した図  [Figure 33] Diagram showing the solid-phase transfection with siRNA alone and its confirmation method. [Fig. 34] Diagram showing the results of the solid-phase transfection with siRNA alone.
[図 35]siRNAの安定性評価に使用した試薬の調製方法を示した図 [Fig. 35] Diagram showing the method of preparing the reagents used to evaluate the stability of siRNA
[図 36]siRNAの測定感度の評価結果を示した図 FIG. 36 shows the evaluation results of siRNA measurement sensitivity.
[図 37]50%ゥシ胎児血清存在下における siRNA濃度の経時変化の結果を示した図 符号の説明  [Fig.37] Diagram showing the results of time-dependent changes in siRNA concentration in the presence of 50% urine fetal serum.
1 混合液 1 mixture
2 プリント装置  2 Printing device
3 固相基板 3 Solid phase substrate
4 細胞懸濁液 4 Cell suspension
5 シャーレ 5 Petri dish

Claims

請求の範囲 The scope of the claims
[1] 遺伝子試料、遺伝子デリバリー材料、細胞接着因子に加え、グルコース、スクロー ス、グリコーゲン、グルタミン、ァノレギニン、ヒスチジン、リシン、トレオニン、トリプトファ ン、パリン、ァラニン、グリシン、プロリン、セリンカもなる群力も選択される少なくとも一 種の化合物を含むものを固相基材に固着してあるトランスフエクシヨンデバイス。  [1] In addition to gene samples, gene delivery materials, and cell adhesion factors, glucose, sucrose, glycogen, glutamine, anoleginine, histidine, lysine, threonine, tryptophan, parin, alanine, glycine, proline, serine can also be selected A transfusion device in which at least one kind of compound is fixed to a solid phase substrate.
[2] 前記遺伝子試料が、一本鎖又は二本鎖のデォキシリボ核酸、リボ核酸、アブタマ一 [2] The gene sample is a single-stranded or double-stranded deoxyribonucleic acid, ribonucleic acid, abutama
、あるいはこれらの化学修飾誘導体力 なる群力 少なくとも一種が選択される請求 項 1記載のトランスフエクシヨンデノ イス。 The transfection denoice according to claim 1, wherein at least one of the group powers of these chemically modified derivatives is selected.
[3] 前記遺伝子デリバリー材料が、カチオン性ポリマー、カチオン性脂質、ミネラルから なる群力 選択される少なくとも一種を含有する請求項 1記載のトランスフ クシヨンデ バイス。 [3] The transfusion device according to [1], wherein the gene delivery material contains at least one selected from the group consisting of a cationic polymer, a cationic lipid, and a mineral.
[4] 前記細胞接着因子が、ゼラチン、フイブロネクチン、コラーゲン、ラミニン、ビトロネク チン、抗インテグリン抗体力もなる群力 少なくとも一種が選択される請求項 1記載の トランスフエクシヨンデバイス。  4. The transfer device according to claim 1, wherein the cell adhesion factor is selected from at least one group force including gelatin, fibronectin, collagen, laminin, vitronectin, and anti-integrin antibody force.
[5] 前記固相基材を構成する材料が、ガラス、合成高分子、金属類、天然高分子から なる群力 選択される請求項 1記載のトランスフエクシヨンデバイス。  5. The transfer device according to claim 1, wherein the material constituting the solid phase substrate is selected from the group force consisting of glass, synthetic polymer, metals, and natural polymer.
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WO2021189355A1 (en) * 2020-03-26 2021-09-30 Chinese Institute For Brain Research, Beijing Amino acid mediated gene delivery and its uses

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