JPWO2005090585A1 - Solid phase gene transfer method using viral vector, composition for the method, and apparatus for the method - Google Patents

Abstract

It is an object of the present invention to develop a gene transfer technique that can increase the speed and efficiency, and can handle a large number of types of genes. Another object of the present invention is to develop a technology for introducing not only genes but also desired substances (for example, peptides, compounds, nucleotide fragments, etc.) into cells. In addition, the present invention is capable of speeding up and efficiency improvement using a virus envelope or virus-derived protein, and a method for introducing many kinds of desired substances, and a solid phase for use in such a substance introducing method. It is an object to develop a composition, and a kit. An object of the present invention is a method for producing a device for introducing a desired substance into a cell, the step of adding a mixture of a viral vector and the substance to be introduced to a solid phase; Adding a solution comprising a surfactant having a moiety.

Description

  The present invention relates to a solid phase, a method, a composition, and a kit for introducing a desired substance into a cell with high efficiency on the solid phase.

  In order to quickly link human gene information revealed by the results of the genome project to disease treatment and drug discovery, it is necessary to identify causative genes and therapeutic genes for various diseases at high speed and with high efficiency. It is indispensable to identify the function of a gene and a protein produced by the gene in an actual cell or living organ with high efficiency for elucidation of the etiology or drug discovery.

  In order to analyze the function of many genes, gene information is integrated using gene arrays and gene chips, etc., and used to analyze how gene expression patterns change during disease. Yes. However, these devices can change gene expression patterns during disease (for example, determine the amount of messenger), but cannot identify what functions these genes have in actual cells.

  The cell level gene analysis technology can analyze the function at the actual cell level, but the cell level gene analysis technology using the conventional manual method increases the speed and efficiency. It is difficult. Gene analysis techniques at the cell level can be broadly divided into (1) gene introduction techniques and (2) analysis techniques for cells into which genes have been introduced. Among these, in particular, the conventional gene transfer technique has problems in efficiency and speedup, and it is difficult to cope with many kinds of genes.

  Therefore, it is desired to develop a gene introduction technique that can increase the speed and efficiency, and that can deal with many kinds of genes.

  A technique for efficiently and rapidly detecting the functions of peptides and low-molecular compounds in cells can be an important tool in drug discovery. However, in the prior art, there has been no technology for rapidly and efficiently detecting the functions of many kinds of substances in cells. Accordingly, it is desired to develop a rapid and highly efficient technique for introducing not only genes but also peptides and compounds into cells and detecting their functions.

  It is an object of the present invention to develop a gene transfer technique that can increase the speed and efficiency, and can handle a large number of types of genes. Another object of the present invention is to develop a technology for introducing not only genes but also desired substances (for example, peptides, compounds, nucleotide fragments, etc.) into cells.

  In addition, the present invention is capable of speeding up and efficiency improvement using a virus envelope or virus-derived protein, and a method for introducing many kinds of desired substances, and a solid phase for use in such a substance introducing method. It is an object to develop a composition, and a kit.

  The present invention has been accomplished by finding a method for gene transfer at high efficiency and at high speed as a result of intensive studies on a solid phase gene transfer method using a virus envelope or virus-derived protein.

Accordingly, the present invention provides the following.
1. A device for introducing a desired substance into a cell, comprising:
(1) solid phase;
(2) viral vectors;
(3) a surfactant having a sugar moiety,
Wherein the viral vector and a surfactant having the sugar moiety are disposed on the solid phase.
2. 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.
3. Item, wherein the substance to be introduced is at least one substance selected from the group consisting of a nucleic acid encoding a gene to be introduced, a peptide, a sugar chain, a low molecular weight compound, a proteoglycan, a glycopeptide, a lipid, and a metal ion. 2. The device according to 2.
4). Item 2. The device according to Item 1, wherein the introduced substance comprises a nucleic acid selected from the group consisting of siRNA, antisense and decoy of the introduced gene.
5. Item 2. The device according to Item 1, wherein the substance to be introduced is a nucleic acid encoding a gene to be introduced.
6). Item wherein the viral vector is (i) a viral envelope, (ii) a liposome comprising at least one of viral envelope proteins, or (iii) a mixture of a viral envelope and a liposome comprising at least one of viral envelope proteins. The device according to 1.
7). Item 7. The device according to Item 6, wherein the virus envelope is a virus envelope derived from a wild type virus or a recombinant virus.
8). The viral envelope is retroviridae, togaviridae, coronaviridae, flaviviridae, paramyxoviridae, orthomyxoviridae, bunyaviridae, rhabdoviridae, poxviridae, herpesviridae, baculoviridae, 7. The device according to item 6, wherein the device is derived from a virus belonging to a family selected from the group consisting of the family Hepadnaviridae.
9. Item 8. The device according to Item 7, wherein the virus is derived from a virus belonging to the Paramyxoviridae family.
10. Item 9. The device according to Item 8, wherein the virus is HVJ.
11. Item 11. The device according to Item 10, wherein the virus envelope protein is a protein selected from the group consisting of F protein, HN protein, NP protein, and combinations thereof.
12 Surfactants having the sugar moiety of the formula _{A- (CH 2) n-CH} 3
The device according to item 1, wherein A is a monosaccharide, disaccharide, trisaccharide, tetrasaccharide or pentasaccharide, and n = 7-11.
13. Item 13. The device according to Item 12, wherein A is a monosaccharide or a disaccharide.
14 14. A device according to item 13, wherein A is a disaccharide.
15. 14. The device according to item 13, wherein A is a glucoside group or a maltoside group.
16. Item 16. The device according to Item 15, wherein A is a maltoside group.
17. 13. The device according to item 12, wherein n = 9-11.
18. Item 18. The device according to Item 17, wherein n = 9.
19. Item wherein the surfactant having a sugar moiety is a surfactant selected from the group consisting of octyl maltoside, decyl maltoside, dodecyl maltoside, octyl glycoside, decyl glucoside, and dodecyl glucoside, and mixtures thereof. The device according to 1.
20. Item 20. The device according to Item 19, wherein the surfactant having a sugar moiety is decyl maltoside.
21. Item 2. The device according to Item 1, wherein the concentration of the surfactant having a sugar moiety is 0.0001% by volume or more and less than CMC.
22. Item 22. The device according to Item 21, wherein the concentration of the surfactant is 0.0009% to 0.28% by volume.
23. The device according to item 1, further comprising (5) a protamine sulfate solution, wherein the protamine sulfate solution is disposed on the solid phase.
24. The device according to item 1, further comprising (5) polyethyleneimine, wherein the polyethyleneimine is disposed on the solid phase.
25. 25. A device according to item 24, wherein the polyethyleneimine is a linear polyethyleneimine.
26. The device of item 1, further comprising (6) a stabilizer, wherein the stabilizer is disposed on the solid phase.
27. Item 26. The device of item 26, wherein the stabilizer is trehalose, maltose, raffinose, fructose, sucrose, glucose, lactose, polyvinylpyrrolidone, polyethylene glycol, methylcellulose, glycine, mannitol, dimethyl sulfoxide (DMSO). ) Or a mixture thereof.
28. 28. The device of item 27, wherein the stabilizer is selected from the group consisting of trehalose, maltose, raffinose, fructose or mixtures thereof.
29. Item 29. The device according to Item 28, wherein the stabilizer is trehalose.
30. Item 2. The device of item 1, wherein the solid phase is coated with a protein selected from the group consisting of fibronectin, collagen, laminin, gelatin, polyornithine and polylysine, and mixtures thereof.
31. 31. A device according to item 30, wherein the solid phase is coated with fibronectin.
32. Item 2. The device according to Item 1, wherein the cell is a mammalian cell.
33. Item 2. The device of item 1, wherein the device is lyophilized.
34. 25. A device according to item 24, which is freeze-dried.
35. 27. A device according to item 26, which is freeze-dried.
36. An apparatus for introducing a gene into a cell comprising:
(1) means for supporting a substance introduction device having at least 1500 wells;
(2) means for incubating the substance introduction device;
(3) means for adding a desired solution to a desired well of the substance introduction device; and (4) a centrifuge;
An apparatus comprising:
37. further,
(5) means for measuring a signal selected from the group consisting of fluorescence, absorbance, luminescence, radiation and turbidity in a desired well;
40. The apparatus of item 36 comprising.
38. A kit for introducing a gene into a cell comprising:
(1) solid phase;
(2) a viral vector; and (3) a solution comprising a surfactant having a sugar moiety,
A kit comprising:
39. The kit according to Item 38, further comprising (4) protamine sulfate.
40. The kit according to Item 38, further comprising (4) polyethyleneimine.
41. 40. The kit according to Item 38, wherein the polyethyleneimine is a linear polyethyleneimine.
42. (5) The kit according to item 38, further comprising a stabilizer.
43. Item 44. The kit according to Item 42, wherein the stabilizer is trehalose, maltose, raffinose, fructose, sucrose, glucose, lactose, polyvinylpyrrolidone K90, polyethylene glycol, methylcellulose, glycine, mannitol, dimethylsulfoxide ( DMSO) or a mixture thereof.
44. 44. A kit according to item 43, wherein the stabilizer is selected from the group consisting of trehalose, maltose, raffinose, fructose or a mixture thereof.
45. 45. A kit according to item 44, wherein the stabilizer is trehalose.
46. Furthermore, (6) The kit of item 38 or item 42 including the process of freeze-drying.

  Accordingly, it is understood that these and other advantages of the present invention will be apparent to those of ordinary skill in the art upon reading and understanding the following detailed description with reference to the accompanying drawings.

  According to the present invention, high speed and high efficiency are possible, and a technique for introducing many kinds of desired substances is provided. 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 weight compounds, proteoglycans, glycopeptides, lipids, metal ions, and the like. Furthermore, with this technology, for example, the functions of human genes and various biopolymers estimated to be 30,000 to 40,000 types in actual cells can be analyzed at high speed and with high efficiency. A highly integrated device is provided. For example, an HVJ vector in which a biopolymer such as a gene or protein is encapsulated is immobilized on a device that is processed with a highly integrated microwell on a slide. Analyzing makes it possible to simultaneously evaluate the functions of a large number of human genes in living cells.

[FIG. 1] FIG. 1 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. 2 is a graph showing the selection of surfactants 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 is introduced according to the method of the present invention. The X-axis indicates the horizontal well number (1-12), and the Y-axis indicates the horizontal well number (A-H).
FIG. 4A is a diagram showing high reproducibility when a pGL3 expression plasmid is introduced according to the method of the present invention. The X-axis indicates the horizontal well number (1-12), and the Y-axis indicates the horizontal well number (A-H).
FIG. 4B is a view showing high reproducibility when a pGL3 expression plasmid is introduced according to the method of the present invention. The X-axis indicates the horizontal well number (1-12), and the Y-axis indicates the horizontal well number (A-H).
[FIG. 5] FIG. 5 is a graph showing the difference in gene transfer efficiency when plates coated with various substances are used.
[FIG. 6] FIG. 6 shows the results of gene transfer efficiency when protamine sulfate and polyethylimine are used. “No enhancer” indicates the result of an experiment using neither protamine sulfate nor polyethylimine.
FIG. 7 shows the results of gene transfer efficiency when protamine sulfate and polyethylimine are used.
FIG. 8A is a result showing gene transfer efficiency when various polyethylimines are used. FIG. 8A shows gene transfer efficiency when PEI-L 25K is used. The X axis is the PEI-L 25K concentration at the time of introduction.
FIG. 8B is a result showing gene transfer efficiency when various polyethylimines are used. FIG. 8B shows gene transfer efficiency when PEI-L 2.5K is used. The X axis is the PEI-L 2.5K concentration at the time of introduction.
[FIG. 8C] FIG. 8 shows the results of gene transfer efficiency when various polyethylimines are used. FIG. 8C shows gene transfer efficiency when PEI-B 25K was used. The X axis is the PEB-L 25K concentration at the time of introduction.
FIG. 8D is a result showing gene transfer efficiency when various polyethylimines are used. FIG. 8D shows the gene transfer efficiency when PEI-B 750K is used. The X axis is the PEB-L 750K concentration at the time of introduction.
FIG. 9A shows the results showing the cytotoxicity of various polyethylimines.
FIG. 9B shows the results showing the cytotoxicity of various polyethylimines.
[FIG. 10] FIG. 10 is a result showing stability by freeze-drying.
[FIG. 11] FIG. 11 shows the results showing that gene transfer efficiency is maintained when stored for a long time after lyophilization.
[FIG. 12] FIG. 12 shows the results showing that gene transfer efficiency is maintained when stored for a long time after lyophilization using different stabilizers.
[FIG. 13] FIG. 13 is a result showing that gene transfer efficiency is maintained when stored for a long time after lyophilization using different stabilizers.
FIG. 14 is a result showing that the storage stability in each well is uniform.
[FIG. 15] FIG. 15 shows the results showing that, even when siRNA is introduced, gene introduction efficiency is maintained after long-term storage as in the case of DNA.
[FIG. 16] FIG. 16 shows the results showing that, even when siRNA is introduced, gene introduction efficiency is maintained after long-term storage as in the case of DNA.
FIG. 17 is a result showing that the freeze-drying method is not related to the coating of the plate.
[FIG. 18] FIG. 18 shows the results of a gene introduction experiment using a plate freeze-dried with a virus vector not containing a nucleic acid to be introduced.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Therefore, it should be understood that the expression of the singular includes the concept of the plural unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition of terms)
A “cell” as used herein is defined in the same way as the broadest meaning used in the art, and is a structural unit of a tissue of a multicellular organism, surrounded by a membrane structure that isolates the outside world, Refers to a living organism having self-regenerative ability and having genetic information and its expression mechanism.

  As used herein, “stabilizing agent” refers to a drug that improves the storage stability of a cell culture container of the present invention containing a gene transfer vector when stored by lyophilization. The stabilizer of the present invention is selected from the group consisting of trehalose, maltose, raffinose, fructose, sucrose, glucose, lactose, polyvinyl pyrrolidone, polyethylene glycol, methyl cellulose, glycine, mannitol, dimethyl sulfoxide (DMSO) or a mixture thereof. But not limited to these materials. Stabilizers preferably include trehalose, maltose, raffinose, fructose or mixtures thereof. More preferably, the stabilizer of the present invention is trehalose.

  Conditions for freeze-drying the cell culture container (also referred to as array or plate) of the present invention are well known to those skilled in the art.

  As used herein, polyethyleneimine (abbreviated as PEI) includes both linear and branched types. The structure of linear PEI is shown as-[NH-CH2-CH2] x-. Here, x is an arbitrary integer. Examples of the linear PEI include, for example, PEI-L (PEI-L 2.5K) having a molecular weight of 2.5 kDa and PEI-L (PEI-L 25K) having a molecular weight of 2.5 kDa. . Branched PEI is shown as-[NH-CH2-CH2] y- [N (CH2-CH2-NH2) -CH2-CH2] z-. Here, y is an arbitrary integer. Here, z is an arbitrary integer independent of y. For example, preferred branched PEIs are PEI-B with a molecular weight of 25 kDa (PEI-B 25K) and PEI-B with a 750 kDa (PEI-B 750K).

  As used herein, “virus envelope vector” refers to a vector in which a foreign gene is encapsulated in a virus envelope, or a vector in which a foreign gene is encapsulated in a component containing a protein derived from the virus envelope. The virus used for the preparation of the viral vector may be a wild type virus or a recombinant virus.

  In the present invention, the viruses used for the preparation of the virus envelope or virus envelope-derived protein include retroviridae, togaviridae, coronaviridae, flaviviridae, paramyxoviridae, orthomyxoviridae, bunyavirus Viruses belonging to the family selected from the group consisting of Family, Rhabdoviridae, Poxviridae, Herpesviridae, Baculoviridae, and Hepadnaviridae are included, but are not limited thereto. Preferably, a virus belonging to the Paramyxoviridae family is used, and more preferably, HVJ (Sendai virus) is used.

  Examples of the virus envelope-derived protein include, but are not limited to, HVJ F protein, HN protein, NP protein, and M protein.

  In the present specification, “HVJ” and “Sendai virus” may be used interchangeably. For example, in the present specification, “HVJ envelope” and “Sendai virus envelope” are used as terms having the same meaning.

  As used herein, “Sendai virus” refers to a virus belonging to the genus Paramyxovirus belonging to the Paramyxoviridae family and having a cell fusion action. Virus particles have an envelope and a polymorphism with a diameter of 150-300 nm. The genome is a minus-strand RNA having a length of about 15500 bases. It has RNA polymerase, is unstable to heat, aggregates almost all kinds of red blood cells, and is hemolytic.

  As used herein, “HAU” refers to the activity of a virus capable of aggregating 0.5% of chicken erythrocytes, and 1 HAU corresponds to approximately 24 million virus particles (Okada, Y. et al., Biken Journal 4, 209-213, 1961).

  Animal cells used as host cells include mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, CHO cells that are Chinese hamster cells, BHK cells, African green monkey kidney cells, human leukemia cells, HBT5637 ( JP-A-63-299), human colon cancer cell lines, and the like. Mouse myeloma cells such as ps20, NSO, rat myeloma cells such as YB2 / 0, human fetal kidney cells such as HEK293 (ATCC: CRL-1573), human leukemia cells such as BALL-1, Africa Examples of green monkey kidney cells include COS-1 and COS-7, and examples of human colon cancer cell lines include HCT-15.

  As used herein, the terms “solid phase”, “substrate” and “support” are used interchangeably herein and refer to the material (preferably solid) from which the array of the invention is constructed. The material of the substrate can be any solid, either covalently or noncovalently, that has the property of binding to the biomolecules used in the present invention or that can be derivatized to have such properties. Materials.

  As such a material for use as a substrate, any material capable of forming a solid surface can be used, for example, glass, silica, silicon, ceramic, silicon dioxide, plastic, metal (including alloys) Natural and synthetic polymers (eg, polystyrene, cellulose, chitosan, dextran, and nylon), including but not limited to: The substrate may be formed from a plurality of layers of different materials. For example, inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used. In addition, polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin Organic materials such as polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, and polysulfone can be used. In the present invention, a membrane used for blotting such as a nylon membrane, a nitrocellulose membrane, and a PVDF membrane can also be used. A nylon membrane is preferred. This is because when a nylon membrane is used, the result 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.

  In this specification, “chip” or “microchip” refers to a micro integrated circuit that has various functions and is a part of a system. In this specification, the “DNA chip” includes a substrate and DNA, and at least one DNA (for example, cDNA fragment) is arranged on the substrate. In this specification, the “protein chip” includes a substrate and a protein, and at least one protein (for example, a polypeptide or an oligopeptide) is arranged on the substrate. “DNA chip” and “protein chip” are included herein as “microchip” or simply “chip”. “Microarray” refers to a chip in which one or more biomolecules (for example, oligonucleotides or peptides such as cDNA fragments) are arranged and arranged.

  Biomolecules used in the present invention (for example, oligonucleotides such as DNA or peptides) can be collected from living organisms, and can be chemically synthesized by methods known to those skilled in the art. For example, oligonucleotides can be prepared by automated chemical synthesis using either a DNA synthesizer or a peptide synthesizer marketed by Applied Biosystems or the like. 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. No. 4,500,707 and Caruthers (1985); US Pat. No. 4,668,777, Caruthers et al. (1987).

Any number of biomolecules (eg, DNA or peptides) can be placed on the substrate, but typically up to 10 ⁸ biomolecules per substrate, and in other embodiments up to 10 ⁷ biomolecules. Up to 10 ⁶ biomolecules, up to 10 ⁵ biomolecules, up to 10 ⁴ biomolecules, up to 10 ³ biomolecules, or up to 10 ² biomolecules can be placed. . In these cases, the size of the substrate is preferably smaller. In particular, the spot size of a biomolecule (eg DNA or peptide) can be as small as the size of a single biomolecule (this can be on the order of 1-2 nm). The minimum substrate area is determined in some cases by the number of biomolecules on the substrate.

When used in the present invention, the “surfactant having a sugar moiety” has a surfactant having a “sugar moiety” in the molecule. Examples of the surfactant include an anionic surfactant, a cationic surfactant, a nonionic surfactant, and an amphoteric surfactant. Examples of the saccharide moiety of the surfactant having a saccharide moiety include, but are not limited to, monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, and octasaccharide. The sugar moiety may or may not be branched. The sugar moiety is preferably a monosaccharide to pentasaccharide, more preferably a monosaccharide or a disaccharide. Examples of the sugar moiety include, but are not limited to, glucoside, galactoside, maltoside, thiomaltoside, and thioglucoside. The surfactant having a sugar moiety is, for example, a compound represented by A- (CH) _n —CH ₃ , where A is a sugar moiety and — (CH) _n —CH ₃ is other than a sugar moiety. It is a hydrophobic part. In the hydrophobic part, 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, It can be 10 or less, 9 or less, 8 or less, or 7 or less. A preferable range of n is 7-11. For example, the surfactant having this sugar moiety is selected from the group consisting of octyl maltoside, decyl maltoside, dodecyl maltoside, octyl glycoside, decyl glucoside, and dodecyl glucoside, and mixtures thereof. A preferred surfactant having a sugar moiety is decyl maltoside.

(Description of Preferred Embodiment)
(Preparation of substance introduction device)
In the present invention, the preparation of a substance introduction device on which a viral vector is immobilized can be carried out by the following steps:
(1) adding a mixture of a viral vector and a substance to be introduced to the solid phase; and (2) adding a solution containing a surfactant having a sugar moiety to the solid phase.
In the above method, the substance to be introduced includes 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, small molecules Also included are compounds, proteoglycans, glycopeptides, lipids, metal ions. When the substance to be introduced is a nucleic acid encoding a gene, the introduced gene can be derived from any living body and can be DNA, RNA, or a nucleotide analog. One or more types of genes may be introduced.

  In the above method, the cell into which the gene is introduced is not particularly limited, but is preferably an animal cell, more preferably a mammalian cell including a human cell. When using an introduction method using a virus envelope or an introduction method using a liposome containing at least one protein of the virus envelope, the viruses used to prepare the virus envelope are retroviridae, togaviridae, coronavirus Belonging to a family selected from the group consisting of: Family, Flaviviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Rhabdoviridae, Poxviridae, Herpesviridae, Baculoviridae, and Hepadnaviridae It can be derived from a virus. Preferably, the virus is a Paramyxoviridae virus, especially HVJ.

  Methods for introducing candidate nucleic acids into cells using liposomes containing at least one protein of the viral envelope are also available. Proteins used in this method include, but are not limited to, F protein, HN protein, NP protein, M protein or combinations thereof.

  The mixture of the viral vector and the substance to be introduced can be prepared, for example, by centrifuging a solution containing the viral vector to cause precipitation, and suspending the precipitate with the introduced substance solution. Examples of the centrifugation conditions include, but are not limited to, 15000 × g, 4 ° C., and 15 minutes. For example, in the case of mass preparation, centrifugation conditions of 15000 × g and 4 ° C. for 60 minutes can also be used.

  Alternatively, a mixture of the virus vector solution and the substance solution to be introduced may be prepared in a separate container in advance, and the mixture may be added to the solid phase. Further, instead of adding the mixture of the virus vector and the substance to be introduced to the solid phase, the virus vector solution and the substance solution to be introduced may be separately added to the solid phase.

  If necessary, a substance introduction device that is a solid phase containing a viral vector, a substance to be introduced, and a surfactant having a sugar moiety is allowed to stand at room temperature. Incubation time may preferably be 5 minutes, 7 minutes, 10 minutes, 12 minutes, 15 minutes, 17 minutes, 20 minutes, 25 minutes, 30 minutes.

  If necessary, protamine sulfate (PS) or polyethyleneimine (PEI) is added to increase gene transfer efficiency. The concentration of protamine sulfate used is preferably 1 μg / ml to 1 mg / ml, more preferably 10 μg / ml to 250 μg / ml, most preferably 100 μg / ml. The concentration of polyethyleneimine used is preferably 0.1 μg / ml to 300 μg / ml, more preferably 0.5 μg / ml to 200 μg / ml, most preferably 1 μg / ml to 100 μg / ml.

  Further, if necessary, after adding protamine sulfate and / or polyethyleneimine, the substance introduction device is centrifuged. The centrifugation conditions are, for example, 1643 × g, 4 ° C., and 30 minutes, but are not limited thereto.

(Gene transfer into cells using the substance transfer device of the present invention)
Using the substance introduction device prepared as described above, a foreign gene can be introduced into a cell by the following method.

  A cell suspension is introduced into a substance introduction device to which a viral vector is immobilized. If necessary, centrifuge the device. The conditions of the centrifuge are typically 182 × g, 32 ° C., and 30 minutes, but are not limited thereto. Thereafter, if necessary, the device is shaken and centrifuged. The conditions of the centrifuge are typically 182 × g, 32 ° C., and 30 minutes, but are not limited thereto. If necessary, centrifugation at 182 × g, 32 ° C. for 30 minutes can be further performed twice.

(Application of gene transfer method to cells using the substance transfer device of the present invention)
Using the above-described gene introduction method, it is possible to perform functional analysis and screening of many types of genes.

(A. Analysis of gene function)
Many genes have been isolated and identified with the progress of the genome project. However, the function of many genes is still unknown. Therefore, it is very useful to develop a simple method for identifying the function of a gene whose function is unknown. For example, by using the gene transfer technique of the present invention, a large number of cell populations in which a large number of genes are introduced into the substance transfer device of the present invention are prepared, and the function of the cell population is analyzed to obtain a desired functional group. A cell into which a nucleic acid having a property has been introduced is selected, and as a result, a gene having a desired function is identified. The desired functions include, for example, encoding a gene that induces angiogenesis, encoding a tumor suppressor gene, encoding a bone formation enhancing gene, encoding an apoptosis-inducing gene, and encoding a cytokine secretion gene. Encoding a neuronal dendritic induction gene, encoding an arteriosclerosis suppressor gene, encoding a diabetes suppressor gene, encoding an autoimmune disease suppressor gene, encoding an Alzheimer's disease suppressor gene, It is selected from the group consisting of encoding a Parkinson's disease suppressor gene, encoding a neuron protection gene, and combinations thereof.

  Also, this selection is preferably performed based on the host cell trait that is altered 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 promotion of 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. Functional properties intended in the above analysis method include cell growth promotion or suppression, cell differentiation or dedifferentiation, marker protein expression or expression suppression, marker mRNA expression or expression suppression, membrane potential change, Examples include, but are not limited to, depolarization, apoptosis, canceration, growth arrest, morphological change, and size change.

  In the above analysis method, various well-known methods and commercially available kits can be used as a method for extracting nucleic acid from a gene-introduced cell.

(B. Screening)
The gene transfer method of the present invention can be applied to screening. For example, when preparing 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, (3) introduced. Application to screening is possible by using a gene library as a substance. By preparing a cell population containing the gene library on the device using the gene library, selecting a cell having a desired function from the cells, and isolating and identifying the nucleic acid in the cell, A gene having a desired function can be screened. In addition, when a cell population that is found to be positive by screening contains a plurality of types of genes, it is possible to isolate and identify genes having a desired function by purifying the substances.

  This screening method can target not only plasmid nucleic acids but also proteins, peptides, low molecular compounds, nucleotide fragments, and the like.

  In addition, since the substance introduction device can reduce the immobilization region of the virus vector, biomolecule screening can be performed not only in a 96-well plate but also in a 384-well plate and a 1536-well plate. is there.

Furthermore, the substance introduction device of the present invention is suitable for automation. Kits for performing the methods of the present invention are also provided in the present invention.
(1. Preparation of virus envelope vector)
Various methods are known as virus envelope vectors. For example, the present inventors have developed a hybrid gene transfer vector by combining a virus and a non-viral vector, and a fusion-forming virus liposome having a fusion-forming envelope derived from Japanese hemagglutinating virus (HVJ; Sendai virus). (Kaneda, Biogenic Amines, 14: 553-572 (1998); Kaneda et al., Mol. Med. Today, 5: 298-303 (1999)). In this delivery system, DNA-loaded liposomes are fused with UV-inactivated HVJ to form HVJ liposomes (400-500 nm in diameter) that are fusion-forming virus-liposomes. The advantage of fusion-mediated delivery is that transfection of DNA is protected from endosomal and lysosomal degradation in receptor cells. Up to 100 kb of DNA is incorporated into HVJ liposomes and delivered to mammalian cells. RNA, oligonucleotides and drugs are also efficiently introduced into cells in vitro and in vivo. HVJ-liposomes have not been shown to induce significant cell damage in vivo.

Repeated transfections have been successful in vivo due to the low immunogenicity of HVJ (Hirano et al., Gene Ther., 5: 459-464 (1998)). This vector system has been improved and anion-type and cation-type HVJ-liposomes have been developed for more efficient gene delivery (Saeki et al., Hum. Gene Ther., 8: 1965-1972 (1997)). Many gene therapy strategies have been successful with this HVJ-liposome system (Dzau et al., Proc. Natl. Acad. Sci. USA, 93: 11421-11425 (1996); Kaneda et al., Mol. Med. Today, 5: 298-303 (1999)). In addition, several attempts have been made to construct HVJ-derived synthetic virosomes (Wu et al., Neuroscience Lett., 190: 73-76 (1995); Ramani et al., FEBS Lett., 404: 164). -168 (1997); Ramani et al., Proc. Natl. Acad. Sci. USA, 95: 11886-11890 (1998)).
If it is necessary to inactivate the virus for the preparation of the vector, various known methods can be used. Typical inactivation methods include UV irradiation, treatment with an alkylating agent, treatment with β-propiolactone, treatment with a surfactant, and partial degradation of the envelope by enzyme treatment. It is not limited to.

Preparation methods that improve the virus envelope vectors listed above are also known. A typical example is shown below. The method for preparing a viral envelope vector described below is an exemplification, and the present invention is not limited to the vector prepared by the following preparation method.
(1.1. Preparation of a viral vector in which a foreign gene is encapsulated in a component containing a protein derived from the viral envelope)
A viral vector containing a protein derived from the viral envelope is a viral vector comprising liposomes obtained by reconstituting an HVJ (Sendai virus) F fusion protein and an HN fusion protein, and an amount of the HVJ genome detectable by RT-PCR. Examples include viral vectors that do not contain RNA.

  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. The recombinantly produced fusion protein is processed in vitro by a protease or in a mammalian cell host by an endogenous protease.

A viral vector containing a protein derived from the viral envelope is prepared, for example, by a method comprising the following steps:
Isolating the fusion protein from the HVJ virus that has not been irradiated with UV,
Reconstituting the fusion protein in the presence of surfactant and lipid to prepare reconstituted particles;
A step of preparing liposomes filled with a desired nucleic acid, and a step of preparing reconstituted particles and the liposomes.

  The surfactant used in the above method is not limited to a specific surfactant, but is preferably octyl glycoside, Triton-X100, CHAPS or NP-40, or a mixture thereof.

  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 the molecule, and (2) exists in the living body or is derived from the living body. Any molecule that does Preferred lipids include, but are not limited to, phosphatidylcholine, phosphatidylserine, cholesterol, sphingomyelin, and phosphatidic acid.

The method for preparing liposomes is well known, and for example, the following method can be used: (A) A phospholipid thin film is previously prepared in a first test tube. Nitrogen gas saturated with water at 55 ° C. is introduced into the test tube and fully hydrated. When hydration is complete, the film becomes transparent;
(B) Gently add the buffer solution of the second test tube to the test tube A, introduce nitrogen gas, seal (for example, by parafilm), and maintain a 37 ° C. thermostatic chamber (to perform the experiment at a constant temperature) Left for about 2 hours in
(C) Prepare giant liposomes by gently shaking. When liposomes are formed, the solution becomes thin and turbid. The following measurement is performed using this.
(D) A sample is taken on a glass slide and the form is confirmed with a fluorescence microscope (1000 times).

  A method for preparing a protein required for preparing a viral vector in which a foreign gene is encapsulated in a component containing a protein derived from a viral envelope is well known.

  For example, HVJ envelope protein can be purified using natural HVJ as a source, or recombinantly expressed HVJ envelope protein can be purified. Well-known protein purification methods include, but are not limited to, ammonium sulfate precipitation, isoelectric focusing, and column purification. When purifying a protein by a column, various columns are selected according to the properties of the desired protein and contaminants. Examples of the column for protein purification include, but are not limited to, an anion exchange column, a cation exchange column, a gel filtration column, and an affinity column.

Alternatively, a viral vector containing a protein from the viral envelope is prepared by a method that includes the following steps:
A step of recombinantly expressing HVJ F protein and HN protein;
A process of processing F protein with protease,
Isolating F protein and HN protein;
Reconstituting F protein and HN protein in the presence of surfactant and lipid to prepare reconstituted particles;
A step of preparing a liposome filled with nucleic acid, and a step of forming a reconstituted particle and the liposome.

A viral vector containing a protein derived from the viral envelope is also prepared by a method comprising the following steps:
-Recombinantly expressing F and HN proteins in a host cell that expresses a protease that processes the F protein;
Isolating F protein and HN protein;
Reconstituting F protein and HN protein in the presence of surfactant and lipid to prepare reconstituted particles;
A step of preparing a liposome filled with a desired nucleic acid, and a step of fusing the reconstituted particle and the liposome.
(1.2. Preparation of virus vector in which foreign gene is encapsulated in virus envelope)
Examples of a method for preparing a viral vector in which a foreign gene is encapsulated in a virus envelope include a method including the following steps:
1) mixing the virus with a foreign gene, and 2) freezing and thawing the mixture, or further mixing the mixture with a surfactant.

Alternatively, a viral envelope-derived viral vector is prepared by a method that includes the following steps:
A process of inactivating the virus,
A step of mixing an inactivated virus with a foreign gene, and a step of freezing and thawing the mixed solution,
Are provided.

In a further aspect of the invention, a method of preparing an inactivated viral envelope vector for gene transfer comprising:
A step of inactivating the virus, and a step of mixing the inactivated virus with a foreign gene in the presence of a surfactant,
Are provided.
(1.3. Liposome vector)
As the liposome vector, a liposome prepared from a lipid generally used for lipofection can be used. For example, lipids such as lipofect AMINE 2000 can be used.
(Cell selection)
In the present invention, various cells can be used as cells into which substances are introduced. Such cells are preferably mammals, more preferably cells from the species from which the candidate nucleic acid is derived.

  The present invention has been described above by exemplifying preferred embodiments. Hereinafter, the present invention will be described based on examples with reference to the drawings. However, it should be noted that the following examples are provided only for illustrative purposes. Accordingly, the scope of the present invention is not limited to the specific embodiments used in the description or the following examples, and is limited only by the scope of the claims.

(Example 1: Preparation and use of a viral vector in which a foreign gene is encapsulated in a component containing a virus envelope-derived protein)
(Preparation of virus)
The HVJ, Z strain was prepared as previously described (Kaneda, Cell Biology: A Laboratory Handbook, JE Celis (Ed.), Academic Press, Orlando, Florida, vol. 3, pp. 50-57 (1994). )) Purified by differential centrifugation. Purified HVJ was resuspended in balanced salt solution (BSS: 137 mM NaCl, 5.4 mM KCl, 10 mM Tris-HCl, pH 7.5) and virus titer was determined by measuring absorbance at 540 nm. The optical density at 540 nm corresponds to 15,000 hemagglutinating units (HAU) and correlates with fusion activity.

(Extraction of F and HN fusion proteins from HVJ)
Nonidet P-40 (NP-40) and phenylmethylsulfonyl fluoride (PMSF) dissolved in ethanol were added to 20 ml (1,750,000) of purified HVJ suspension at final concentrations of 0.5% and 2 mM, respectively. HAU). This mixture was incubated for 30 minutes at 4 ° C. with rotation. This suspension was then centrifuged at 100,000 g for 75 minutes at 4 ° C. to remove insoluble protein and viral genome (Uchida et al., 1979). The supernatant was dialyzed against 5 mM phosphate buffer (pH 6.0) for 3 days, and the remaining NP-40 and PMSF were washed away by changing the buffer every day. The dialyzed solution was centrifuged at 100,000 g for 75 minutes at 4 ° C. to remove insoluble material. The supernatant solution was added to a 10 mM phosphate buffer (pH 5.2) containing 0.3 M sucrose and 1 mM KCl based on the method described previously (Yoshima et al., J. Biol. Chem., 256: 5355-5361 (1981)). ) Was applied to an ion exchange column of CM-Sepharose CL6B (Pharmacia Fine Chemicals, Uppsala, Sweden). The flow through and 0.2M NaCl eluate were collected. Both fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to analyze protein components. Gels were stained with Coomassie brilliant blue and the ratio of each protein was assessed using computerized densitometry (NIH Image; Apple Computers, Cupertino, CA, USA).

(Recombinant expression)
An HVJ fusion protein can also be prepared by incorporating a gene encoding the fusion protein into an expression vector and expressing it in an appropriate host cell. The amino acid sequences of F protein and HN protein are known.

  As an expression vector that can be used in various host cells, various commercially available vectors can be used.

Expression vectors encoding fusion proteins can be introduced into cells and any of the various methods known and described in the art (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed, Vol 1 to 3 , Cold Spring Harbor Laboratory
Press, New York (1989), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1994), each of which is incorporated herein by reference). Of fusion proteins can be produced. Examples of methods for introducing a recombinant expression vector into prokaryotic or eukaryotic cells include transformation or transfection methods such as electroporation.

  When recombinant F protein was expressed in E. coli, it was expressed as an inactive F0 form. In order to convert the inactive F0 form protein expressed in E. coli to the active F1 form, trypsinization at 37 ° C. for 30 minutes with 0.0004-0.001% trypsin was required.

  A polypeptide corresponding to the trypsinized activated F1 protein can be expressed in E. coli using an expression vector comprising a gene encoding the truncated activated F1 amino acid sequence. The truncated F1 protein needs to contain at least 26 amino acid residues from the 117th phenylalanine to the 142nd alanine. When the truncated protein forms an inclusion body, it is easily possible for those skilled in the art to obtain the active protein by refolding the inclusion body (Robert F. Kelly and Marjorie E. Winkler, Genetic Engineering, (1990) vol. 12, see pages 1-19).

  Expression of F protein using host cells that can be replicated by HVJ (eg, rodent tracheal epithelial cells; chicken embryos; monkey kidney primary culture cells; human fetal lung, kidney, amnion primary culture cells) In that case, the expressed full-length F protein is cleaved by the host cell's endogenous protease and as a result is activated, so that the active F protein can be expressed and isolated. Alternatively, a host cell that expresses Triptase clara (Kido et al., Molecular Cells 9, 235-244 (1999)) as an endogenous enzyme (eg, rat tracheal epithelial cell) or a recombinantly expressed host cell Can also be used.

  Methods for selecting and constructing these expression vectors, methods for introduction into host cells, methods for expression in host cells, and methods for recovering expressed proteins are well known to those skilled in the art.

(Purification of fusion protein from HVJ)
For purification of the fusion protein, NP-40 treated HVJ lysate was clarified by ultracentrifugation. Supernatant proteins were analyzed by SDS-PAGE before further purification. This supernatant contained many proteins from HVJ. The supernatant was then applied to ion exchange chromatography. The 52 kDa and 72 kDa proteins were mainly eluted in the flow-through fraction. By mobility on SDS-PAGE, these two proteins were identified as F1 and HN, respectively (Okada, Methods in Enzymology, N. Duznes (Ed.), Academic Press, San 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 obtained efficiently. And a further 60 kDa protein that appeared to be an NP protein of HVJ appeared. As a result, only the flow-through fraction was used as a source of fusion protein for further experiments. 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 viral envelope. A previous paper (Nakanishi et al., Exp. Cell Res., 142: 95-101 (1982)) reports that this ratio is necessary for efficient fusion of HVJ.

(Preparation of virus vector)
A lipid mixture of 3.56 mg phosphatidylcholine and 0.44 mg cholesterol was dissolved in chloroform and the lipid solution was evaporated in a rotary evaporator (Uchida et al., J. Cell. Biol. 80: 10-20 (1979). )). The dried lipid mixture was completely dissolved by vortexing in 2.0 ml of the protein solution (1.6 mg) from the flow-through fraction containing 0.85% NP-40. The solution was then dialyzed against 10 mM phosphate buffer (pH 7.2) containing 0.3 M sucrose and 1 mM KCl to remove NP-40. Dialysis was performed for 6 days with daily buffer changes. This dialyzed solution was agarose beads (Bio-Gel A-50m) equilibrated with 10 mM phosphate buffer (pH 5.2) containing 0.3 M sucrose and 1 mM KCl (Bio-Rad Laboratories, Hercules, CA, USA). Fractions with an optical density greater than 1.5 at 540 nm were collected as reconstituted fusion particles and fused with nucleic acid-loaded liposomes prepared from 10 mg lipid as described below to prepare viral vectors.

(Luciferase gene expression in transfected cells derived from human HEK293 strain)
In order to confirm the gene transfer activity of the virus vector prepared by the above method, human HEK293 strain and luciferase gene were used as follows.

pCMV-luciferase (7.4 kb) was transferred from pGEM-luc (Promega Corp., Madison, WI, USA) to HindIII in pcDNA3 (5.4 kb) (Invitrogen, San Diego, CA, USA). And by cloning at the BamHI site. A viral vector containing about 40 μg of pCMV-luciferase was constructed as described above and 1/10 volume of this viral vector (about 1.5 × 10 ¹¹ particles / ml, DNA concentration about 40 μg / ml). (100 μl) was incubated with 2 × 10 ⁵ cells from the human 293 cell line (human embryonic kidney: HEK). The same amount of luciferase DNA was introduced into 2 × 10 ⁵ HEK293 cells using HVJ liposomes. Cells were harvested 24 hours after transfection and the luciferase activity assay was confirmed as described elsewhere (Saeki et al., Hum. Gene Ther., 8: 1965-1972 (1997)).

(Example 2: Preparation and use of HVJ envelope vector by freeze-thawing)
(Preparation of viral vectors and their use)
(1: Preparation of HVJ envelope vector by freeze-thawing)
Using a luciferase gene as a foreign gene, the recombinant HVJ virus was freeze-thawed at various times and then introduced into cultured cells.

To 500 μl of TE, 750 μg of luciferase expression vector pcOriPLuc (Saeki and Kaneda et al., Human Gene Therapy, 11, 471-479 (2000)) and various concentrations of HVJ virus were mixed. The HVJ virus concentration was adjusted to 10, 25, 50, 100 HAU / μl. This solution was divided into 12 parts, and each piece 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 a medium of BHK-21 cells (24 well dish, 4 × 10 ⁴ cells / dish, 0.5 ml DMEM, 10% FCS), 37 ° C., 5% CO _2. After 20 minutes of reaction, the plate was washed with PBS, 0.5 ml of a new culture solution was added, and cultured for 24 hours.

  After removing the medium and adding 500 μl of 1 × Cell Culture Lys Reagent (Promega) onto the cells to lyse the cells, the cells were transferred to a microtube and centrifuged. From 20 μl of the obtained supernatant, Promega Luciferase Assay System and Lumat Luciferase activity was measured using LB9501 Luminophotometer. The measurement was performed 3 times for each solution, and the average value was obtained.

  As a result, the luciferase activity increased as the number of times of freezing and thawing of the recombinant HVJ virus increased, and 10 times or more of luciferase expression was observed in 20 times of freezing and thawing compared to 3 times of freezing and thawing. From this result, it was confirmed that the number of times of freezing and thawing of the recombinant HVJ virus is preferably 5 times or more, more preferably about 15 to 20 times under the conditions used in this example.

(2: Gene transfer efficiency of HVJ envelope vector prepared by freeze-thawing)
The same recombinant HVJ virus as in 1 above was thawed and thawed 30 times, and then the efficiency of gene transfer into the cells was examined using the same number of viruses added to the host cells.

  As a result, for example, when the X axis is 500 HAU, the addition amount of the solution having a virus concentration of 10 HAU / μl is 50 μl, and the solution of 100 HAU / μl is 5 μl. The solution with a virus concentration of 100 HAU / μl had a gene expression efficiency reduced by about 50% compared to the case with a concentration of 10-50 HAU / μl. From this result, it was confirmed that the recombinant virus concentration is preferably in the range of 10 to 50 HAU / μl under the conditions of this example.

  The recombinant HVJ virus was thawed 29 times and then thawed 30 times, stored in that frozen state for 1 week, thawed and added to the cells. As a result, the recombinant HVJ virus stored frozen for 1 week also showed the same level of luciferase gene expression as that of the virus that had been thawed thirty times continuously.

(Example 3: Preparation and use of inactivated HVJ envelope vector (HVJ-E) using surfactant)
(1: Growth of HVJ)
HVJ can be generally used by inoculating a fertilized egg of a chicken by inoculation with a seed virus. However, cultured cells such as monkeys and humans, and a system for continuously infecting cultured tissues with a virus (cultivating a hydrolase such as trypsin). Any of those that are propagated by using a solution added to the liquid and those that are propagated by infecting a cultured cell with a cloned viral genome and causing persistent infection can be used.

  In this example, HVJ was propagated as follows.

  HVJ (species Z), which has been propagated using SPF (Specific pathogen free) fertilized eggs, separated and purified, is dispensed into a cell storage tube, and 10% DMSO is added and stored in liquid nitrogen And prepared.

  The chicken eggs immediately after fertilization were received and placed in an incubator (SHOWA-FURANKI P-03 type; containing about 300 chicken eggs) and bred for 10 to 14 days under conditions of 36.5 ° C. and humidity of 40% or more. In the dark room, use an ophthalmoscope (light bulb light exits through a window with an aperture of about 1.5 cm) to check embryo survival and air chamber and chorioallantoic membrane. About 5 mm above, the location of the virus injection was marked with a pencil (select the location excluding the thick blood vessels). Seed virus (taken from liquid nitrogen) 500 times with polypeptone solution (mixed with 1% polypeptone, 0.2% NaCl, adjusted to pH 7.2 with 1M NaOH, autoclaved and stored at 4 ° C.) Diluted to 4 ° C. Eggs were sterilized with isodine and alcohol, a small hole was made in the virus injection site, and 0.1 ml of diluted seed virus was injected into the chorioallantoic cavity using a 1 ml syringe with a 26 gauge needle. Dissolved paraffin (melting point: 50-52 ° C.) was placed on the hole using a Pasteur pipette to close it. The eggs were placed in an incubator and bred for 3 days under conditions of 36.5 ° C. and humidity of 40% or more. The inoculated eggs were then placed at 4 ° C. overnight. The air chamber part of the next limit egg was divided with tweezers, a 10 ml syringe with an 18 gauge needle was placed in the chorioallantoic membrane, the chorioallantoic fluid was aspirated, collected in a sterile bottle, and stored at 4 ° C.

(2: Purification of HVJ)
HVJ can be purified by purification methods by centrifugation, column purification methods, or other purification methods known in the art.
(2.1: Purification method by centrifugation)
Briefly, the grown virus solution was collected, 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% w / v). It should be noted that the virus should be handled as gently as possible during purification and stored at 4 ° C.

  In this example, specifically, HVJ was purified by the following method.

  About 100 ml of HVJ-containing chorioallantoic fluid (collecting HVJ-containing chicken egg chorioallantoic fluid and storing at 4 ° C.) was placed in two 50 ml centrifuge tubes with a wide-mouthed Komagome pipette (Saeki, Y., and Kaneda, Y : Protein modified liposomes (HVJ-liposomes) for the delivery of genes, volume of genesis and proteins. Cell Biology; 27th edition, C. Biology; A. laboratory handbook. 135, 1998) and centrifuged in a low speed centrifuge at 3000 rpm for 10 minutes at 4 ° C. (brake off) to remove egg tissue pieces.

  After centrifugation, the supernatant was dispensed into four 35 ml centrifuge tubes (for high-speed centrifugation) and centrifuged with an angle rotor at 27,000 g for 30 minutes (accelerator and brake on). The supernatant was removed, and about 5 ml of BSS (10 mM Tris-HCl (pH 7.5), 137 mM NaCl, 5.4 mM KCl; autoclaved and stored at 4 ° C.) (can be PBS instead of BSS) was added to the precipitate, It left still at 4 degreeC as it was. The precipitate was loosened by pipetting gently with a wide-mouth Komagome pipette and collected in one tube, and similarly centrifuged at 27,000 g for 30 minutes with an angle rotor. Excluding the supernatant, about 10 ml of BSS was added to the precipitate, and the mixture was allowed to stand at 4 ° C. overnight. The precipitate was loosened by gently pipetting with a wide-mouth Komagome pipette, and centrifuged at 3000 rpm for 10 minutes at 4 ° C. (brake off) with a low-speed centrifuge to remove the tissue fragments and virus clumps that could not be removed. The supernatant is placed in a new sterile tube and stored as purified virus at 4 ° C.

  0.9 ml of BSS was added to 0.1 ml of this virus solution, absorption at 540 nm was measured with a spectrophotometer, and the virus titer was converted to hemagglutination activity (HAU). An absorption value 1 at 540 nm corresponded to approximately 15,000 HAU. HAU is considered to be approximately proportional to the fusion activity. In addition, erythrocyte agglutinating activity may be actually measured using chicken erythrocyte fluid (0.5%) (Animal Cell Utilization Manual, REALIZE INC. (Uchida, Oishi, Furuzawa) P259-268, 1984. See

Furthermore, purification of HVJ using a sucrose density gradient can be performed as necessary. Specifically, the virus suspension is placed in a centrifuge tube overlaid with 60% and 30% sucrose solutions (autoclaved), and density gradient centrifugation is performed at 62,800 × g for 120 minutes. After centrifugation, the band seen on the 60% sucrose solution layer is collected. 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 sterilization) and 0.5M EDTA solution (autoclave sterilization) to the virus suspension to a final concentration of 10% and 2-10 mM, respectively, and gently at −80 ° C. Freeze and finally store in liquid nitrogen (freezing can be done in 10 mM DMSO instead of glycerol and 0.5 M EDTA solution).
(2.2: Purification method by column and ultrafiltration)
Instead of the purification method by centrifugation, purification of HVJ using a column is also applicable to the present invention.

  Briefly, purification was performed using concentration by ultrafiltration with a filter with a molecular weight cut-off of 50,000 (about 10 times) and elution by ion exchange chromatography (0.3 M to 1 M NaCl).

  Specifically, in this example, HVJ was purified by column using the following method.

  After the chorioallantoic fluid was collected, it was filtered through an 80 μm to 10 μm membrane filter. 0.006-0.008% BPL (final concentration) was added to chorioallantoic fluid (4 ° C., 1 hour) to inactivate HVJ. BPL was inactivated by incubating chorioallantoic fluid at 37 ° C. for 2 hours.

The solution was concentrated about 10 times by tangential flow ultrafiltration using 500 KMWCO (A / G Technology, Needham, Massachusetts). As a buffer solution, 50 mM NaCl, 1 mM MgCl ₂ , 2% mannitol, 20 mM Tris (pH 7.5) was used. The HAU assay gave excellent results with almost 100% HVJ recovery.

  HVJ was purified by column chromatography (buffer solution: 20 mM Tris HCl (pH 7.5), 0.2-1 M NaCl) by QSepharoseFF (Amersham Pharmacia Biotech KK, Tokyo). The recovery was 40 to 50%, and the purity was 99% or more.

  The HVJ fraction was concentrated by tangential flow ultrafiltration using 500 KMWCO (A / G Technology).

(3: Inactivation of HVJ)
When inactivation of HVJ was required, it was performed by ultraviolet irradiation or alkylating agent treatment as described below.

(3.1: UV irradiation method)
1 ml of the HVJ suspension was placed in a petri dish having a diameter of 30 mm and irradiated with 99 or 198 millijoules / cm ² . Gamma irradiation can also be used (5-20 gray), but complete inactivation does not occur.

(3.2: Treatment with alkylating agent)
Immediately before use, 0.01% β-propiolactone was prepared in 10 mM KH ₂ PO. During the work, it was kept at a low temperature and worked quickly.

  Β-propiolactone was added to a suspension of HVJ immediately after purification to a final concentration of 0.01% and incubated on ice for 60 minutes. Thereafter, it was incubated at 37 ° C. for 2 hours. Dispense 10,000 HAU per tube into an Eppendorf tube, centrifuge at 15,000 rpm for 15 minutes, and store the precipitate at -20 ° C. Regardless of the inactivation method described above, the precipitate can be stored at -20 ° C., and the DNA can be directly incorporated by treatment with a surfactant to prepare a vector.

(4: Creation of HVJ envelope vector)
92 μl of a solution containing 200 to 800 μg of foreign DNA was added to the preserved HVJ and well suspended by pipetting. This solution can be stored at −20 ° C. for at least 3 months. When protamine sulfate was added to DNA before mixing with HVJ, the expression efficiency was enhanced by a factor of 2 or more.

  This mixed solution was placed on ice for 1 minute, 8 μl of octylglycoside (10%) was added, the tube was shaken on ice for 15 seconds, and left on ice for 45 seconds. The treatment time with the surfactant is preferably 1 to 5 minutes. Besides octyl glycoside, Triton-X100 (t-octylphenoxypolyethoxyethanol), CHAPS (3-[(3-colamidopropyl) -dimethylammonio] -1-propanesulfonic acid), NP-40 (nonylphenoxypoly) Surfactants such as ethoxyethanol 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.

  1 ml of cold BSS was added and immediately centrifuged at 15,000 rpm for 15 minutes. 300 μl of PBS or physiological saline was added to the resulting precipitate 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.

(5: Gene transfer into cells using HVJ envelope vector)
(Gene transfer method)
1,000 HAU was placed in an Eppendorf tube (30 μl), and 5 μl of protamine sulfate (1 mg / ml) was added. The medium of BHK-21 cells (200,000 per well and spread in 6 wells the day before) was changed, and 0.5 ml of medium (10% FCS-DMEM) was added per well. To each well, add a mixture of the above-mentioned vector (equivalent to 1,000 HAU) and protamine sulfate, mix the vector and cells well by shaking the plate back and forth, left and right, and in a 5% CO ₂ incubator at 37 ° C. for 10 minutes. I left it alone.

The medium was changed and left at 37 ° C. in a 5% CO ₂ incubator overnight (16 hr to 24 hr) to examine gene expression. In the case of luciferase (pcLuci; luciferase gene having a CMV promoter), the cells were lysed with 0.5 ml of Cell Lysis Buffer (Promega), and the activity in a 20 μl solution was measured using a luciferase assay kit (Promega). In the case of green fluorescent protein (pCMV-GFPE; Promega), it was directly observed with a fluorescence microscope, 5 to 8 fields were observed at 400 magnifications, and the ratio of cells that emitted fluorescence was calculated.

Example 4: Preparation and use of liposome vectors
(Preparation of liposome vector)
The liposome vector of the present invention was prepared by using 20-24 μg of cDNA derived from the cDNA library and 24-72 μl of lipofect AMINE 2000 (Lipofectamine 2000 (Invitrogen life technologies 9 (Carlsbad, California 92008))) reagent in 1.2 ml of serum-free medium. The mixture is rapidly mixed and incubated at room temperature for 20 minutes to form a liposome-nucleic acid complex.

(Transfection with liposome vectors)
Host cells are added to each well of a 96 well plate with appropriate media and cultured. When transfecting in the presence of serum, add 12.5 μl of the complex directly to each well of a 96 well plate and mix. When transfection is performed in the absence of serum, the medium containing serum is removed and replaced with medium without serum before adding the complex.
After culturing in a CO ₂ incubator for 4 to 12 hours, the medium is changed. After incubation for a predetermined time, the assay is performed.

(Example 5: Gene transfer using substance introduction devices prepared using various surfactants)
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.

(Preparation of cells, plasmid DNA and HVJ-E)
BHK-21 cells (babyhamsterney cells) were purchased from American Type Culture Collection (ATCC, Rockville, MD). The cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% FBS. In order to evaluate the transfection efficiency, pGL3 (Promega), which is a luciferase expression vector, was used. The plasmid was prepared using a Qiagen column (Hilden, Germany). The HVJ virus was amplified and the HVJ-E vector was prepared as described in Example 3.

(Preparation of HVJ-E containing nucleic acid)
16.7 HAU of HVJ-E was centrifuged at 15000 g for 15 minutes at 4 ° C. After removing the supernatant, the suspension was suspended in 1.33 μg (0.67 μl) of the pGL3 plasmid solution, and 49.33 μl of PBS (−) was further added. This mixture was added to one well of a 96-well microtiter plate and 15 μl of the surfactant shown in FIGS. 1 and 2 was added to the mixture. The mixture was incubated for 15 minutes, then 85 μl of 0.21 mg / ml protamine sulfate (dissolved in PBS (−)) was added. The plate was centrifuged at 3000 rpm (1643 × g), 30 minutes, 4 ° C., and further incubated at 4 ° C. overnight.

(Transfection)
For transfection, 80 μl of the supernatant in the wells of the plate to which the vector containing the nucleic acid prepared above was added was removed, 2.5 × 10 ⁴ cells were added, and then 1000 rpm for 30 minutes and 32 ° C. twice. Centrifuged. The cells were cultured overnight at 34 ° C. using a medium supplemented with 10% FBS. 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 FIG. 1 and FIG.

  As shown in FIG. 1, luciferase activity was detected when each of dodecyl maltoside, octyl glycoside, and digitonin was used. The intensity of activity was strongest for dodecyl maltoside, then strong for octyl glycoside, and weakest for digitonin.

As shown in FIG. 2, octyl glycoside, octyl maltoside, decyl maltoside, dodecyl maltoside, digitonin, and Triton-X were used. Luciferase activity was detected when octyl glycoside, octyl maltoside, decyl maltoside, dodecyl maltoside, and digitonin were used. On the other hand, when Triton-X was used, luciferase activity was not detected. From this result, it is considered that the surfactant needs to have a sugar moiety for gene introduction. On the other hand, whether the hydrophobic part is an alkyl group or a steroid, gene transfer activity was confirmed, and therefore it is considered that the hydrophobic part does not have an essential structural feature. However, when the hydrophobic moiety is an alkyl group, the gene transfer efficiency was higher than that of a steroid. When the hydrophobic moiety is an alkyl group represented by the formula — (CH) ₂ n—CH ₃ , high gene transfer efficiency was brought about when n = 7 to 11. The gene transfer efficiency was highest when n = 9. Of the individual surfactants used, the order of strong activity was decyl maltoside, dodecyl maltoside, octyl maltoside, octyl glycoside, and digitonin.

  The concentration below CMC was examined with reference to FIG. An effective surfactant in this method is dodecyl maltoside, and a peak was observed at 1/4 CMC. Therefore, in FIG. 2, a similar surfactant was examined in the 1/4 CMC set in FIG. For octyl glucoside and digitonin, cytotoxicity was observed, so the concentration with the least effect was selected. In order to compare with the conventional method, Triton X-100 used the concentration used in the conventional method.

(Example 6: Gene transfer using substance introduction devices prepared using various surfactants)
A total of 4208 HAU HVJ-E was centrifuged at 15000 g for 15 minutes at 4 ° C. After removing the supernatant, 2 mg / ml pGL3 (167.8 μl) was added to suspend the vector. 4.05 ml of PBS (−) was added to the mixture, and 16.7 μl of pGL3 / HVJ-E / PBS mixture was added to each 96-well plate using a 96 auto dispenser machine (ADS-384, Biotech). Dispensed into wells (HVJ-E: 16.7 HAU /
Well, pGL3: 1.33 ug / well). 0.01% dodecyl maltoside was added to each of the 96 wells to 5 μl / well. Plates were incubated for 15 minutes, then PBS (−) was added to 22.3 μl / well, and 1 mg / ml protamine sulfate / PBS (−) was added to 6.0 μl / well. . The solution was added to the plate and the plate was centrifuged at 3000 rpm for 30 minutes at 4 ° C. and further incubated at 4 ° C. overnight.

A cell solution of 1.67 × 10 ⁵ cells / ml is prepared for transfection, and 2 is added to the plate to which the vector containing the nucleic acid prepared above is added using a 96 auto dispenser machine (ADS-384, Biotech). .5 × 10 ⁴ cells (150 μl) were added and centrifuged at 1000 rpm for 30 minutes and 32 ° C. twice. The cells were cultured overnight at 34 ° C. using a medium supplemented with 10% FBS. 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 FIG. 3 and FIG.

  As shown in FIG. 3, the gene transfer method of the present invention showed high reproducibility. The average values of fluorescence intensity (RU) were 2080518 and 1667747, and the standard deviations were 377928.9 and 324477. The relative standard deviations (C.V.%) are 18.2% and 19.5%, and the high reproducibility of the present invention is indicated by numerical values (FIGS. 4A and B).

(Example 7: Gene transfer efficiency when using a solid phase coated with various proteins)
Overall, 20995 HAU HVJ-E was centrifuged at 15000 g for 15 minutes at 4 ° C. After removing the supernatant, the vector was suspended in 157 μl of 2 mg / ml pGL3 plasmid solution. 47.1 μl of 1% Triton X-100 was added and centrifuged at 15000 g for 15 minutes at 4 ° C. After removing the supernatant, the vector was suspended in 1662.4 μl of PBS (−), and 237.5 μl of 1 mg / ml protamine sulfate (dissolved in PBS (−)) was added. Next, 6.1 ml of PBS (−) was added and this solution was added to each of the 24-well plates shown in FIG. 5 (200 μl / well). The plate was centrifuged at 3000 rpm (1643 × g), 30 minutes, 4 ° C. and further incubated at 4 ° C. overnight.

For transfection, 100 μl of the supernatant in the well of the plate to which the vector containing the nucleic acid prepared above was added was removed, 2.5 × 10 ⁵ cells (1 ml) were added, and then at 1000 rpm for 30 minutes at 32 ° C. And centrifuge twice. The cells were cultured overnight at 34 ° C. using a medium supplemented with 10% FBS. Luciferase activity was detected using a luciferase assay kit (Promega) and fluorescence intensity was detected with an image reader ARVOmx (Wallac).

The plate shown in FIG. 5 was used. The coating on each plate is as follows:
Corning: No coating (Corning)
SUMILON: No coating (manufactured by Sumitomo Bakelite),
PLL: Poly-L lysine coating (manufactured by Sumitomo Bakelite),
Gelatin: Gelatin coating (manufactured by Sumitomo Bakelite),
C-1: Collagen 1 coating (manufactured by Sumitomo Bakelite),
B. D. : No coating (BD Bioscience)
PLO / LM: Poly L ornithine / laminin coating (BD Bioscience),
PDL / LM: poly D lysine / laminin coating (BD Biosciences),
Laminin: coating (made by BD Bioscience),
C-1: Collagen 1 coating (manufactured by BD Bioscience),
C-4: Collagen 4 coating (manufactured by BD Biosciences), and Fibrectin: Fibronectin coating (manufactured by BD Biosciences).

  As shown in FIG. 5, the gene transfer efficiency was highest when a plate coated with fibronectin was used.

(Example 8: Isolation and analysis of a gene encoding a protein having a desired function)
By using the present invention, it is possible to isolate a target gene having a desired functional property. For example, using the gene transfer vector prepared in Example 3 of the present specification and the expression plasmid library, the gene is introduced into the cells on the device according to the method described in Example 5, and the desired cells are selected from the cells in the wells. It is possible to isolate a gene encoding a protein having a desired function by determining a cell having the above function.

  In this case, not only the gene transfer vector prepared in Example 3 of the present specification but also any “virus envelope vector” and “liposome vector” can be used for the gene isolation method exemplified in this example. It is.

  A method for isolating a gene encoding vascular endothelial growth factor using the method of this example is exemplified below.

A human heart cDNA library (GIBCO BRL; plasmid obtained by ligating human heart-derived cDNA to a plasmid pSPORT having a CMV promoter) introduced into E. coli DH12S. A plasmid is prepared from E. coli. 200 μg of the plasmid is enclosed in a 10000 HAU HVJ-E gene transfer vector (the gene transfer vector prepared in Example 3 of the present invention, 3 × 10 ⁹ particles). As host cells, about 5000 cells of human aortic endothelial cells HAEC (Sanko Junyaku) are added to each well of a 96-well microtiter plate together with the medium, and the cells cultured overnight are used. To each well containing host cells, 1/100 volume of HVJ-E is added and left at 37 ° C. for 30 minutes, and then the medium is changed.

  The used medium is in a low nutrient state with a serum concentration of 1%, and the culture is performed for 1 week in this state. Under these conditions, HEAC growth is not confirmed.

  Two weeks later, a cell proliferation assay is performed. Cell Titer 96 (Promega) is used as a reagent, and the redox state of mitochondria is regarded as a change in the color tone of the reagent and used as an index of cell proliferation.

  Well color depth is an indicator of cell proliferation. The darkest well is a well in which cells that have undergone the most active cell growth are present. 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 exhibiting the most proliferative action using the Qiagen DNeasy Tissue Kit. The nucleic acid to be prepared includes plasmid DNA, which is competent E. coli. into E. coli (DH5α; Takara Shuzo) using the heat shock method.

  This E.I. E. coli is seeded on a plate medium containing ampicillin to form colonies. About 20 to 200 colonies can be obtained from DNA prepared from one well. Plasmid DNA (pDNA) is extracted from each colony, and the presence of the gene fragment in the plasmid is confirmed by restriction enzyme treatment. Usually, about 60 to 70% of the prepared plasmid is a plasmid having an insert.

  Next, plasmid DNA is purified using Qiagen's Endo Free Plasmid Maxi Kit, the purified plasmid is encapsulated in HVJ-E, and introduced again into HAEC cells, and the same cell proliferation experiment is performed. In this cell proliferation experiment, a plasmid that showed significantly high cell proliferation is a candidate plasmid expected to contain a nucleic acid encoding vascular endothelial growth factor.

  The above results demonstrate that by using the present invention, a gene having a desired functional property could be quickly and easily isolated.

(Example 9: Method for isolating mutant nucleic acid having desired functional properties)
By using the present invention, it is possible to isolate a mutant gene having a desired functional property.

  Using the viral vector prepared in Example 3 of the present specification, a gene having a desired function is isolated. For the gene isolation method exemplified in this example, not only the virus vector prepared in Example 3 of the present specification but also any “virus envelope vector” and “liposome vector” can be used. .

  As a starting material, a nucleic acid encoding a specific gene is selected. A plasmid is prepared in which the nucleic acid is operably linked to a sequence that functions as a promoter in the cell. 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 5000 cells are added to each well of a 96-well macrotiter plate along with the medium and cultured overnight. After culture, the mutant host cells in each well are screened for the desired function. A cell exhibiting a desired function is taken out, and a nucleic acid is prepared from the cell in the well exhibiting the most preferable property using a DNeasy Tissue Kit from Qiagen. The nucleic acid to be prepared includes plasmid DNA, which is competent E. coli. into E. coli (DH5α; Takara Shuzo) using the heat shock method.

  This E.I. E. coli is seeded on a plate medium containing ampicillin to form colonies. Usually, about 20 to 200 colonies can be obtained from DNA prepared from one well. Plasmid DNA is extracted from each colony, and the presence of the gene fragment in the plasmid is confirmed by restriction enzyme treatment.

  Next, Qiagen's Endo Free Plasmid Maxi Kit is used to purify the plasmid DNA, the purified plasmid is encapsulated in HVJ-E, reintroduced into HAEC cells, and tested for similar functional properties. In this cell proliferation experiment, it is proved that the plasmid showing the preferable functional property is a plasmid containing the mutated nucleic acid having the desired functional property.

(Example 10: Improvement of encapsulation efficiency of desired substance in HVJ)
In the above examples, protamine sulfate (PS) was used as the conventional encapsulating enhancer for HVJ, but in this example, polyethyleneimine (PEI) was used instead of PS. As a result, compared to PS, (1) an increase in the efficiency of introduction of biopolymers into cells and (2) expansion of cell versatility were demonstrated. In addition, PEI has a linear type and a branched type. (1) The linear type can maintain high introduction efficiency in a wide range of concentrations, and (2) the linear type has lower cytotoxicity. Proven. The results are shown below.

There are two types of PEI known as a cationic polymer, linear and branched. This time, two types of PEI were used, two types each having a different molecular weight, each of a linear type (PEI-L) and a branched type (PEI-B). The structure of linear PEI is shown as — [NH—CH ₂ —CH ₂ ] _x — (X is an arbitrary integer), and in this example, PEI-L (PEI-L) having a molecular weight of 2.5 kDa. 2.5K) and 25 kDa PEI-L (PEI-L 25K) were used. The branched PEI is shown as — [NH—CH ₂ —CH ₂ ] _y — [N (CH ₂ —CH ₂ —NH ₂ ) —CH ₂ —CH ₂ ] _z — (y and z are arbitrary integers). In this example, PEI-B having a molecular weight of 25 kDa (PEI-B 25K) and PEI-B having a molecular weight of 750 kDa (PEI-B 750K) were used.

  For BAEC cells, the method of Example 6 above was modified as follows, and instead of using PS, PEI was used. Specifically, it is as follows.

  HVJ-E was centrifuged at 15000 g for 15 minutes at 4 ° C. to add 4.5 HAU per well of a 96 well plate. After removing the supernatant, it was suspended in an appropriate amount of HBSS (Hank's Balanced Salt Solution), and 5 μl (4.5 HAU) of the HVJ-E / HBSS mixed solution was added to the 96-auto dispenser machine in 96 wells. Dispense into each well of the plate. A DNA solution (0.25 mg / mL pEGFP-C1 5 μl / well) was added to each of 96 wells and mixed. Thereafter, 3 μl / well of 0.01% dodecyl maltoside was added to each of 96 wells, mixed and the plate was incubated for 15 minutes. Next, PEI was added at 12 μl / well (concentration and type differed depending on the cell and biopolymer used), and after mixing, HBSS was added at 25 μl / well and mixed. The plate was centrifuged at 3000 rpm for 30 minutes at 4 ° C. and further incubated at 4 ° C. overnight.

For transfection, a 6.7 × 10 ⁴ cell / ml BACE cell solution was prepared, and a 96 auto dispenser machine was used to add 1 × 10 ⁴ cells ( 150 μl) was added and centrifuged at 1000 rpm for 30 minutes and 32 ° C. twice. The cells were cultured at 34 ° C. for 50 hours using a medium supplemented with 10% FBS. 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 FIG. 6 and FIG.

  As shown in FIG. 6, the efficiency of introducing plasmid DNA into cells was improved as compared with the case of using PS in all PEI used this time. Furthermore, as shown in FIG. 7, gene transfer to GB-1 cells and SK-BR-3 cells that could not be detected when PS was used was also demonstrated. In addition, when PEI is used, gene transfer into floating cells is similarly efficient.

  Next, gene transfer efficiency using four types of PEI under the above conditions was compared. The result is shown in FIG. When PEI was used with HVJ-E, PEI-L 25K was demonstrated to be most suitable for BAEC cells. The optimum PEI type varies depending on the cell. When PEI-L (linear type) was used, the range of PEI concentration showing high introduction efficiency was wide, and when PEI-B (branched type) was used, the range of concentration range showing high introduction efficiency was narrow. Similar trends are seen in other cells.

  Next, the cytotoxicity using 4 types of PEI was compared. Specifically, the influence of various PEIs on cell proliferation when BAEC cells were cultured for 76 hours was measured. As a result, as shown in FIG. 9, when used with HVJ-E, PEI-B was more toxic to cells than PEI-L. Cytotoxicity became stronger as the molecular weight of PEI increased. Both PEI-L and PEI-B showed the highest introduction efficiency in the vicinity of the limit concentration where cytotoxicity appeared. The same tendency can be seen in other cells.

(Example 11: Lyophilization protocol)
Viral vectors that can introduce various substances into cells are very effective in elucidating the pathogenesis and drug discovery. Therefore, in particular, it is necessary to stably and efficiently introduce the viral vector encapsulating the introduced substance into the cell after storing it for a long period of time. Technology that maintains efficiency is required for long-term storage. Therefore, according to the present invention, activity retention by long-term storage is achieved as follows.

(Freeze drying protocol)
A virus vector (HVJ-E) encapsulating a gene (reporter gene) and the like was lyophilized in each well of a 96-well plate and stored according to each condition. Then, the gene etc. were introduce | transduced into the cell by dispensing the cell to each well of the preserve | saved plate. The stability test of lyophilized HVJ-E was measured by a reporter assay (luciferase assay) using the reporter gene expressed in the cells as an indicator. An exemplary protocol is as follows.
1. Suspend the virus vector in distilled water.
2. DNA or siRNA (in addition, RNA, antibody, peptide, functional protein) in automatic encapsulation device (ADS384 (device name: Multistage Dispense Station ADS-384-8, manufacturer: Biotech Co., Ltd. (Tokyo))) , Sugar chain, lipid, semiconductor or complex thereof) and surfactant vector dodecyl maltoside (in addition, Triton X-100 etc.) on the plate (plate coating types: fibronectin, collagen I, collagen IV) , Tissue culture treatment, gelatin, poly D lysine, laminin, poly L ornithine).
3. A substance selected from the group consisting of stabilizers (trehalose, maltose, raffinose, fructose, sucrose, glucose, lactose, polyvinyl pyrrolidone, polyethylene glycol, methyl cellulose, glycine, mannitol, dimethyl sulfoxide (DMSO) or mixtures thereof. The stabilizer is preferably trehalose, maltose, raffinose, fructose, or a mixture thereof, and is centrifuged at 3000 revolutions.
4. According to the freeze-drying program, freeze-dry using a vacuum freeze-drying apparatus (FTS).
5. The freeze-dried plate is sealed together with an oxygen scavenger and a desiccant (Omi Chemical) in a polyethylene bag, further sealed in an aluminum pack, and stored at each temperature.
6. After storage, the activity (RLU) was measured with a plate reader (Perkin Elmer) by a reporter assay (luciferase assay (Promega)) and evaluated.

  At that time, the conditions of the type and concentration of the stabilizer, storage temperature, and storage period were set and evaluated.

(Preparation of freeze-dried plate)
A lyophilized plate containing 14.4 HAU HVJ-E per well and 0.25 μg of plasmid was made by the following procedure.

  HVJ-E was centrifuged at 15000 g for 15 minutes at 4 ° C., and the supernatant was removed. It was suspended in distilled water to obtain an HVJ-E suspension. The subsequent operations were performed by automation using an ADS384 apparatus.

  HVJ-E suspension (14.4 HAU) was added to each well of a 96 well microtiter plate (5 μl / well). Then, a solution containing 0.25 μg of DNA (0.05 μl at a concentration of 0.05 mg / ml) was added and mixed. Dodecyl maltoside (0.01% 3/10 times amount; in this example, 3 μl) was added and mixed. Next, protamine sulfate (12 μl at a concentration of 0.125 mg / ml; 1.5 μg / well in this example) was added and mixed. The various stabilizers were then added at the indicated concentrations and mixed. In this example, 5 μl was actually added. In the case of trehalose, 5 μl of 3% solution was added to a final concentration of 0.5%. The plate was lyophilized in a vacuum lyophilizer FTS (FTS Systems, Inc. 3538 Main Street, PO Box 158 StoneRidge, NY 12484 USA).

(1. Stability by freeze-drying)
Using the stabilizer trehalose (concentration: 0.5%), the efficiency of introduction of the pGL3 plasmid into BHK cells was compared between when lyophilized and after lyophilization. As a result, as shown in FIG. 10, when the lyophilized untreated specimen and the lyophilized specimen were compared by luciferase activity (RLU), they were hardly deteriorated by the lyophilization / storage treatment. From this, it was shown that freeze-drying is effective for the stability of long-term storage.

(2. Test for storage temperature and duration)
Genes to 2.5 × 10 ⁴ BHK cells using 2.5 μg plasmid pGL3, 14.4 HAU HVJ-E: JP49, and 0.5% trehalose after various storage temperatures and storage periods After introduction, the cells were cultured for 24 hours at 34 ° C., and the gene introduction efficiency was measured. The storage temperature was -80 degrees, -20 degrees, 4 degrees, 25 degrees (60% Rh), and the storage periods were 1 month and 6 months. As a result, as shown in FIG. 11, the luciferase activity (RLU) did not deteriorate at all even when stored at each temperature for 6 months. From this, it was shown that lyophilization enables stable storage over a long period regardless of the storage temperature.

(3. Testing for stabilizers and shelf life)
Trehalose and maltose were used as the stabilizer. As a result, as shown in FIG. 12, even when stored for 6 months, it was demonstrated that the introduction activity was sufficiently maintained if a stabilizer was added. By using 0.5% stabilizer at -20 degree storage for 6 months, the activity could be maintained 70 times or more.

(4. Testing for stabilizers)
Various stabilizers (trehalose, maltose, raffinose, fructose) were used at various concentrations (0.03% to 1.5%) to evaluate the storage stability after 1 month storage at -20 ° C. . The stabilizer was evaluated at a sample concentration before freeze-drying of 0.03% to 1.5%. The result is shown in FIG.

  In each stabilizer, a concentration of 0.001% to 3% is preferable, but the following concentrations were particularly preferable.

concentration(%)
Trehalose 0.1-0.25
Maltose 0.1-0.25
Raffinose 0.03-0.13
Fructose 0.1-0.5
(5. Uniform stability provided by the stabilization of the present invention)
The plate in which the same specimen was dispensed in all 96 wells was freeze-dried and introduced into cells, and a graph was created for all specimens in each plate. The results are shown in FIG. As shown in FIG. 14, the luciferase activity was almost equal in 96-well specimens, whether stored for 1 month at −20 ° C. or stored for 1 month at −80 ° C. As shown in the results, there was almost no difference between the wells due to lyophilization, and it was demonstrated that they can be used without problems in drug discovery and clinical development.

(6. Examples of introduced substances other than DNA)
Using siRNA, retention of gene transfer activity in freeze-dried and stored plates was examined. A viral vector (14.4 HAU) encapsulating siRNA (10 pmol) was lyophilized on a fibronectin plate (Human Fibronectin Cellware 96-well Plate (Becton Dickinson)), stored for one month, and then a reporter assay was performed. As cells, 2.5 × 10 ⁴ BHK cells were used.

  In this experiment, siRNA (siRNA against pGL2) and pGL2 (plasmid encoding firefly luciferase gene, Promega) were used. For the reporter assay, Dual-Glo Luciferase Assay System (Promega) was used. As a result, as shown in FIG. 15, expression suppression activity was retained during storage at −20 degrees to 25 degrees. FIG. 16 is a graph showing gene suppression activity on the Y axis for each of the samples immediately after plate preparation and after storage for 1 month at −20 ° C. under the same conditions as in the experiment of FIG. For the suppressive effect of siRNA on the target gene itself, deterioration due to lyophilization was not observed. From this result, it was demonstrated that HVJ-E containing siRNA can be stored for a long time.

(7. Types of immobilized plates)
A viral vector (14.4 HAU) encapsulating a luciferase gene was lyophilized onto various plates, and a reporter assay was performed. Three types of plates were used: a collagen-treated 96-well plate (Becton Dickinson), a fibronectin-treated 96-well plate (Becton Dickinson), and a tissue culture-treated 96-well plate (Becton Dickinson). As cells, 2.5 × 10 ⁴ BHK cells were used. As a result, as shown in FIG. 17, luciferase activity was obtained in any of the plates. 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.

(Example 12: Application of freeze-drying method)
A conventional gene introduction agent capable of introducing a gene into a cell has several steps to introduce the gene into the cell. Furthermore, the gene introduction agent mixed with the gene was not effective for long-term storage. Therefore, the development of an array / device for gene introduction that can be stably and efficiently introduced into cells after storage for a long period of time is desired. In the present invention, a cell is introduced by dropping a desired gene onto a plate on which a viral vector is immobilized and seeding the cells, and the gene is introduced only by an epoch-making simple and unprecedented operation. Make it possible.

(1. Preparation and use of the plate of this example)
The plate of this example is preferably prepared by the following procedure (A). This plate is preferably used as in procedure (B) below. Moreover, the evaluation is performed like a procedure (C), for example.

(A. Preparation of plate)
The plate of this example is prepared as follows.
1. Suspend the virus vector in distilled water.
2. Virus vector suspension together with protamine sulfate on plate (type of coating coating: fibronectin, collagen I, collagen IV, tissue culture treatment, gelatin, poly D lysine, laminin, poly L One of ornithine or a mixture thereof, preferably fibronectin). Next, selected from the group consisting of stabilizers (trehalose, maltose, raffinose, fructose, sucrose, glucose, lactose, polyvinylpyrrolidone, polyethylene glycol, methylcellulose, glycine, mannitol, dimethyl sulfoxide (DMSO) or mixtures thereof Including, but not limited to, substances, preferably stabilizers including trehalose, maltose, raffinose, fructose or mixtures thereof).
3. Centrifuge at 3000 rpm to immobilize on the plate.
4. According to the freeze-drying program, freeze-dry using a vacuum freeze-drying apparatus (FTS).
5. The freeze-dried plate is sealed together with an oxygen scavenger and a desiccant (Omi Chemical) in a polyethylene bag, further sealed in an aluminum pack, and stored at each temperature.

(B. How to use the plate)
The plate of the present invention prepared as described above is used as follows.
6. DNA or siRNA (in addition to RNA, antibodies, peptides, functional proteins, sugar chains, lipids, semiconductors) with an automatic encapsulation device (ADS384) (just before gene introduction into cells) against the stored plate Is dropped into each well, the gene is encapsulated in a viral vector, and the cells are seeded to introduce the gene into the cells.

(C. Evaluation of gene transfer efficiency)
The plate of the present invention is evaluated as follows.
7. Evaluation is made by measuring the activity with a plate reader (Perkin Elmer) by a reporter assay (luciferase assay (Promega)).

(2. Preparation and use of the plate of this example)
A lyophilized plate containing 14.4 HAU HVJ-E per well was made by the following procedure.

  Using an automatic encapsulation device (ADS384), 14.4 HAU of HVJ-E was added to a 96-well microtiter plate, centrifuged at 15000 g for 15 minutes at 4 ° C., and the supernatant was removed. It was suspended in distilled water to obtain an HVJ-E suspension. The HVJ-E suspension was then mixed on a fibronectin coated plate with protamine sulfate (12 μl at a concentration of 0.125 mg / ml). Next, a stabilizer (trehalose, concentration: 0.5%) was mixed. The plate was immobilized by centrifugation at 3000 rpm for 30 minutes at 4 ° C. The plate was lyophilized in a vacuum lyophilizer FTS.

  5 μl (0.25) and 10 μl (0.5) of plasmid pGL3-CMV (0.05 mg / mL) were added dropwise to a lyophilized plate (including HVJ-E). Further, as a control, 5 μl (0.25) and 10 μl (0.5) of plasmid pGL3-CMV (0.05 mg / mL) were dropped into wells on a plate not containing HVJ-E. The result is shown in FIG. The horizontal axis represents the amount of plasmid (μg), and the vertical axis represents luciferase activity (RLU).

  When there is no viral vector, no luciferase activity is observed even when the gene is dropped, but when the plasmid solution is dropped onto a freeze-dried plate on which the viral vector is immobilized, up to about 40-fold luciferase activity is confirmed. It was done. This means that a plate containing no nucleic acid to be introduced is preserved, and a nucleic acid solution is added to a freeze-dried plate containing HVJ-E just before the introduction of the nucleic acid, enabling highly efficient gene introduction. Prove that. According to the present invention, a simple gene transfer plate that can be produced simply by adding a plasmid solution can be prepared.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  The present invention provides a gene transfer technique that can increase the speed and efficiency, and can handle a large number of genes. The present invention also provides a technique for introducing not only genes but also desired substances (for example, peptides, compounds, nucleotide fragments, etc.) into cells.

  In addition, the present invention is capable of speeding up and efficiency improvement using a virus envelope or virus-derived protein, and a method for introducing many kinds of desired substances, and a solid phase for use in such a substance introducing method. , Compositions, and kits are provided.

Claims (46)

A device for introducing a desired substance into a cell, comprising:
(1) solid phase;
(2) viral vectors;
(3) a surfactant having a sugar moiety,
Wherein the viral vector and a surfactant having the sugar moiety are disposed on the solid phase. The device of claim 1, further comprising (4) a substance to be introduced, wherein the introduced substance is disposed on the solid phase. The substance to be introduced is at least one substance selected from the group consisting of a nucleic acid encoding a gene to be introduced, a peptide, a sugar chain, a low molecular weight compound, a proteoglycan, a glycopeptide, a lipid, and a metal ion. Item 3. The device according to Item 2. The device according to claim 1, wherein the introduced substance comprises a nucleic acid selected from the group consisting of siRNA, antisense and decoy of the introduced gene. The device according to claim 1, wherein the substance to be introduced is a nucleic acid encoding a gene to be introduced. The viral vector is (i) a viral envelope, (ii) a liposome comprising at least one viral envelope protein, or (iii) a mixture of a viral envelope and a liposome comprising at least one viral envelope protein. Item 2. The device according to Item 1. The device according to claim 6, wherein the viral envelope is a viral envelope derived from a wild type virus or a recombinant virus. The viral envelope is retroviridae, togaviridae, coronaviridae, flaviviridae, paramyxoviridae, orthomyxoviridae, bunyaviridae, rhabdoviridae, poxviridae, herpesviridae, baculoviridae, The device of claim 6, wherein the device is derived from a virus belonging to a family selected from the group consisting of: and Hepadnaviridae. The device according to claim 7, wherein the virus is derived from a virus belonging to the Paramyxoviridae family. The device of claim 8, wherein the virus is HVJ. 11. The device of claim 10, wherein the viral envelope protein is a protein selected from the group consisting of F protein, HN protein, NP protein, and combinations thereof. Surfactants having the sugar moiety of the formula _{A- (CH 2) n-CH} 3
The device according to claim 1, wherein A is a monosaccharide, disaccharide, trisaccharide, tetrasaccharide or pentasaccharide, and n = 7-11. The device according to claim 12, wherein A is a monosaccharide or a disaccharide. The device of claim 13, wherein A is a disaccharide. The device according to claim 13, wherein A is a glucoside group or a maltoside group. The device according to claim 15, wherein A is a maltoside group. The device of claim 12, wherein n = 9-11. The device of claim 17, wherein n = 9. The surfactant having the sugar moiety is a surfactant selected from the group consisting of octyl maltoside, decyl maltoside, dodecyl maltoside, octyl glycoside, decyl glucoside, and dodecyl glucoside, and mixtures thereof. Item 2. The device according to Item 1. 20. The device of claim 19, wherein the surfactant having a sugar moiety is decyl maltoside. The device of claim 1, wherein the concentration of the surfactant having a sugar moiety is 0.0001% by volume or more and less than CMC. The device of claim 21, wherein the concentration of the surfactant is from 0.0009 vol% to 0.28 vol%. The device of claim 1, further comprising (5) a protamine sulfate solution, wherein the protamine sulfate solution is disposed on the solid phase. The device of claim 1, further comprising (5) polyethyleneimine, wherein the polyethyleneimine is disposed on the solid phase. 25. The device of claim 24, wherein the polyethyleneimine is a linear polyethyleneimine. The device of claim 1, further comprising (6) a stabilizer, wherein the stabilizer is disposed on the solid phase. 27. The device of claim 26, wherein the stabilizer is trehalose, maltose, raffinose, fructose, sucrose, glucose, lactose, polyvinylpyrrolidone, polyethylene glycol, methylcellulose, glycine, mannitol, dimethyl sulfoxide ( DMSO) or a mixture thereof. 28. The device of claim 27, wherein the stabilizer is selected from the group consisting of trehalose, maltose, raffinose, fructose or mixtures thereof. 30. The device of claim 28, wherein the stabilizing agent is trehalose. The device of claim 1, wherein the solid phase is coated with a protein selected from the group consisting of fibronectin, collagen, laminin, gelatin, polyornithine and polylysine, and mixtures thereof. 32. The device of claim 30, wherein the solid phase is coated with fibronectin. The device of claim 1, wherein the cell is a mammalian cell. The device of claim 1, which is lyophilized. 25. The device of claim 24, which is lyophilized. 27. The device of claim 26, wherein the device is lyophilized. An apparatus for introducing a gene into a cell comprising:
(1) means for supporting a substance introduction device having at least 1500 wells;
(2) means for incubating the substance introduction device;
(3) means for adding a desired solution to a desired well of the substance introduction device; and (4) a centrifuge;
An apparatus comprising: further,
(5) means for measuring a signal selected from the group consisting of fluorescence, absorbance, luminescence, radiation and turbidity in a desired well;
37. The apparatus of claim 36. A kit for introducing a gene into a cell comprising:
(1) solid phase;
(2) a viral vector; and (3) a solution comprising a surfactant having a sugar moiety,
A kit comprising: The kit according to claim 38, further comprising (4) protamine sulfate. Furthermore, the kit of Claim 38 containing (4) polyethyleneimine. The kit according to claim 38, wherein the polyethyleneimine is a linear polyethyleneimine. The kit according to claim 38, further comprising (5) a stabilizer. 43. The kit of claim 42, wherein the stabilizer is trehalose, maltose, raffinose, fructose, sucrose, glucose, lactose, polyvinylpyrrolidone K90, polyethylene glycol, methylcellulose, glycine, mannitol, dimethyl sulfoxide. A kit selected from the group consisting of (DMSO) or a mixture thereof. 44. The kit of claim 43, wherein the stabilizer is selected from the group consisting of trehalose, maltose, raffinose, fructose or mixtures thereof. 45. The kit of claim 44, wherein the stabilizing agent is trehalose. Furthermore, the kit of Claim 38 or Claim 42 including the process of (6) freeze-drying.

Images