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WO2005090585A1 - Procede de transfert de gene sur une phase solide utilisant un vecteur de virus, composition pour le procede et appareil pour le procede - Google Patents

Procede de transfert de gene sur une phase solide utilisant un vecteur de virus, composition pour le procede et appareil pour le procede Download PDF

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Publication number
WO2005090585A1
WO2005090585A1 PCT/JP2005/004136 JP2005004136W WO2005090585A1 WO 2005090585 A1 WO2005090585 A1 WO 2005090585A1 JP 2005004136 W JP2005004136 W JP 2005004136W WO 2005090585 A1 WO2005090585 A1 WO 2005090585A1
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Prior art keywords
virus
cells
gene
protein
hvj
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PCT/JP2005/004136
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English (en)
Japanese (ja)
Inventor
Hitoshi Kotani
Toshihiro Nakajima
Akito Maeda
Kuniko Mizuhata
Yasufumi Kaneda
Shohei Akahane
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Genomidea Inc.
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Priority to JP2006519405A priority Critical patent/JPWO2005090585A1/ja
Publication of WO2005090585A1 publication Critical patent/WO2005090585A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Solid phase gene transfer method using viral vector composition for the method, and device for the method
  • the present invention relates to a solid phase for introducing a desired substance into cells with high efficiency on a solid phase.
  • gene information is integrated using gene arrays and gene chips to analyze how gene expression patterns change during disease. It is used.
  • these devices can change gene expression patterns during disease (eg, determine the amount of messenger), but cannot identify the functions of those genes in actual cells.
  • Genetic analysis technology at the cell level is capable of analyzing functions at the actual cell level. Genetic analysis technology at the cell level using the conventional manual method is faster and more efficient. It is difficult to perform a dashi. Gene analysis techniques at the cell level can be broadly classified into (1) gene transfer techniques and (2) analysis techniques for transfected cells. Among these, in particular, the conventional gene transfer technique has problems in efficiency and high-speed dangling, and it is difficult to cope with many types of genes.
  • desired substances for example, peptides, compounds, nucleotide fragments, etc.
  • the present invention also provides a method for introducing a variety of desired substances, which can be performed at high speed and with high efficiency by using a virus envelope or a virus-derived protein, and is used for such a substance introduction method.
  • the challenge is to develop solid phases, compositions, and kits for performing these tasks.
  • the present invention has been achieved as a result of diligent studies on a solid-phase gene transfer method using a virus envelope or a virus-derived protein, and as a result, found a method for high-efficiency and high-speed gene transfer.
  • the present invention provides the following.
  • a device for introducing a desired substance into cells comprising:
  • a device wherein the viral vector and the surfactant having the sugar moiety are disposed on the solid phase.
  • the device according to item 1 further comprising (4) a substance to be introduced, wherein the substance to be introduced is disposed on the solid phase.
  • the substance to be introduced is a nucleic acid, a peptide, a sugar chain, or a nucleic acid encoding a gene to be introduced.
  • the device according to item 2 wherein the molecular compound, proteodarican, glycopeptide, lipid, and metal ion force are at least one substance selected from the group consisting of:
  • the substance to be introduced contains a nucleic acid selected from the group consisting of siRNA, antisense and decoy of the gene to be introduced.
  • the vinoresbatter comprises (i) a viral envelope, (ii) a ribosome containing at least one viral envelope protein, or (iii) a viral envelope, and a ribosome containing at least one viral envelope protein.
  • the virus envelope is retroviridae, togaviridae, coronaviridae, flaviviridae, paramyxoviridae, orthomyxoviridae, bunyaviridae, rhabdoviridae, boxviridae, herpesviridae, baculovirus.
  • Item 7 The device according to Item 6, wherein the device is derived from a virus belonging to the family that is also selected from the family of viridae and hepadnaviridae.
  • virus envelope protein is an F protein, an HN protein, an NP protein, or a protein whose combination or group power is also selected.
  • the surfactant having a sugar moiety has the formula
  • A is a monosaccharide or a disaccharide. 14. The device according to item 13, wherein A is a disaccharide.
  • A is a dalcoside group or a maltoside group.
  • Surfactant power having the sugar moiety octyl maltoside, decyl maltoside, dodecyl maltoside, otatyl dalicoside, decyl darcoside, and dodecyl darcoside, as well as a mixture of these surfactants
  • the device according to item 1 further comprising (5) a protamine sulfate solution, wherein the protamine sulfate solution is disposed on the solid phase.
  • the stabilizing agent is trehalose, manoletose, raffinose, fructose, sucrose, glucose, ratatose, polyvinylinolebi mouth lidone, polyethylene glycol, methylcellulose, glycine, mannitol, dimethyl.
  • DMSO sulfoxide
  • the stabilizer is also selected from a group consisting of trehalose, maltose, raffinose, fructose or a mixture thereof.
  • a device for introducing a gene into a cell comprising:
  • An apparatus comprising:
  • Item 36 The apparatus according to Item 36, comprising:
  • a kit for introducing a gene into cells comprising:
  • a kit comprising:
  • kit according to item 38 further comprising (4) protamine sulfate.
  • kit according to item 38 further comprising (4) polyethyleneimine.
  • kit according to item 38 further comprising (5) a stabilizer.
  • kits according to item 42 wherein the stabilizing agent is trehalose, maltose, raffinose, fructose, sucrose, glucose, ratatose, polyvinylpyrrolidone K90, polyethylene glycol, methylcellulose, glycine, mannitol.
  • the strength of the group is selected from the group consisting of dimethyl sulfoxide (DMSO) or a mixture thereof.
  • kit according to item 38 or 42 further comprising (6) a step of freeze-drying.
  • Substances introduced into cells according to the present invention include not only nucleic acids such as nucleotide fragments, DNA, and RNA, but also proteins, sugar chains, low-molecular compounds, proteodaricans, glycopeptides, lipids, and metal ions. And the like.
  • this technology enables fast and highly efficient analysis of the functions of human genes and various biopolymers estimated to be 30,000 to 40,000 in actual cells.
  • a highly integrated device is provided.
  • a HVJ vector encapsulating biopolymers such as genes and proteins is immobilized on a device with micro-wells processed on a slide with high integration, and multiple samples are introduced after introduction into cells in micro-wells.
  • a detection device By analyzing with a detection device, it is possible to simultaneously evaluate the functions of a large number of human genes in living cells.
  • FIG. 1 is a graph showing the selection of surfactants for automation, showing the results of gene transfer using various surfactants at various concentrations according to the method of the present invention. .
  • FIG. 2 is a graph showing the selection of a surfactant for automation, and shows the results of gene transfer using various surfactants at various concentrations according to the method of the present invention. .
  • FIG. 3 is a view showing high reproducibility when a pGL3 expression plasmid was introduced according to the method of the present invention.
  • the X axis indicates the horizontal cell number (1-12), and the Y axis indicates the horizontal cell number (A-H).
  • FIG. 4A is a view showing high reproducibility when a pGL3 expression plasmid is introduced according to the method of the present invention.
  • the X-axis shows the horizontal shell number (1-12), and the Y-axis shows the horizontal shell number (A-H).
  • FIG. 4B is a diagram showing high reproducibility when a pGL3 expression plasmid was introduced according to the method of the present invention.
  • the X-axis shows the horizontal shell number (1-12), and the Y-axis shows the horizontal shell number (A-H).
  • FIG. 5 is a view showing a difference in gene transfer efficiency when plates coated with various substances are used.
  • FIG. 6 shows the results showing gene transfer efficiency when protamine sulfate and polyethylimine were used. "None" indicates the results of an experiment using neither protamine sulfate nor polyethylimine.
  • FIG. 7 shows the results showing gene transfer efficiency when protamine sulfate and polyethylimine were used.
  • FIG. 8 shows the results showing gene transfer efficiency when various polyethylimines were used.
  • FIG. 8A shows the gene transfer efficiency when PEI-L 25K was used.
  • the X-axis is the PEI-L 25K concentration at the time of introduction.
  • FIG. 8 shows the results of gene transfer efficiency when various polyethylimines were used. It is.
  • FIG. 8B shows the gene transfer efficiency when PEI-L2.5K was used.
  • the X-axis is the ⁇ —L2.5 ⁇ concentration at the time of introduction.
  • FIG. 8 shows the results showing gene transfer efficiency when various polyethylimines were used.
  • FIG. 8C shows the gene transfer efficiency when using ⁇ — ⁇ 25 ⁇ .
  • the X-axis is the ⁇ —L 25 ⁇ concentration at the time of introduction.
  • FIG. 8D shows the results of gene transfer efficiency when various polyethylimines were used.
  • FIG. 8D shows the gene transfer efficiency when ⁇ - ⁇ 750 ⁇ is used.
  • the X-axis is the concentration of L-750 at the time of introduction.
  • FIG. 9 ⁇ shows the results showing the cytotoxicity of various polyethylimines.
  • FIG. 9 ⁇ shows the results showing the cytotoxicity of various polyethylimines.
  • FIG. 10 shows the results showing stability by freeze-drying.
  • FIG. 11 shows the results showing that gene transfer efficiency is maintained when the cells are freeze-dried and stored for a long period of time.
  • Fig. 12 shows the results showing that gene transfer efficiency is maintained when lyophilized using different stabilizers and stored for a long period of time.
  • FIG. 13 shows the results showing that gene transfer efficiency is maintained when lyophilized using different stabilizers and stored for a long period of time.
  • FIG. 14 shows the results showing that the storage stability of each cell was uniform.
  • FIG. 15 shows the results showing that, even when siRNA was introduced, the gene introduction efficiency was maintained after long-term storage as in the case of DNA.
  • FIG. 16 shows the results showing that, even when siRNA is introduced, the gene introduction efficiency is maintained after long-term storage as in the case of DNA.
  • FIG. 17 shows the results showing that the freeze-drying method is not related to plate coating.
  • FIG. 18 shows the results of a gene transfer experiment using a plate obtained by lyophilizing a virus vector containing no nucleic acid to be introduced and lyophilizing it.
  • cell is defined in the same broad sense as used in the art, and is a constituent unit of a tissue of a multicellular organism, and is a membrane that isolates the outside world.
  • the term "stabilizer” refers to an agent that improves the storage stability of a cell culture container of the present invention containing a gene transfer vector when the container is stored by lyophilization.
  • the stabilizer of the present invention include trehalose, maltose, raffinose, fructose, sucrose, glucose, ratatose, polyvinylpyrrolidone, polyethylene glycol, methylcellulose, glycine, mannitol, dimethylsulfoxide (DMSO) or a mixture thereof. Examples include, but are not limited to, substances from which both force and group force are selected.
  • Stabilizers preferably include trehalose, maltose, raffinose, fructose or mixtures thereof. More preferably, the stabilizer of the present invention is trehalose.
  • polyethyleneimine includes both linear and branched types.
  • the structure of linear PEI is shown as-[NH-CH2-CH2] x-.
  • X is any integer.
  • preferable linear PEI include PEI-L (PEI-L2.5K) having a molecular weight of 2.5 kDa and PEI-L (PEI-L 25K) having a molecular weight of 25 kDa.
  • Branched PEI is — [NH-CH2-CH2] y- [N (CH 2—CH2—NH2) —CH2—CH2] z—.
  • y is any integer.
  • z is any integer independent of y.
  • preferred branched PEIs are PEI-B with a molecular weight of 25 kDa (PEI-B 25K) and PEI-B with a molecular weight of 750 kDa (PEI-B 750K).
  • viral envelope vector refers to a vector in which a foreign gene is encapsulated in a viral envelope, or a foreign gene is encapsulated in a component containing a protein derived from a viral envelope.
  • Vector ⁇ The virus used for the production of the virus vector may be a wild-type virus or a recombinant virus.
  • the virus used for the preparation of the virus envelope or the protein derived from the virus envelope includes retroviridae, togaviridae, coronaviridae, flaviviridae, and paramyxoviridae.
  • Viruses belonging to the family selected from the group consisting of: Orthomyxoviridae, Byadinores, La Grape Inenoles, Boxwinores, Henolesininoles, Noculoiviridae, and Hepadnaviridae. Listed powers are not limited to these.
  • a virus belonging to the family Nomyxoviridae is used, more preferably, HVJ (Sendai virus).
  • Virus envelope-derived proteins include, for example, HVJ F protein, HN protein, NP protein, and M protein.
  • HVJ HVJ
  • Sendai virus can be used interchangeably.
  • envelope of HVJ envelope of Sendai virus
  • envelope of Sendai virus are used as words having the same meaning.
  • Sendai virus refers to a virus that belongs to the genus Paramyxovirus in the family Paramyxovirus and has a cell fusion effect. Virus particles have an envelope and show a polymorphism of 150 to 300 nm in diameter. The genome is a minus-strand RNA of about 15500 bases in length. It has an RNA polymerase, is thermally unstable, agglutinates almost all types of red blood cells, and is hemolytic.
  • HAU refers to the activity of a virus capable of aggregating 0.5% of avian erythrocytes, and 1 HAU corresponds to approximately 24 million virus particles (Okada, Y. et al.). , Bike n Journal 4, 209—213, 1961).
  • animal cells used as host cells include mouse 'myeloma cells, rat's myeloma cells, mouse' hybridoma cells, Chinese'no cells, CHO cells as B. muster cells, BHK cells, and African green monkeys.
  • Examples include kidney cells, human leukemia cells, HBT5637 (JP-A-63-299), and human colon cancer cell lines.
  • African green monkeys such as ps20 and NSO for mouse myeloma cells, YB2Z0 for rat myeloma cells, HEK293 (ATCC: CRL-1573) for human fetal kidney cells, and BALL-1 for human leukemia cells
  • kidney cells include COS-1 and COS-7
  • human colon cancer cell lines include HCT-15.
  • solid phase As used herein, the terms “solid phase,” “substrate,” and “support” are used interchangeably herein and refer to the material from which the array of the present invention is constructed (preferably, Solid).
  • the material of the substrate may have the property of binding to the biomolecule used in the present invention, or may be derivatized to have such property, either covalently or non-covalently. , Any solid material.
  • any material that can form a solid surface can be used, and examples thereof include glass, silica, silicon, ceramic, silicon dioxide, plastic, and metal (alloy). ), Natural and synthetic polymers (eg, polystyrene, cellulose, chitosan, dextran, and nylon), including but not limited to:
  • the substrate may be formed from multiple layers of different materials.
  • inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used.
  • nylon membrane In the present invention, nylon membrane, nitrocellulose membrane, PVDF membrane, etc.
  • a film used for blotting can also be used.
  • Nylon membranes are preferred. This is because, when a Nippon membrane is used, the results can be analyzed using a simple analysis system. However, when analyzing a high-density material, it is preferable to use a material having hardness, such as glass.
  • the term “chip” or “microchip” refers to a microminiature integrated circuit that has various functions and becomes a part of a system.
  • the “DNA chip” includes a substrate and DNA, and at least one DNA (eg, a cDNA fragment) is disposed on the substrate.
  • the term “protein chip” includes a substrate and a protein, and at least one protein (eg, polypeptide or oligopeptide) is disposed on the substrate.
  • “DNA chip” and “protein chip” are included herein as “microchip” or simply “chip”.
  • a “microarray” is one in which one or more biomolecules (eg, oligonucleotides such as cDNA fragments or peptides) are arranged and arranged on such a chip!
  • biomolecules for example, oligonucleotides such as DNA or peptides
  • those collected from a living body can be used, and the biomolecules can be obtained by a method known to those skilled in the art.
  • oligonucleotides can be prepared by automated chemical synthesis using either a DNA synthesizer or a peptide synthesizer, such as those marketed by Applied Biosystems. Compositions and methods for the synthesis of automated oligonucleotides are described, for example, in US Pat. No. 4,415,732, Caruthers et al. (1983); US Pat. Nos. 4,500,707 and Caruthers (1985). U.S. Patent No. 4,668,777, Caruthers et al. (1987).
  • the substrate any number of biomolecules (e.g., DNA or peptide) may be provided, usually, per one substrate 1, 10 to 8 biomolecule 10 7 In other embodiments until bIOLOGICAL molecule, 10 6 biological molecule, 10 to 5 biological molecules, no more than 10 4 biomolecules, 10 3 to biomolecules, or 10 up to two biomolecules number of biomolecules Can be arranged.
  • the size of the substrate is preferably smaller.
  • the spot size of a biomolecule eg, DNA or peptide
  • the minimum board area is V, in some cases, is determined by the number of biomolecules on the substrate.
  • surfactant having a sugar moiety includes a surfactant having a “sugar moiety” in the molecule.
  • Surfactants include ionic surfactants, cationic surfactants, non-ionic surfactants, and amphoteric surfactants.
  • Sugar moieties of surfactants having a sugar moiety include, but are not limited to, monosaccharides, disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, heptasaccharides, and octasaccharides. The sugar moiety may or may not be branched.
  • the sugar moiety is preferably a monosaccharide monopentasaccharide, more preferably a monosaccharide or disaccharide.
  • examples of the sugar moiety include, but are not limited to, dalcoside, galactoside, maltoside, thiomaltoside, and thiodarcoside.
  • a surfactant having a sugar moiety is, for example, a compound represented by A- (CH) 2 -CH, where A is a sugar n 3
  • And-(CH) -CH is a hydrophobic moiety other than the sugar moiety. Its hydrophobic part is n 3
  • n is 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, and 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 Below, 8 or less, or 7 or less.
  • the preferred range of n is 7-11.
  • the surfactant having the sugar moiety is selected from the group consisting of octyl maltoside, decyl maltoside, dodecyl maltoside, otatildaricoside, decyldarcoside, dodecyldarcoside, and mixtures thereof.
  • a preferred surfactant having a sugar moiety is decyl maltoside.
  • the preparation of the substance introduction device in which the virus vector is immobilized can be carried out by the following steps:
  • substances to be introduced include not only nucleic acids such as nucleic acids, proteins, peptides, nucleotide fragments, nucleotide fragments, DNA, RNA, nucleotides, nucleosides and derivatives thereof, but also proteins, sugar chains, Also included are low molecular weight compounds, proteodaricans, glycopeptides, lipids and metal ions.
  • nucleic acids such as nucleic acids, proteins, peptides, nucleotide fragments, nucleotide fragments, DNA, RNA, nucleotides, nucleosides and derivatives thereof, but also proteins, sugar chains, Also included are low molecular weight compounds, proteodaricans, glycopeptides, lipids and metal ions.
  • the introduced gene is derived from any organism. And can be DNA, RNA or nucleotide analogs.
  • the gene to be introduced may be one kind or plural kinds.
  • the cell into which the gene is introduced is not particularly limited, but is preferably an animal cell, and more preferably a mammalian cell including a human cell.
  • the virus used to prepare the viral envelope may be a retroviridae, a togaviridae, Group selection with coronaviridae, flaviviridae, paramyxoviridae, orthomyxoviridae, bunyaviridae, rhabdoviridae, boxviridae, herpesviridae, baculoviridae, and hepadnaviridae It may be derived from a virus belonging to the family to be treated.
  • the virus is a Paramyxoviridae virus, especially HVJ.
  • a method for introducing a candidate nucleic acid into a cell using a ribosome containing at least one protein of a virus envelope is also available.
  • Proteins used in this method include, but are not limited to, F protein, HN protein, NP protein, M protein or a combination thereof.
  • the mixture of the virus vector and the substance to be introduced is prepared, for example, by centrifuging a solution containing the virus vector to form a precipitate, and suspending the precipitate using the substance solution to be introduced.
  • Conditions for this centrifugation include, for example, 15000 X g, 4 ° C, and 15 minutes.
  • centrifugation conditions of 15000 X g at 4 ° C for 60 minutes can be used.
  • a mixture of the virus vector solution and the substance solution to be introduced may be prepared in another container in advance, and the mixture may be added to the solid phase.
  • a virus vector solution and a substance solution to be introduced may be separately added to the solid phase.
  • the substance introduction device which is a solid phase containing a virus vector, a substance to be introduced, and a surfactant having a sugar moiety is allowed to stand at room temperature.
  • the incubation time can preferably be 5 minutes, 7 minutes, 10 minutes, 12 minutes, 15 minutes, 17 minutes, 20 minutes, 25 minutes, 30 minutes.
  • protamine sulfate (PS) or polyethylenimine (PEI) is added to increase the gene transfer efficiency.
  • the concentration of protamine sulfate used is preferably 1 ⁇ g / ml-lmg / mU, more preferably 10 ⁇ g / ml-250 ⁇ g / m, most preferably 100 g / ml.
  • the concentration of polyethyleneimine used is preferably 0.1 g / ml-300 ⁇ g / ml, more preferably 0.5 g / ml-200 ⁇ g / ml, most preferably 1 ⁇ gZ One ml is 100 ⁇ gZ ml.
  • the substance introduction device is centrifuged.
  • the conditions for this centrifugation are, for example, 1643 X g, 4 ° C, and 30 minutes, but are not limited thereto.
  • a foreign gene can be introduced into cells by the following method.
  • the cell suspension is introduced into the substance introduction device in which the virus vector is immobilized. Centrifuge the device if necessary. The conditions of the centrifuge are typically, but not limited to, 182 ⁇ g, 32 ° C., for 30 minutes. Thereafter, if necessary, the device is shaken and centrifuged. The conditions of the centrifuge are typically, but not limited to, 182 ⁇ g, 32 ° C., 30 minutes. If necessary, two more centrifugations at 182 X g at 32 ° C for 30 minutes can be performed.
  • a large number of cell populations are prepared by introducing a large number of genes into the substance introduction device of the present invention, and the functions of the cell populations are analyzed to obtain a desired function.
  • Cells into which a nucleic acid having a specific property has been introduced are selected. As a result, a gene having a desired function is identified.
  • Desired functions include, for example, encoding a gene that induces angiogenesis, encoding a tumor suppressor gene, encoding a gene that enhances bone formation, encoding a gene that induces apoptosis, secreting cytoin.
  • a gene encoding a neurite-inducing gene for neurons, encoding an arterial stiffness inhibitor gene, encoding a diabetes suppressor gene, encoding an autoimmune disease suppressor gene, Alzheimer's disease suppressor gene , Encoding a Parkinson's disease suppressor gene, encoding a neuronal protection gene, and a combination of these.
  • this selection is preferably performed based on the characteristics of the host cell that are changed by the expression of the candidate nucleic acid. For example, when isolating a gene encoding a growth factor from a candidate nucleic acid, the desired functional property is to promote the growth of a particular cell or any cell.
  • the above analysis method can be applied to any functional property as long as the target functional property can be recognized.
  • the functional properties intended in the above analysis method include promotion or suppression of cell proliferation, cell differentiation or dedifferentiation, expression or suppression of marker protein expression, suppression of expression or expression of marker mRNA, change in membrane potential, Examples include, but are not limited to, depolarization, apoptosis, canceration, growth arrest, morphological changes, size changes, and the like.
  • the gene transfer method of the present invention can be applied to screening.
  • a substance introduction device comprising (1) a solid phase; (2) a viral vector; (3) a substance to be introduced; and (4) a surfactant having a sugar moiety
  • the (3) By using a gene library as a substance, it can be applied to screening.
  • a gene library By preparing a cell population containing a gene library on a device using a gene library, selecting cells having a desired function from the cells, and isolating and identifying nucleic acids in the cells A gene having a desired function can be screened. Also, If the cell population that is found to be positive by cleaning contains multiple types of genes, it is possible to isolate and identify a gene having a desired function by purifying the substance. is there.
  • This screening method can be applied not only to plasmid-like nucleic acids, but also to proteins, peptides, low molecular weight compounds, nucleotide fragments, and the like.
  • the substance introduction device can reduce the size of the immobilization region of the virus vector, screening of biomolecules can be performed not only in 96-well plates, but also in 384-well plates and 1536-well plates. It is possible to do.
  • the substance introduction device of the present invention is suitable for automation.
  • a kit for performing the method of the present invention is also provided in the present invention.
  • virus envelope vectors Various methods are known as virus envelope vectors.
  • the present inventors have developed a hybrid gene transfer vector by combining a virus and a non-viral vector, and have developed a fusion-forming virus having a fusion-forming envelope derived from Japanese hemagglutinating virus (HVJ; Sendai virus).
  • HVJ Japanese hemagglutinating virus
  • Ribosomes have been constructed (Kaneda, Biogenic Amines, 14: 553-572 (1998); Kaneda et al., Mol. Med. Today, 5: 298-303 (1999)).
  • HVJ ribosomes 400-500 nm in diameter
  • An advantage of fusion-mediated delivery is that transfatating DNA also protects endosomal and lysosomal degradation in recipient cells.
  • Up to 100 kb of DNA is incorporated into the HVJ ribosome and delivered to mammalian cells.
  • RNA, oligonucleotides and drugs are also efficiently introduced into cells in vitro and in vivo. The HVJ ribosome was not shown to induce significant cell damage in vivo.
  • Methods for preparing the above-listed viral envelope vectors are also known. Representative examples are shown below. The method for preparing a virus envelope vector described below is an exemplification, and the present invention is not limited to the vector prepared by the following method.
  • a viral vector containing a protein derived from the viral envelope includes a HVJ (Sendai virus) F fusion protein and a ribosome reconstituted with an HN fusion protein.
  • HVJ Sendai virus
  • Viral vectors that do not contain genomic RNA are included.
  • the F fusion protein and HN fusion protein used for the preparation of such a viral vector may be a natural HVJ-derived protein or a recombinantly expressed protein. Recombinantly produced fusion proteins are processed by proteases in vitro or by endogenous proteases in mammalian cell hosts.
  • a viral vector containing a protein derived from the viral envelope is prepared, for example, by a method comprising the following steps:
  • the surfactant used in the above method is not limited to a specific surfactant, but is preferably otatildaricoside, Triton-X100, CHAPS or NP-40, or these. Is a mixture of
  • the lipid used in the above method is not limited to a specific lipid, and (1) has a long-chain fatty acid or a similar hydrocarbon chain in a molecule, and (2) exists in a living body, or Any molecule derived from a living organism may be used.
  • Preferred lipids include, but are not limited to, phosphatidylcholine, phosphatidylserine, cholesterol, sphingomyelin, and phosphatidic acid.
  • Ribosome preparation methods are well known, and for example, the following methods can be used.
  • the HVJ envelope protein can be purified using natural HVJ as a source, or the recombinantly expressed HVJ envelope protein can be purified.
  • Known methods for purifying proteins include, for example, ammonium sulfate precipitation, isoelectric focusing, Lam purification methods include, but are not limited to.
  • Lam purification methods include, but are not limited to.
  • purifying a protein using a column various columns are selected depending on the properties of the desired protein and the properties of contaminants. Columns for protein purification include, but are not limited to, anion exchange columns, cation exchange columns, gel filtration columns, and affinity columns.
  • a viral vector containing a protein derived from the viral envelope is prepared by a method comprising the following steps:
  • a viral vector containing a protein derived from the viral envelope is also prepared by a method comprising the following steps:
  • a method of preparing a virus vector having a foreign gene encapsulated in a virus envelope includes, for example, a method comprising the following steps:
  • a viral vector derived from the viral envelope is prepared by a method comprising the following steps:
  • a method for preparing an inactivated virus envelope vector for gene transfer comprising:
  • lipids generally used for lipofection can be used.
  • a lipid such as lipofect AMINE 2000 can be used.
  • various cells can be used as cells into which a substance is introduced.
  • a cell is preferably a mammal, more preferably a cell from the species from which the candidate nucleic acid is derived.
  • Example 1 Preparation and use of a virus vector in which a foreign gene is encapsulated in a component containing a protein derived from a virus envelope) (Preparation of virus)
  • HVJ hemagglutinin units
  • Nonidet P-40 (NP-40) and fermethylsulfur-fluoride (PMSF) dissolved in ethanol were purified to a final concentration of 0.5% and 2 mM, respectively, in 20 ml of HVJ suspension (l, 750, 000 HAU). The mixture was incubated at 4 ° C. for 30 minutes with no rotation. The suspension was then centrifuged at 100,000 g at 4 ° C. for 75 minutes to remove insoluble proteins and the viral genome (Uchida et al., 1979). The supernatant was dialyzed against 5 mM phosphate buffer (pH 6.0) for 3 days, and the buffer was changed every day to wash off residual NP-40 and PMSF.
  • the dialyzed solution was centrifuged at 100,000 g for 75 minutes at 4 ° C. to remove insoluble substances.
  • the supernatant was subjected to a 10 mM phosphate buffer (pH 5) containing 0.3% sucrose and ImM KC1 according to the method described previously (Yoshima et al., J. Biol. Chem., 256: 5355-5361 (1981)).
  • HVJ fusion proteins can also be prepared by incorporating the gene encoding the fusion protein into an expression vector and expressing it in a suitable host cell. F tongue The amino acid sequences of protein and HN protein are known.
  • Expression vectors that encode the fusion protein can be introduced into cells, and are any of a variety of methods known and described in the art (eg, Sambrook et al., Molecular Cloning: A Laboratoy Manual ⁇ 2nd Ed). , Vols 1 to 3, Cold Spring Harbor Laboratory
  • Methods for introducing a recombinant expression vector into prokaryotic or eukaryotic cells include, for example, a transformation or transfection method such as an electoporation method.
  • the polypeptide corresponding to the trypsin-treated activated F1 protein can be expressed in Escherichia coli using an expression vector containing a gene encoding the shortened amino acid sequence of activated F1.
  • the truncated F1 protein needs to include at least 26 amino acid residues from ferrule at position 117 to alanine at position 142. If the truncated protein forms an inclusion body, one skilled in the art can easily obtain the active protein by refolding the inclusion body (Robert F. Kelley and Marjorie E. Winkler). , Genetic Engineerings (1990) vol. 12, pages 11-19).
  • Cells capable of replicating HVJ are used as host cells.
  • HVJ eg, rodent tracheal epithelial cells; chicken embryos; primary cultured cells of monkey kidney; primary cultured cells of human fetal lung, kidney, and amnion
  • the expressed full-length F protein is Since it is cleaved by the mouth thease and as a result is activated, it is possible to express and isolate the active form F protein.
  • host cells that express Triptase clara can also be used.
  • Triptase clara eg, rat tracheal epithelial cells
  • hosts that express recombinantly Cells can also be used.
  • the lysate of HVJ treated with NP-40 was clarified by ultracentrifugation.
  • the protein in the supernatant was separated by SDS-PAGE before further purification.
  • This supernatant contained many proteins from HVJ.
  • this supernatant was applied to ion exchange chromatography.
  • the 52 kDa and 72 kDa proteins eluted primarily in the flow through fraction. These two proteins were identified as F1 and HN, respectively, by mobility on SDS-PAGE (Okada, Methods in Enzymology, N. Duzgnes (Ed.), Academic Press, Diegoan Diego, vol. 221). , pp. 18-41 (1993)).
  • the faint band below the 52 kDa protein was considered to be a degradation product of the fusion proteins (F1 and HN). This is because these proteins were not reproducible between different experiments.
  • the protein was further eluted with 0.2M NaCl. However, the fusion protein was not as efficient as it could be. Then, an additional 60 kDa protein appeared to be the NP protein of HVJ.
  • Densitometry showed that the concentration ratio of F1 to HN in the flow-through fraction was 2.3: 1. This was consistent with the ratio of both proteins in the virus envelope.
  • An earlier paper (Nakanishi et al., Exp. Cell Res., 142: 95-101 (1982)) reports that this ratio is necessary for efficient fusion of HVJ.
  • the dialyzed solution was agarose beads (Bio—Gel A—50 m) equilibrated with 10 mM phosphate buffer (pH 5.2) containing 0.3 M sucrose and ImM KC1 (Bio—Rad Laboratories, Hercules , CA, USA). Fractions with an optical density at 540 nm greater than 1.5 were collected as reconstituted fusion particles and fused with a nucleic acid-filled liponome that was also prepared at 10 mg lipid force as described below to prepare a viral vector.
  • pCMV luciferase (7.4 kb)
  • pGEM luciferase gene from luc (Promega Corp., Madison, Wis., USA)
  • pcDNA3 5.4 kb (Invitrogen, San Diego, CA, USA)
  • a viral vector containing about 40 g of pCMV-luciferase was constructed as described above, and the viral vector (about 1.5 ⁇ 10 11 particles Zml, DNA concentration about 40 ⁇ g / ml) of 1Z10
  • the quantity (100 1) was incubated with 2 ⁇ 10 5 cells from the human 293 cell line (human fetal kidney: HEK).
  • luciferase DNA was transfected into 2 ⁇ 10 DNAEK293 cells using HVJ ribosomes. Twenty-four hours after transfection, the cells were collected, and the luciferase activity was confirmed as described elsewhere (Saeki et al., Hum. Gene Ther., 8: 1965-1972 (1997)).
  • the recombinant HVJ virus was freeze-thawed at various times and then transfected into cultured cells.
  • 500 ⁇ l of TE was mixed with 750 ⁇ g of luciferase expression vector pcOriPLuc (Saeki and Kaneda et al., Human Gene Therapy, 11, 471-479 (2000)) and various concentrations of HVJ virus.
  • the HVJ virus concentration was adjusted to 10, 25, 50, and 100 HAUZ1. This solution was divided into 12 parts, each of which was frozen with dry ice and then thawed up to 30 times.
  • the solution that has been frozen and thawed a predetermined number of times is added to the medium of BHK-21 cells (24 ⁇ eldish, 4 ⁇ 10 4 cells / dish, 0.5 ml DMEM, 10% FCS), and then added to the medium. After reacting with 5% CO for 20 minutes, wash with PBS and culture again
  • the solution was added to 0.5 ml and cultured for 24 hours.
  • luciferase activity was increased as the number of freeze-thaw cycles of the recombinant HVJ virus was increased, and luciferase expression was observed 10 times or more in 20 freeze-thaw cycles compared to 3 freeze-thaw cycles. . From these results, it was confirmed that, under the conditions used in this example, the number of freeze-thaw cycles of the recombinant HVJ virus was preferably 5 or more, and more preferably about 15 to 20 times.
  • the amount of the solution added with the virus concentration of 10 HAUZ ⁇ 1 is 50 ⁇ 1, and the amount of the solution of 100 HAU / ⁇ 1 is 5 ⁇ 1.
  • a solution with a virus concentration of 100 HAU / ⁇ 1 reduced the efficiency of gene expression by about 50% compared to a solution with a concentration of 10-50 HAUZ ⁇ 1. From these results, it was confirmed that the recombinant virus concentration is preferably in the range of 10-50 HAUZ ⁇ 1 under the conditions of this example.
  • the recombinant HVJ virus was thawed 29 times and then thirty times frozen. After preservation in the frozen state for 1 week, it was thawed and added to the cells. As a result, The surviving recombinant HVJ virus also showed the same level of luciferase gene expression as the virus that had been subjected to 30 consecutive freeze-thaw cycles.
  • the HVJ can be generally used by multiplying by inoculating a seed virus into fertilized eggs of chickens. (Added to the solution), and those obtained by infecting cultured cells with a cloned virus genome to cause persistent infection and then growing are all available.
  • HVJ was propagated as follows.
  • HVJ seed virus is propagated using fertilized eggs of SPF (Specific pathogen free), separated and dispensed.
  • SPF Specific pathogen free
  • the purified HVJ (species Z) is dispensed into a cell storage tube, and 10% DMSO is added to Stored and prepared in nitrogen.
  • Seed virus (removed from liquid nitrogen) with a polypeptone solution (mixed with 1% polypeptone and 0.2% NaCl, adjusted to pH 7.2 with 1M NaOH, sterilized by autoclave, and stored at 4 ° C) was diluted 500-fold and placed at 4 ° C.
  • Eggs are disinfected with isodine and alcohol, a small hole is drilled through the virus injection area with a 1000-thread penetrator, and 0.1 ml of diluted seed virus is injected into the allantoic cavity using an lml syringe with a 26-gauge needle. did.
  • the melted paraffin (melting point 50-52 ° C) was placed over the hole using a Pasteur pipette and closed.
  • the eggs were placed in an incubator and reared for 3 days at 36.5 ° C and a humidity of 40% or more.
  • the inoculated eggs were then placed overnight at 4 ° C.
  • divide the air chamber of the egg with tweezers put a 10 ml syringe with an 18 gauge needle into the chorioallantoic membrane, aspirate the allantoic fluid, collect in a sterile bottle, and keep at 4 ° C Existed.
  • HVJ can be purified by a purification method by centrifugation, a purification method by a column, or other purification methods known in the art.
  • the grown virus solution was recovered and the tissue and cell debris in the culture solution and chorioallantoic fluid were removed by low-speed centrifugation.
  • the supernatant was purified by high speed centrifugation (27,500 ⁇ g, 30 minutes) and ultracentrifugation (62,800 ⁇ g, 90 minutes) using a sucrose density gradient (30-60% wZv). Care should be taken to keep the virus as mild as possible during purification and store at 4 ° C.
  • HVJ was purified by the following method.
  • HVJ-containing chorioallantoic fluid collected the HVJ-containing avian eggs and store at 4 ° C
  • a wide-mouth Komagome pipette Saeki, Y , And Kan eda, Y: Protein modinea liposomes (HVJ—liposomes) for the delivery of genes, oligonucleotides and proteins.
  • HVJ Protein modinea liposomes
  • the precipitate was loosened by gentle pipetting using a wide-mouth Komagome pipette, collected in a single tube, and centrifuged at 27, OOOg for 30 minutes using an angle rotor. Except for the supernatant, about 10 ml of BSS was added to the precipitate, and the mixture was similarly allowed to stand at 4 ° C. Gently pipette with a wide-mouthed Komagome pipe to loosen the precipitate, centrifuge at 3000 rpm for 10 minutes at 4 ° C with a low-speed centrifuge (brake off), and remove virulent tissue fragments ⁇ virus aggregation Look at the lumps, V. Put the supernatant in a new sterile tube and store at 4 ° C as purified virus.
  • HAU hemagglutination activity
  • the absorption value 1 at 540 nm corresponded to approximately 15, OOOHAU.
  • HAU is considered to be approximately proportional to the fusion activity.
  • Hemagglutination activity may also be measured using- ⁇ avian erythrocyte fluid (0.5%)! / ⁇ (Manual for practical use of animal cells, REALIZE INC. (Edited by Uchida, Oishi, Furuzawa) P259 — See 268, 1984).
  • purification of HVJ using a sucrose density gradient can be performed as necessary. Specifically, the virus suspension is placed on a centrifugal tube overlaid with 60% and 30% sucrose solutions (autoclave sterilization), and subjected to density gradient centrifugation at 62,800 ⁇ g for 120 minutes. After centrifugation, collect the band on the 60% sucrose solution layer. The collected virus suspension is dialyzed overnight at 4 ° C using BSS or PBS as an external solution to remove sucrose. If not used immediately, add glycerol (autoclave sterilized) and 0.5M EDTA solution (autoclave sterilized) to the virus suspension to a final concentration of 10% and 2-10 mM, respectively, at -80 ° C. C. Gently freeze in C, and finally store in liquid nitrogen (freeze storage is also possible with 10 mM DMSO instead of glycerol and 0.5 M EDTA).
  • purification of HVJ by a column is also applicable to the present invention.
  • purification was performed using concentration (approximately 10-fold) by ultrafiltration using a filter having a molecular weight cutoff of 50,000 and elution by ion exchange chromatography (0.3M to 1M NaCl).
  • HVJ was purified by a column using the following method.
  • chorioallantoic fluid After collecting the chorioallantoic fluid, it was filtered through an 80 m-10 m membrane filter. 0.006-0. 008% BPL (final concentration) was added to the chorioallantoic fluid (4 ° C, 1 hour) to inactivate the HVJ. BPL was inactivated by incubating chorioallantoic fluid at 37 ° C for 2 hours.
  • HVJ was purified by a column chromatography method (buffer: 20 mM TrisHCl (pH 7.5), 0.2-1 M NaCl) using QSepharoseFF (Amersham Fanolemasia Biotech KK, Tokyo). The recovery was 40-50% and the purity was over 99%.
  • the HVJ fraction was concentrated by a tangential flow ultrafiltration method using 500KMWCO (A / G Technology).
  • HVJ suspension was placed in a 30 mm petri dish and irradiated with 99 or 198 millijoules Zcm 2 .
  • Gamma ray irradiation is also available (5-20 gray) but does not completely deactivate.
  • the suspension of HVJ was supplemented with ⁇ -propiolatatone to a final concentration of 0.01% and incubated on ice for 60 minutes. After that, the cells were incubated at 37 ° C for 2 hours. Dispense 10,000 HAU per tube into Eppendorf tubes, centrifuge at 15,000 rpm for 15 minutes, and store the precipitate at -20 ° C. Regardless of the inactivation method described above, the precipitate is not stored at -20 ° C, and the DNA is directly incorporated by detergent treatment to create a vector.
  • Triton—X100 t—t-octylphenoxypolyethoxyethanol
  • CHAPS [(3-cholamidopropyl) dimethylammo] —1—propanesulfonic acid
  • NP— Detergents such as 40 (noylphenoxypolyethoxyethanol) may also be used.
  • Preferred final concentrations of Triton-X100, NP-40 and CHAPS are 0.24-0.80%, 0.04-0.12% and 1.2-2.0%, respectively.
  • Cold BSS was supplemented with lml of calo, and immediately centrifuged at 15, OOOrpm for 15 minutes.
  • PBS or physiological saline was added to the resulting precipitate in a volume of 300 1 and suspended by vortexing and pipetting.
  • the suspension can be used directly for gene transfer, or can be used for gene transfer after storage at 20 ° C.
  • This HVJ envelope vector maintained comparable gene transfer efficiency after storage for at least 2 months.
  • a 1,000 HAU portion was placed in an Eppendorf tube (30 ⁇ l), and protamine sulfate (lmgZm1) 51 was added. Replace the medium with BHK-21 cells (previously 200,000 cells per well, spread over 6 wells) and add 0.5 ml of medium (10% FCS-DMEM) per well. did. To each well, add a mixture of the above vector (equivalent to 1,000 HAU) and protamine sulfate, shake the plate back and forth and left and right to mix the vector and cells well, and place in a 5% CO incubator at 37 ° C. Left for 10 minutes.
  • luciferase a luciferase gene having a CMV promoter
  • lyse the cells with 5 ml of Cell Lysis Buffer (Promega) O, and use the luciferase assay kit (Promega) to determine the activity in a 20 ⁇ l solution. It measured using.
  • pCMV-GFPE green fluorescent protein
  • the ribosome vector of the present invention is prepared by combining a cDNA derived from a cDNA library of 20 to 24; zg and a reagent of 24 to 72 ⁇ l of lipofect AMINE 2000 (Invitrogen life technologies (Carlsbad, California 92008)).
  • the host cells are added to each well of a 96-well plate with an appropriate medium, and cultured. If transfection is performed in the presence of serum, add 12.5 conjugate directly to each well of a 96-well plate and mix. When performing transfusion in the absence of serum, remove the serum-containing medium before adding the complex, and replace with a serum-free medium.
  • a substance introduction device was prepared by immobilizing an HVJ-E vector containing a desired nucleic acid on a solid phase using the following method.
  • BHK-21 cells (babyhamsterkidney cells) were purchased from the American Type Culture Collection (ATCCRockville, MD). The cells were cultured in Dulbecco's modified Idal medium supplemented with 10% FBS. To evaluate the transfusion efficiency, pGL3 (Promega), a luciferase expression vector, was used. Plasmids were prepared using Qiagen columns (Hilden, Germany). The HVJ virus was amplified and the HVJ-E vector was prepared as described in Example 3.
  • luciferase activity was detected when each of dodecyl maltoside, otatildaricoside, and digitonin was used.
  • the intensity of the activity was the strongest for dodecyl maltoside, followed by digitonin, which was the strongest for otatildaricoside.
  • the order of the highest activity was decyl maltoside, dodecyl maltoside, octyl maltoside, otatildaricoside, digitonin.
  • the gene transfer method of the present invention showed high reproducibility.
  • the mean values of the fluorescence intensity (RU) are 2080518 and 1667747, with a standard deviation of 377798.9 and And 324477.
  • the relative standard deviations (CV%) were 18.2% and 19.5%, indicating the high reproducibility of the present invention by numerical values (FIGS. 4A and B).
  • the plate shown in Fig. 5 was used.
  • the coating of each plate is as follows: Corning: no coating (manufactured by Corning),
  • PLL Poly L lysine coating (Sumitomo Bakelite),
  • Gelatin gelatin coating (Sumitomo Bakelite),
  • C-1 Collagen 1 coating (Sumitomo Bakelite),
  • PLO / LM Poly L-ol-tin Z laminin coating (manufactured by BD Bioscience)
  • PDL / LM Poly D lysine Z laminin coating (manufactured by BD Bioscience)
  • C-1 Collagen 1 coating (manufactured by BD Biosciences)
  • C 4 Collagen 4 coating (manufactured by BD Biosciences)
  • Fibronectin Fibronectin coating (manufactured by BD Biosciences).
  • Example 8 Isolation and analysis of a gene encoding a protein having a desired function
  • a target gene having a desired functional property can be isolated.
  • the cells are introduced into the cells on the device according to the method described in Example 5, and the By determining a cell having a desired function from the above, it is possible to isolate a gene encoding a protein having a desired function.
  • the method for isolating the gene exemplified in the present example includes not only the gene transfer vector prepared in Example 3 of the present specification but also any "viral envelope vector” and " “Ribosomal vectors” can be used.
  • a human heart cDNA library (GIBCO BRL; a plasmid in which human heart-derived cDNA was linked to a plasmid pSPORT having a CMV promoter) was introduced into E. coli DH12S, and a plasmid was prepared from the E. coli. I do. Plasmid 200 mu g, enclosed in HV JE gene transfer vectors 10000HAU (gene transfer vector prepared in Example 3 of the present invention, 3 X 1 0 9 particle) . As host cells, approximately 5000 human aortic endothelial cells HAEC (Sanko Junyaku) are added to each well of a 96-well microtiter plate together with a medium, and the cells are cultured overnight. To each cell containing the host cells, add 1/100 of the above HVJ-E, leave at 37 ° C for 30 minutes, and replace the medium.
  • the medium used is a low nutrient condition with a serum concentration of 1%, and culture is performed under this condition for one week. Under these conditions, HEAC growth is not confirmed.
  • the color depth of the well is an indicator of cell proliferation.
  • the darkest jewel is the jewel where the most actively growing cells are located. Therefore, the entire microtiter plate is read by a plate reader, and the cell growth is graphed by a computer. From this graph, nucleic acids are prepared from the cells in the two wells that have the highest proliferation activity, using the DNeasy Tissue Kit from Qiagen. Since the prepared nucleic acid contains plasmid DNA, this is introduced into E. coli (DH5 o; Takara Shuzo) using a heat shock method.
  • This E. coli is inoculated on a plate medium containing ampicillin to form a colony. About 20-200 colonies can be obtained from DNA prepared from one well. For each colony, plasmid DNA (pDNA) is extracted, and the presence of a gene fragment in the plasmid is confirmed by restriction enzyme treatment. Usually, the plasmid has about 60-70% of the plasmid force insert of the prepared plasmid.
  • pDNA plasmid DNA
  • the plasmid DNA was purified using Qiagen's Endo Free Plasmid Maxi Kit, the purified plasmid was encapsulated in HVJ-E, and introduced again into HAEC cells. Do.
  • a plasmid showing significantly higher cell growth is a plasmid expected to contain a nucleic acid encoding vascular endothelial growth factor.
  • Example 3 of the present specification Using the viral vector prepared in Example 3 of the present specification, a gene having a desired function is isolated.
  • the gene isolation method exemplified in this example not only the virus vector prepared in Example 3 of this specification but also any ⁇ virus envelope vector '' and ⁇ ribosome vector '' can be used. It is.
  • nucleic acid encoding a specific gene is selected.
  • the nucleic acid is then To produce a plasmid operably linked to a sequence that functions as a promoter.
  • the plasmid is introduced into a first host cell according to Example 3.
  • the first host cell into which the nucleic acid has been introduced is mutagenized, and about 5,000 cells are added to each well of a 96-well macrotiter plate together with a medium, and cultured overnight. After culturing, the mutant host cells in each cell are screened for the desired function. The cells exhibiting the desired function are removed, and nucleic acids are prepared from the cells in the wells exhibiting the most preferable properties using Qiagen's DNeasy Tissue Kit.
  • the prepared nucleic acid contains plasmid DNA, which is introduced into E. coli (DH5 o; Takara Shuzo) using a heat shock method.
  • This E. coli is seeded on a plate medium containing ampicillin to form a colony. Usually about 20-200 colonies can be obtained from DNA prepared from a single well. For each colony, plasmid DNA is extracted, and the presence of a gene fragment in the plasmid is confirmed by restriction enzyme treatment.
  • plasmid DNA was purified using Qiagen's Endo Free Plasmid Maxi Kit, the purified plasmid was encapsulated in HVJ-E, and introduced again into HAEC cells. test. In this cell growth experiment, it was revealed that the plasmid exhibiting preferable functional properties was a plasmid containing a mutant nucleic acid having the desired functional properties.
  • protamine sulfate PS was used for the conventional encapsulation in the HVJ, but in this embodiment, polyethyleneimine (PEI) was used instead of PS.
  • PEI polyethyleneimine
  • PEI known as a cationic polymer includes a linear type and a branched type.
  • PEI-L linear type
  • PEI-B branched type
  • the structure of linear PEI is shown as — [NH—CH—CH] — (X
  • PEI-L having a molecular weight of 2.5 kDa (PEI-L2.5K) and PEI-L having a molecular weight of 25 kDa (PEI-L 25K) were used.
  • Branched PEI is [NH— CH— CH
  • PEI-B having a molecular weight of 25 kDa PEI-B 25K
  • PEI-B having a molecular weight of 750 kDa PEI-B 750K
  • Example 6 For BAEC cells, the method of Example 6 above was modified as follows, and PEI was used instead of PS. Specifically, it is as follows.
  • HVJ-E was centrifuged at 15000g for 15 minutes at 4 ° C in order to add 4.5 HAU per 1-well of a 96-well plate. After removing the supernatant, suspend the cells in an appropriate amount of HBSS (Hank's Balanced Salt Solution). Dispensed to each well of the well plate. Each 96-well was mixed with a DNA solution (0.25 mg / mL pEGFP-C1 in 5 lZ-well) and mixed. Thereafter, each of 96 wells was mixed with 0.01% dodecyl maltoside in 31 / well and mixed, and the plate was incubated for 15 minutes.
  • HBSS Hort's Balanced Salt Solution
  • PEI was added at 12 ⁇ l (different in concentration and type depending on the cell and biopolymer used), and after mixing, 25 ⁇ l of HBSS was added and mixed. The plate was centrifuged at 3000 rpm for 30 minutes at 4 ° C and further incubated overnight at 4 ° C.
  • 6.7 ⁇ 10 4 cells Zml of BACE cell solution was prepared, and IX was placed on a plate supplemented with the vector containing the nucleic acid prepared above using a 96 auto dispenser machine.
  • 10 4 cells (150 1) were added to the mixture and centrifuged twice at 32 ° C. for 30 minutes at 100 rpm. The cells were cultured in a medium supplemented with 10% FBS at 34 ° C for 50 hours. Luciferase activity was detected using a luciferase assay kit (Promega) and fluorescence intensity was detected with an image reader ARVOmx (Wallac). The results are shown in Figs.
  • FIG. 8 shows the results.
  • PEI-L 25K proved to be most suitable for BAEC cells.
  • the optimal PEI type varies depending on the cell.
  • PEI-L linear type
  • PEI-B branched type
  • the width of the concentration range that shows high introduction efficiency is narrow. . Similar trends are seen in other cells.
  • cytotoxicity using the four types of PEI was compared. Specifically, the effects of various PEIs on cell proliferation when BAEC cells were cultured for 76 hours were measured. As a result, as shown in FIG. 9, when HVJ-E was used, PEI-B was more toxic to cells than PEI-L. The cytotoxicity increased as the molecular weight of PEI increased. Both PEI-L and PEI-B showed the highest transduction efficiencies near the limit concentration at which cytotoxicity appeared. This shows the same tendency in other cells.
  • Viral vectors that can introduce various substances into cells are very effective in elucidating the etiology and in drug discovery. Therefore, in particular, it is necessary to stably and efficiently introduce the virus vector containing the introduced substance into cells after storing it for a long period of time. Long-term storage requires technologies that maintain efficiency. Thus, according to the present invention, activity retention by long-term storage is achieved as follows.
  • HVJ-E virus vector containing a gene (reporter gene) and the like was freeze-dried in each well of a 96-well plate, and stored under each condition. Thereafter, the cells were dispensed into each well of the stored plate to introduce genes and the like into the cells. Using the reporter gene expressed in the cell as an indicator, the stability test of lyophilized HVJ-E was measured by a reporter assay (Luciferase Atsay).
  • An exemplary protocol is as follows.
  • ADS384 Equipment name: Multistage Dispense Station ADS-3 84-8, Manufacturer: Biotech (Tokyo)
  • Surfacts DNA or siRNA otherwise RNA, antibodies, peptides, functional proteins, sugar chains, lipids, semiconductors or their complexes
  • Vector suspension with the agent dodecyl maltoside other than Triton X-100 etc.
  • a plate type of plate coating: fibronectin, collagen I, collagen IV, tissue culture treatment, gelatin, poly D-lysine, laminin, Mix with poly-L-ortin.
  • Stabilizing agents trehalose, maltose, raffinose, fructose, sucrose, glucose, ratatose, polyvinylpyrrolidone, polyethylene glycol, methinolecellulose, glycine, mannitol, dimethyl sulfoxide (DMSO) or a mixture thereof
  • Stabilizers preferably include trehalose, maltose, raffinose, fructose or a mixture thereof), and the mixture is centrifuged at 3000 rpm. I do.
  • a lyophilized plate containing 14.4 HAU of HVJ-E per well and 0.25 g of plasmid was prepared by the following procedure.
  • HVJ-E was centrifuged at 15000 g for 15 minutes at 4 ° C, and the supernatant was removed. The suspension was suspended in distilled water to obtain an HVJ-E suspension. The subsequent operation was performed by automation using an ADS384 device.
  • the HVJ-E suspension (14.4 HAU) was added to each well of a 96-well microtiter plate (5 ⁇ l / well). Then, 0.25 ⁇ g (DNA (at a concentration of 0.05 mg / ml, 5 ⁇ l ) was added and mixed. Dodecyl maltoside (0.01%, 3Z, 10 times; in this example, 31) was added and mixed. Next, protamine sulfate (121 at a concentration of 0.125 mg Zml; 1.5 gZ well in this example) was added and mixed. Next, various stabilizers were added at the indicated concentrations and mixed. In this example, 51 was actually added.
  • Trehalose and maltose were used as stabilizers. As a result, as shown in FIG. 12, it was proved that even when stored for 6 months, the induction activity was sufficiently maintained when the stabilizer was added. By using 0.5% stabilizer at -20 ° C for 6 months, the activity could be maintained more than 70 times.
  • Fig. 13 shows the results.
  • each stabilizer a concentration of 0.001% to 3% is preferable, and particularly, the following concentrations are preferable.
  • Lyophilization was performed on the plate in which the same specimen was dispensed in all 96 wells, and the cells were introduced into cells. Graphs were prepared for all the specimens in each plate, and the results are shown in FIG. As shown in FIG. 14, even when stored at 20 ° C. for one month or at 80 ° C. for one month, the luciferase activity was almost equal to that of a 96-well sample. Was. As shown in the results, it was demonstrated that the difference between freeze-drying between gels was almost negligible and could be used for drug discovery and clinical development without any problems.
  • siRNA Using siRNA, we examined the retention of gene transfer activity on freeze-dried and preserved plates.
  • the virus vector (14.4 HAU) containing the siRNA (lOpmol) is freeze-dried on a fibronectin plate (Human Fibronectin Cellware 96-well Plate (Betaton Dickinson)), stored for one month, and then stored in a reporter assay. Was done.
  • As the cells 2.5 ⁇ 10 4 BHK cells were used.
  • siRNA siRNA against pGL2
  • pGL2 plasmid encoding firefly luciferase gene, Promega
  • a reporter assay a Dual-Glo Luciferase Assay System (Promega) was used.
  • FIG. 15 shows 16 is a graph showing, on the Y-axis, gene suppression activity immediately after plate preparation and after storage for one month at ⁇ 20 ° C. under the same conditions as in the experiment of FIG.
  • the suppression effect of the siRNA on the target gene itself did not show any deterioration due to freeze-drying.
  • the virus vector (14.4 HAU) containing the luciferase gene was freeze-dried on various plates, and a reporter assay was performed.
  • Three types of plates are used: collagen-treated 96 ⁇ L plate (Betaton Dickinson), fibronectin-treated 96 ⁇ L plate (Bettaton Dickinson), tissue culture treatment 96 ⁇ L plate (Bettaton Dickinson) Was.
  • As the cells 2.5 ⁇ 10 4 BHK cells were used.
  • luciferase activity was obtained in each plate. The activity after storage was higher in the order of collagen-treated 96-well plate, fibronectin-treated 96-well plate, and tissue culture-treated 96-well plate.
  • gene transfer agents that can transfer genes into cells have involved several steps to transfer genes into cells. Furthermore, gene-mixed gene transfer agents have been effective for long-term storage. Therefore, it is desired to develop a gene transfer array device that can be stably, efficiently, and easily introduced into cells after storage for a long period of time. According to the present invention, gene transfer is carried out by dropping a desired gene onto a plate on which a virus vector has been immobilized and seeding the cells to perform cell transfer. To be able to do.
  • the plate of this example is preferably prepared by the following procedure (A). This plate is preferably used as in the following procedure (B). The evaluation is performed, for example, as in a procedure (C).
  • the plate of this example is prepared as follows.
  • ADS384 automatic encapsulation device
  • a stabilizing agent trehalose, manoletose, raffinose, funolectose, sucrose, gnoleose, ratatose, polyvinylpyrrolidone, polyethylene glycol, methinolecellulose, glycine, mannitol, dimethyl sulfoxide (DMSO) or a mixture thereof
  • Stabilizers include, but are not limited to, substances selected from the group consisting of mixtures, preferably trehalose, maltose, raffinose, fructose or mixtures thereof.
  • the plate of the present invention prepared as described above is used as follows.
  • DNA or siRNA on the stored plate with an automatic encapsulation device (ADS384) (just before gene transfer into cells) Is dropped into each well, the gene is encapsulated in a virus vector, and then the cells are seeded to introduce the gene into the cells.
  • ADS384 automatic encapsulation device
  • the plates of the present invention are evaluated as follows.
  • Lyophilized plates containing 14.4 HAU of HVJ-E per well were prepared by the following procedure. [0171] Using an automatic encapsulation device (ADS384), add 14.4 HAU of HVJ-E to a 96-well microtiter plate, centrifuge at 15000 g for 15 minutes at 4 ° C, and discard the supernatant. Removed. The suspension was suspended in distilled water to obtain an HVJ-E suspension. Next, the HVJ-E suspension was mixed with protamine sulfate (0.125 mg / ml at 121) on a fibronectin-coated plate. Next, a stabilizer (trehalose, concentration: 0.5%) was mixed. The plate was centrifuged at 4 ° C. for 30 minutes at 3,000 rotations to fix the plate. The plate was freeze-dried using a vacuum freeze-dryer FTS.
  • ADS384 automatic encapsulation device
  • Plasmid pGL3-CMV (0.05 mg / mL) (5 ⁇ l (0.25), 101 (0.5)) was added dropwise to a freeze-dried plate (including HVJ-E).
  • As a control 5 ⁇ l (0.25) and 10 ⁇ l (0.5) of the plasmid pGL3-CMV (0.05 mg / mL) were added dropwise to a well on a plate containing no HVJ-II.
  • the results are shown in FIG.
  • the horizontal axis shows the amount of plasmid (g), and the vertical axis shows luciferase activity (RLU).
  • the present invention provides a gene transfer technique capable of achieving high speed and high efficiency and capable of handling a large variety of genes.
  • the present invention also provides a technique for introducing not only genes but also desired substances (eg, peptides, compounds, nucleotide fragments, etc.) into cells.
  • the present invention provides a method for introducing a variety of desired substances using a virus envelope or a protein derived from a virus, which can achieve high speed and high efficiency, and is used for such a substance introducing method.
  • the present invention provides solid phases, compositions, and kits for

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Abstract

L'intention est de dEvelopper une technique de transfert de gEne par laquelle on peut obtenir une vitesse ElevEe et une efficacitE ElevEe et applicable A des gEnes d'un grand nombre de types. L'intention est Egalement de dEvelopper une technique servant A transfErer non seulement un gEne mais Egalement une substance souhaitEe (par exemple un peptide, un composE, un fragment de nuclEotide, etc.) dans une cellule. L'intention est en plus de dEvelopper un procEdE en utilisant une enveloppe de virus ou une protEine ayant pour origine un virus, par lequel on peut obtenir une vitesse ElevEe et une efficacitE ElevEe et on peut transfErer des substances souhaitEes d'un grand nombre de types, et une phase solide, une composition et un kit devant Etre utilisEs dans le procEdE de transfert de substances tel que dEcrit ci-dessus. On peut parvenir A atteindre ces objets par un procEdE de construction d'un dispositif servant A transfErer une substance souhaitEe dans une cellule, qui comprend : l'Etape consistant A ajouter un mElange d'un vecteur de virus avec la substance devant Etre transfErEe sur une phase solide ; et l'Etape consistant A ajouter une solution contenant un tensioactif ayant une fraction sucre A la phase solide.
PCT/JP2005/004136 2004-03-19 2005-03-09 Procede de transfert de gene sur une phase solide utilisant un vecteur de virus, composition pour le procede et appareil pour le procede WO2005090585A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001286282A (ja) * 2000-02-02 2001-10-16 Japan Science & Technology Corp 遺伝子導入のためのウイルスエンベロープベクター
WO2003014338A1 (fr) * 2001-08-02 2003-02-20 Anges Mg, Inc. Procede de production d'enveloppes virales inactivees

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001286282A (ja) * 2000-02-02 2001-10-16 Japan Science & Technology Corp 遺伝子導入のためのウイルスエンベロープベクター
WO2003014338A1 (fr) * 2001-08-02 2003-02-20 Anges Mg, Inc. Procede de production d'enveloppes virales inactivees

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KANEDA Y. ET AL: "Hemagglutinating virus of Japan (HVJ) envelope vector as a versatile gene delivery system", MOLECULAR THERAPY, vol. 6, no. 2, August 2002 (2002-08-01), pages 219 - 226, XP002957356 *

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