JP2010273565A - Gene transfer method, method for preparing nucleic acid complex and nucleic acid complex - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for transferring a gene, by which the gene can efficiently be transferred to a cell in a culture medium containing serum, to provide a method for preparing a nucleic acid complex, comprising combining a gene transfer agent with a nucleic acid in a solution containing a serum, and to provide a nucleic acid complex prepared by the preparation method. <P>SOLUTION: The method for transferring a gene, comprises contacting a nucleic acid complex prepared by combining a gene transfer agent with a nucleic acid, with the nucleic acid to transfer the gene to the cell. The gene transfer agent comprises a temperature-sensitive polymer whose physical properties are changed at a prescribed temperature (T), and the nucleic acid complex is contacted with the cell in a culture medium containing a serum at a prescribed temperature (T) or higher. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a gene introduction method for introducing a gene into a cell using a gene introduction agent, a method for preparing a nucleic acid complex which is a complex of a gene introduction agent and a nucleic acid, and a nucleic acid prepared by this nucleic acid complex preparation method. Concerning the complex.

  In recent years, gene therapy research has become more and more important as the molecular genetic factors of human diseases become clear. Gene therapy is aimed at the expression of DNA at the target site, how to make the DNA reach the target site, how to efficiently introduce the DNA into the target site and make it functionally expressed at the site It becomes important. Many vectors, including retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, Sendai viruses, or herpes viruses, have been modified to carry therapeutic genes as vectors for the introduction of foreign DNA. Used in human clinical trials. However, the risk of infection and immune response remains.

  Synthetic polymer vectors and cationic lipid vectors have been researched and developed as gene transfer technologies to replace virus vectors that have problems with safety, quality stability, and manufacturing costs. For example, 2-N, N-dimethylaminoethyl methacrylate A gene introduction agent containing a cationic polymer composed of a cationic vinyl monomer has been reported (Non-patent Document 1).

  As a synthetic polymer vector for transporting DNA into cells, the present applicants have developed a branched structure vector in which a cationic polymer chain radially extends with an aromatic ring as a nucleus, such as benzene, to condense DNA at high density. It was found that small nucleic acid complex microparticles can be formed and genes can be efficiently introduced into cells, and a patent application was filed earlier (Patent Documents 1 and 2 below).

  Applicants also have a thermosensitive-cationic property having a polymer block of N-isopropylacrylamide as the temperature-sensitive polymer and a polymer block of 3-N, N-dimethylaminopropylacrylamide as the cationic polymer block. Using a block copolymer as a gene transfer agent, this temperature-sensitive-cationic block copolymer is applied to a substrate, then the temperature is raised to insolubilize, and a nucleic acid is added thereto to form a cationic polymer chain that undergoes Brownian motion on the substrate. A gene introduction method was proposed in which a nucleic acid was included in an ion binding property and cells were then seeded (Patent Document 3 below).

WO2004 / 092388 JP 2007-70579 A Japanese Patent Application No. 2008-205256

Smedt, S., Demeester, J., Hennik, W., (2000), Cationic polymer based gene delivery system, J Pharmaceutical research 17,113-126

  The gene introduction agent in which the polymer chain extends radially from the benzene ring described in Patent Documents 1 to 3 can be arranged with a higher charge density due to its structure compared to a linear polymer composed of the same monomer unit. It is. Therefore, a complex with a nucleic acid such as DNA or RNA can be more strongly aggregated, and fine polyplex particles having a smaller particle diameter can be formed. For this reason, the cell membrane permeability of the polyplex particles is increased and the gene transfer activity is improved, but there are the following problems.

  When a gene is introduced into cells, a medium containing serum is more suitable as a medium for culturing cells than a medium not containing serum. This is because a medium containing no serum has a large stress on cells due to lack of nutrients, and adversely affects cells after culturing. Stem cells such as iPS cells in which control of cell differentiation is particularly important This is because it is not suitable for the introduction of genes into.

  However, the gene introduction agent containing the above-mentioned cationic polymer does not show high gene introduction activity in a medium containing serum. This is because the gene introduction agent containing the above-mentioned cationic polymer has a high zeta potential even under a neutral condition of about pH 7, and can agglutinate nucleic acids. This is probably because anionic proteins and the like are also adsorbed and aggregated to form macro particles, making it difficult for cells to be taken up into cells and for DNA to be difficult to recognize by transporters.

  Therefore, it is desired to develop a gene introduction method that can efficiently introduce genes into cells even in a medium containing serum.

  This invention is made | formed in view of the said conventional subject, and it aims at providing the gene introduction method which can introduce | transduce a gene into a cell efficiently in the culture medium containing serum.

  Another object of the present invention is to provide a method for preparing a nucleic acid complex in which a gene introduction agent and a nucleic acid are complexed in a solution containing serum, and a nucleic acid complex prepared by this preparation method.

  The gene introduction method of claim 1 is a gene introduction method for introducing a gene into a cell by contacting the cell with a nucleic acid complex obtained by combining a gene introduction agent and a nucleic acid, and the gene introduction agent comprises: It consists of a thermosensitive polymer whose physical properties change at a predetermined temperature (T), and the nucleic acid complex and cells are brought into contact with the serum-containing medium at the predetermined temperature (T) or higher. Is.

  The gene introduction method according to claim 2 is the method according to claim 1, wherein the thermosensitive polymer exhibits a cationic property at a temperature lower than a predetermined temperature (T) and exhibits a hydrophobic property at a temperature higher than the predetermined temperature (T). It is characterized by being.

  The gene introduction method according to claim 3 is the method according to claim 1 or 2, wherein the nucleic acid complex is prepared by mixing a solution containing a gene introduction agent and a nucleic acid at a temperature lower than a predetermined temperature (T), and then bringing the solution to a predetermined temperature (T ) It is obtained by heating the above and combining the gene introduction agent and the nucleic acid.

  The gene introduction method according to claim 4 is the method according to any one of claims 1 to 3, wherein the thermosensitive polymer comprises a branched polymer having a branched chain, and the branched chain is at least 2-N. , N-dimethylaminoethyl methacrylate and / or a derivative polymer block thereof.

  The gene introduction method according to claim 5 is the gene transfer method according to claim 4, wherein the branched polymer uses an iniferter as a compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule. -N, N-dimethylaminoethyl methacrylate and / or its derivative is obtained by light irradiation living polymerization.

  The gene introduction method according to claim 6 is characterized in that, in claim 4 or 5, the branched chain consists only of a polymer block of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof. .

  The gene introduction method according to claim 7 is the method according to any one of claims 4 to 6, wherein the molecular weight of the polymer block of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof contained in the branched polymer is set. 5,000 to 100,000.

  The gene introduction method according to claim 8 is the method according to any one of claims 1 to 7, wherein the molecular weight of the gene introduction agent is 10,000 to 150,000.

  The gene introduction method according to claim 9 is characterized in that, in any one of claims 1 to 8, the predetermined temperature (T) is 30 to 34 ° C.

  The gene introduction method according to claim 10 is the method according to any one of claims 3 to 9, wherein the solution containing the gene introduction agent and the nucleic acid is mixed at 0 to 30 ° C, and then the solution is added to 32 to 100 ° C. It is characterized in that the gene introduction agent and the nucleic acid are complexed by heating.

  The gene transfer method according to claim 11 is characterized in that in any one of claims 1 to 10, the nucleic acid complex and cells are brought into contact at 34 to 45 ° C.

  The method for preparing a nucleic acid complex of the present invention (Claim 12) is a method for preparing a nucleic acid complex in which a nucleic acid and a gene introduction agent are combined by mixing a solution containing the gene introduction agent and the nucleic acid, The gene transfer agent uses a compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule as an iniferter, and contains at least 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof. A branched polymer polymerized by light irradiation living polymerization, a mixing step of mixing the gene introduction agent and the nucleic acid at a temperature of the solution lower than a predetermined temperature (T), and after the mixing step, The solution is heated at a predetermined temperature (T) or higher, and then a serum is added to the solution to combine the gene introduction agent and the nucleic acid.

  The nucleic acid complex of the present invention (Claim 13) is prepared by the method for preparing a nucleic acid complex according to Claim 12.

  The gene transfer method of the present invention uses a thermosensitive polymer whose physical properties change at a predetermined temperature (T) as a gene transfer agent. The complex of this gene transfer agent and a nucleic acid, cells, and serum are used. In the medium containing, it makes it contact above this predetermined temperature (T), and it can introduce | transduce a gene efficiently, without giving stress to a cell in the medium containing serum.

  The gene introduction agent is preferably a thermosensitive polymer that exhibits a cationic property at a temperature lower than a predetermined temperature (T) and exhibits a hydrophobic property at a temperature equal to or higher than the predetermined temperature (T). If it is such a gene introduction agent, the gene introduction agent and the nucleic acid can be combined using the cationic property at a temperature lower than a predetermined temperature (T), and the hydrophobicity, That is, since it changes to nonionic, complexation of a nucleic acid complex and an anionic substance in serum hardly occurs.

  The nucleic acid complex is prepared by mixing a solution containing a gene introduction agent and a nucleic acid at a temperature lower than a predetermined temperature (T), and then heating the solution to a temperature equal to or higher than a predetermined temperature (T). It is preferable that it is obtained by combining (claim 3).

  The temperature-sensitive polymer includes a branched polymer having a branched chain, and the branched chain includes at least a polymer block of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof. Preferably, the branched polymer is a compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule as an iniferter, and at least 2-N, N-dimethyl It is preferable that aminoethyl methacrylate and / or its derivative is subjected to light irradiation living polymerization (claim 5).

  This polymer composed of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof has such a property that the charge of the whole molecule is lowered by heating above a predetermined temperature. After complexing the gene introduction agent and the nucleic acid, heating to bring the charge to near zero, and then mixing the nucleic acid and the complete medium containing serum, the nucleic acid complex and anionicity in the serum Can be prevented from complexing with other proteins.

  The branched chain preferably comprises only a polymer block of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof (Claim 6), and 2-N, N-dimethyl contained in the branched polymer. The molecular weight of the polymer block of aminoethyl methacrylate and / or a derivative thereof is preferably 5,000 to 100,000 (claim 7). The molecular weight of the gene introduction agent is preferably 10,000 to 150,000 (claim 8). When the molecular weight of the polymer block of 2-N, N-dimethylaminoethyl methacrylate and / or its derivative and the gene introduction agent is within the above range, the cells can be more efficiently treated while suppressing the high molecular weight of the gene introduction agent. A gene can be introduced into.

  In the gene introduction method of the present invention, the predetermined temperature (T) is preferably 30 to 34 ° C. (Claim 9).

  Moreover, it is preferable that after the gene introduction agent and the nucleic acid are mixed at 0 to 30 ° C., the gene introduction agent and the nucleic acid are combined at 32 to 100 ° C. (Claim 10). If the temperature when mixing the gene introduction agent and the nucleic acid is within the above range, the nucleic acid and the gene introduction agent are easily mixed uniformly in the solution, and the temperature at which the gene introduction agent and the nucleic acid are combined is within the above range. If so, the gene introduction agent and the nucleic acid can be efficiently combined.

  The nucleic acid complex and the cell are preferably contacted at 34 to 45 ° C. (claim 11).

  In the method for preparing a nucleic acid complex of the present invention, a compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule is used as an iniferter, and at least 2-N, N-dimethylaminoethyl methacrylate and Using a gene transfer agent comprising a branched polymer obtained by light-irradiating living polymer of a derivative thereof and / or its derivative, the gene transfer agent and the nucleic acid are mixed at a temperature lower than a predetermined temperature (T), and the solution is set at a predetermined temperature (T). After warming as described above, serum is added to this solution to complex the gene introduction agent and the nucleic acid (claim 12).

  The nucleic acid complex of the present invention (Claim 13) is a nucleic acid complex prepared by the method for preparing a nucleic acid complex of the present invention.

It is a graph which shows the evaluation result of the gene introduction activity of the gene introduction agent of Example 1 and Comparative Example 1.

  Hereinafter, embodiments of the present invention will be described in detail.

[Gene transfer method and method for producing gene transfer agent used in the gene transfer method of the present invention]
The gene introduction method of the present invention is a gene introduction method for introducing a gene into a cell by contacting the cell with a nucleic acid complex obtained by combining a gene introduction agent and a nucleic acid, and the gene introduction agent is a predetermined gene introduction method. It is composed of a thermosensitive polymer whose physical properties change at temperature (T), and is characterized in that the nucleic acid complex and cells are contacted at a temperature equal to or higher than the predetermined temperature (T) in a medium containing serum. It is.

  The gene introduction agent used in the gene introduction method of the present invention is preferably a thermosensitive polymer that exhibits a cationic property at a temperature lower than a predetermined temperature (T) and exhibits a hydrophobic property at a temperature higher than the predetermined temperature (T). The warm polymer preferably includes a branched polymer having a branched chain, and the branched chain includes at least a polymer block of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof.

  As the branched polymer, a compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule is used as an iniferter, and at least 2-N, N-dimethylaminoethyl methacrylate and / or its A polymer obtained by polymerizing a derivative is preferred.

  In this specification, the iniferter is a polymerization initiator that generates radicals upon irradiation with light, a function as a chain transfer agent, a function that bonds with the growth terminal to stop the growth, and a polymerization when light irradiation stops. It is a molecule that functions as a polymerization initiator / termination agent for stopping.

  As a compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule as an iniferter, three or more N, N-dialkyldithiocarbamylmethyl groups are bonded to the benzene ring as a branched chain. It is preferable that the following is exemplified. That is, the tri-branched compound is obtained by addition reaction of 1,3,5-tri (bromomethyl) benzene and sodium N, N-dialkyldithiocarbamate (sodium N, N-dialkyldithiocarbamate) in ethanol. 1,3,5-tri (N, N-dialkyldithiocarbamylmethyl) benzene, and four-branched compounds include 1,2,4,5-tetrakis (bromomethyl) benzene and N, N-dialkyldithiocarbamic acid 1,2,4,5-tetrakis (N, N-dialkyldithiocarbamylmethyl) benzene obtained by addition reaction of sodium (sodium N, N-dialkyldithiocarbamate) in ethanol, 6-branched compound As hexakis (bromomethyl) benzene and N, N-dialkyldithio Rubamin sodium (sodium N, N-dialkyldithiocarbamate) and the hexakis obtained by addition reaction in ethanol (N, N-dialkyldithiocarbamate carbamylmethyl) include benzene. Here, the alkyl group of the dialkyl moiety contained in the N, N-dialkyldithiocarbamylmethyl group is preferably an alkyl group having 2 to 18 carbon atoms such as an ethyl group, but an aromatic group such as a phenyl group. It may be a hydrocarbon group. That is, not only N, N-dialkyldithiocarbamylmethyl group but also N, N substituted with aliphatic hydrocarbon group and / or aromatic hydrocarbon group including N, N-diaryldithiocarbamylmethyl group and the like. A di-substituted dithiocarbamylmethyl group can achieve the object.

  In the following, the case of performing light irradiation living polymerization using an iniferter having the above-mentioned branched chain will be described as an example, and the gene introduction agent used in the present invention will be described. The agent is not limited to such a thing.

  The above iniferter is almost insoluble in polar solvents such as alcohol, but is easily soluble in nonpolar solvents. As this nonpolar solvent, hydrocarbons and halogenated hydrocarbons are suitable, and in particular, benzene, toluene, chloroform or methylene chloride, particularly toluene is preferred.

  The gene introduction agent used in the present invention can be obtained by polymerizing at least 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof to this iniferter.

  For the iniferter, at least 2-N, N-dimethylaminoethyl methacrylate and / or its derivative may be polymerized, but monomers other than 2-N, N-dimethylaminoethyl methacrylate and its derivative are polymerized. May be. Examples of monomers other than 2-N, N-dimethylaminoethyl methacrylate and derivatives thereof include vinyl monomers such as acrylic acid derivatives and styrene derivatives. Monomers other than the 2-N, N-dimethylaminoethyl methacrylate and derivatives thereof may be used alone or in combination of two or more.

  However, in order to simplify the synthesis and reduce the cost using only one kind of monomer, 2-N, N-dimethylaminoethyl methacrylate and / or the above-mentioned iniferter as a gene introduction agent used in the present invention. Or what superposed | polymerized only the derivative (s) is preferable.

  Iniferter and 2-N, N-dimethylaminoethyl methacrylate and 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof (hereinafter sometimes simply referred to as “monomer”) are reacted with each other. And / or preparing a raw material solution containing a derivative thereof and irradiating it with light to obtain a reaction product in which a monomer is bound to an iniferter. As the solvent of this solution, alkanes, alkenes, aromatics, and halogenated hydrocarbons are preferable, and specific examples include benzene, toluene, chloroform, carbon tetrachloride, and methylene chloride. Among these, toluene or chloroform is preferable. is there.

  The concentration of 2-N, N-dimethylaminoethyl methacrylate and / or its derivative in the raw material solution is preferably 0.1 M or more, for example, 0.1 to 5.0 M. The concentration of the iniferter is preferably about 0.1 to 100 mM.

The wavelength of the irradiated light is preferably 200 to 400 nm. For example, a short arc xenon lamp, a low pressure mercury lamp, a high pressure mercury lamp, or the like can be used. The irradiation time of the light depends on the irradiation intensity, about 10 to 360 minutes are preferred, about 10 minutes to 180 minutes 1μW ^/ cm ² ~10mW ^/ cm 2 as low irradiation intensity is particularly preferred.

  In addition, you may perform a 2nd light irradiation process further after this light irradiation process (1st light irradiation process). That is, the solution containing the reaction product is diluted with an alcohol, preferably an alcohol solution of the above monomer. As the alcohol, methanol or ethanol, particularly methanol is preferable. The monomer concentration in the alcohol solution is preferably about 100 mM to 5 M as the final concentration.

  It is preferable to add 5 to 500 parts by volume of the alcohol solution to 1 part by volume of the reaction product-containing liquid from the first light irradiation step.

  The diluted solution thus diluted with the alcohol solution is subjected to the second light irradiation step, and the monomer is further polymerized with respect to the reaction product. In this case, the irradiation light source may be any light source having a wavelength of 240 to 400 nm. For example, a short arc xenon lamp, a low-pressure mercury lamp, a high-pressure mercury lamp, or the like can be used. The light irradiation time is preferably about 10 minutes to 120 minutes.

  Since the target branched polymer is produced in the reaction solution by such light irradiation, it is purified as necessary to obtain a gene introduction agent.

  The molecular weight of the polymer block composed of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof contained in the obtained gene introduction agent (branched polymer) depends on the number of branched chains, but is 5,000. ˜100,000, in particular, about 8,000 to 50,000 is preferable, and about 5 to 150 polymer blocks comprising 2-N, N-dimethylaminoethyl methacrylate and / or derivatives thereof in the branched chain. It preferably consists of monomer units. If the molecular weight of the polymer block composed of 2-N, N-dimethylaminoethyl methacrylate and / or its derivative is too small, sufficient cohesion of nucleic acids cannot be obtained, and if it is too large, the nucleic acid complex has a high molecular weight. It is disadvantageous.

  In addition, in this specification, molecular weight refers to the number average molecular weight of polyethylene glycol conversion by GPC (gel permeation chromatography).

  The molecular weight of the gene introduction agent is preferably about 5,000 to 150,000, particularly about 5,000 to 100,000, and particularly about 8,000 to 50,000, although it depends on the number of branched chains. If the molecular weight of the gene introduction agent is too small, the polymers are less likely to aggregate in a hydrophobic bond, making it difficult to form a nucleic acid complex. On the other hand, if the molecular weight is too large, it is disadvantageous in terms of degradation, metabolism, excretion, etc., unsuitable for in vivo use, difficult to put into practical use as a pharmaceutical product, and cytotoxicity also appears.

  The gene introduction agent used in the present invention exhibits a high zeta potential of about +15 mV to +40 mV when complexed with a nucleic acid even when the molecular weight is a low molecular weight within the above range.

  In the gene introduction method of the present invention, the predetermined temperature (T) is preferably slightly lower than the body temperature of the human body, for example, about 30 to 34 ° C.

  In the present invention, a solution containing the gene introduction agent and the nucleic acid is mixed at a temperature lower than a predetermined temperature (T), and then the solution is heated to a temperature equal to or higher than the predetermined temperature (T) to obtain the gene introduction agent and the nucleic acid. In particular, the gene introduction agent and the nucleic acid are mixed at about 0 to 30 ° C., particularly about 15 to 25 ° C., and then the solution is heated to mix the gene introduction agent and the nucleic acid. It is preferable to combine at about 32 to 100 ° C, particularly 35 to 50 ° C. For example, a gene introduction agent having a 2-N, N-dimethylaminoethyl methacrylate polymer moiety on a branched chain and a nucleic acid are mixed within the above temperature range, and then heated and combined to form 2 in the branched chain. The —N, N-dimethylaminoethyl methacrylate polymer portion becomes globulized and hydrophobized, so that nucleic acids can be efficiently aggregated.

  Furthermore, the temperature at which the nucleic acid complex is brought into contact with the cells is preferably about the same as or higher than the body temperature of the human body, for example, 34 to 50 ° C, particularly 35 to 40 ° C. If the temperature at which the nucleic acid complex is brought into contact with the cell is within the above range, the gene can be efficiently introduced into the cell.

  The medium containing serum used in the present invention is preferably a liquid medium containing serum, and the concentration of serum contained in such a liquid medium is about 0.1 to 60% by weight, particularly About 5 to 15% by weight is preferable. If the serum concentration of the medium is too low, the effect of containing the serum cannot be obtained sufficiently, and if the concentration is too high, the balance of the optimally designed medium for the cultured cells may be lost, Various components of serum are not preferable because they may have some influence on cultured cells.

  As the serum, fetal bovine serum (FCS) is preferable, but human or animal adult serum or plasma can also be used. And can be used after removing the globulin component. In addition, when the nucleic acid complex of the present invention is applied to a living body, plasma of the same species is preferable.

[Method for preparing nucleic acid complex and nucleic acid complex]
The method for preparing a nucleic acid complex of the present invention comprises a mixing step of mixing the gene introduction agent and the nucleic acid when the temperature of the solution containing the gene introduction agent synthesized as described above and the nucleic acid is less than a predetermined temperature (T). Then, after the mixing step, the solution is heated to a predetermined temperature (T) or higher and then a serum is added to the solution to complex the gene introduction agent and the nucleic acid.

  The predetermined temperature (T) is preferably between 30 to 34 ° C. as in the case of the gene introduction method described above.

  The temperature of the solution in the mixing step (hereinafter referred to as “temperature condition 1”) is preferably slightly lower than the predetermined temperature (T), for example, about 0 to 30 ° C., particularly about 15 to 25 ° C. preferable. If the temperature condition 1 is too low, the nucleic acid and the gene introduction agent are difficult to mix uniformly in the solution. If the temperature condition 1 is too high, the gene introduction agent and the nucleic acid introduction agent are mixed sufficiently uniformly before the temperature condition 1 is too high. Aggregate and cannot form a nucleic acid complex including nucleic acids.

  Further, the temperature of the solution in the composite step (hereinafter referred to as “temperature condition 2”) is preferably slightly higher than the predetermined temperature (T), for example, about 32 to 100 ° C., particularly 35 to 50 ° C. The degree is preferred. If the temperature condition 2 is too low, it is difficult to efficiently aggregate the nucleic acid in the gene introduction agent, and if the temperature condition 2 is too high, there is a possibility that a problem due to the high temperature treatment may occur.

  In order to combine the gene introduction agent (vector) and the nucleic acid, the nucleic acid is added and mixed under a temperature condition 1 with respect to a dispersion having a concentration of about 1 to 1000 μg / mL. In this mixing step, it is preferable to add an excessive amount of the vector to the nucleic acid so that the vector is saturated with respect to the nucleic acid.

  The time for mixing the solution containing the nucleic acid and the gene introduction agent in this mixing step is preferably about 3 to 30 minutes. If the mixing time is too short, the solution will not be uniform, making it difficult for the nucleic acid and the gene introduction agent to associate with each other, and if the mixing time is too long, no further effect will be obtained. , The mixing time is long and not efficient.

  Preferred examples of the nucleic acid include herpes simplex virus thymidine kinase gene (HSV1-TK gene), p53 tumor suppressor gene, BRCA1 tumor suppressor gene and cytokine gene such as TNF-α gene, IL-2 gene, IL-4 gene, HLA- Cytokine genes such as B7 / IL-2 gene, HLA-B7 / B2M gene, IL-7 gene, GM-CSF gene, IFN-γ gene and IL-12 gene, and gp-100, MART-1 and MAGE-1 These cancer antigen peptide genes can be used for cancer treatment.

  In addition, cytokine genes such as VEGF gene, HGF gene and FGF gene, c-myc antisense, c-myb antisense, cdc2 kinase antisense, PCNA antisense, E2F decoy and p21 (sdi-1) gene are used for vascular treatment. Available. Further, not only DNA introduction and gene expression as described above, but also RNA interference that destroys intracellular mRNA can be carried out by introduction of siRNA. Such a series of genes is well known to those skilled in the art.

  After the mixing step, the solution containing the nucleic acid and the gene introduction agent is heated to temperature condition 2 to combine the nucleic acid and the gene introduction agent. Since the polymer block comprising 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof is temperature-sensitive and cationic, the solution containing the nucleic acid and the gene introduction agent is thus treated under temperature conditions. By heating to 2, it can be efficiently complexed with the nucleic acid. That is, since the polymer block composed of 2-N, N-dimethylaminoethyl methacrylate and / or its derivative is weakly cationic, the cohesive force is weak and it is not completely complexed with DNA under neutral conditions. Since they are associated with each other at a distance, DNA can be combined with the gene introduction agent by heating and increasing the hydrophobicity of the polymer block to cause aggregation.

  The time required for this composite step is preferably about 3 to 30 minutes after heating to temperature condition 2. If the time for this conjugation step is too short, the nucleic acid and the gene introduction agent do not form a complex, and if this time is too long, secondary particles of the complex are formed, which is disadvantageous for incorporation into cells. There is a possibility of growing to a large particle size.

  The particle size of the nucleic acid-containing complex is preferably about 50 to 400 nm. If it is smaller than this, there is a possibility that the action of the enzyme may reach the nucleic acid inside the nucleic acid-containing complex, or it may be filtered out by the kidney. Moreover, when larger than this, there exists a possibility that it may become difficult to introduce | transduce into a cell.

  The nucleic acid is used in such a form that when introduced into a cell, the function can be expressed in the cell. For example, in the case of DNA, it is used as a plasmid in which the DNA is placed so that the DNA is transcribed in the introduced cell and functionally expressed through production of the polypeptide encoded thereby. Preferably, a promoter region, a start codon, DNA encoding a protein having a desired function, a stop codon, and a terminator region are sequentially arranged.

  If desired, two or more nucleic acids can be included in one plasmid.

  In the present invention, “cells” that are desirable as a target for introduction of nucleic acids are those in which functional expression of the nucleic acids is desired. Examples thereof include cardiomyocytes, smooth muscle cells, fibroblasts, skeletal muscle cells, vascular endothelial cells, bone marrow cells, bone cells, blood cell stem cells, blood cell cells and the like. In addition, monocytes, dendritic cells, macrophages, histocytes, Kupffer cells, osteoclasts, synovial A cells, microglia, Langerhans cells, epithelioid cells, multinucleated giant cells, etc., gastrointestinal epithelial cells / tubule epithelium Such as cells.

  The nucleic acid-containing complex using the vector of the present invention can be administered to a living body by any method.

  As the administration method, intravenous or intraarterial injection is particularly preferable, but intramuscular, adipose tissue, subcutaneous, intradermal, intralymphatic, intralymphatic, body cavity (pericardial cavity, thoracic cavity, abdominal cavity, cerebrospinal cavity) Etc.) In addition to administration into the bone marrow, administration into the diseased tissue is also possible.

  The pharmaceutical comprising this nucleic acid-containing complex as an active ingredient is further a pharmaceutically acceptable carrier (osmotic pressure regulator, stabilizer, preservative, solubilizer, pH adjuster, thickener, etc.) as necessary. It is possible to mix with. Any known carrier can be used.

  Moreover, the medicine containing this nucleic acid-containing complex as an active ingredient also includes those containing two or more nucleic acid-containing complexes with different types of nucleic acids. Such a medicine having a plurality of therapeutic purposes is particularly useful in the field of diversifying gene therapy.

  As for the dosage, the dosage administered to animals, particularly humans, varies depending on various factors such as the target nucleic acid, the method of administration, and the specific site to be treated. However, the dosage should be sufficient to produce a therapeutic response.

  This nucleic acid-containing complex is preferably applied to gene therapy. Applicable diseases vary depending on the type of nucleic acid included in the complex, but in addition to diseases in the cardiovascular region that cause lesions such as peripheral arterial disease, coronary artery disease, restenosis after arterial dilation, cancer (malignant Melanoma, brain tumor, metastatic malignant tumor, breast cancer, etc.), infectious diseases (HIV, etc.), single genetic diseases (cystic fibrosis, chronic granulomas, α1-antitrypsin deficiency, Gaucher disease, etc.) .

  Further, an aqueous solution of the gene introduction agent combined with the nucleic acid is attached to a substrate by coating or the like, and dried as necessary, thereby forming a polymer coating or the like carrying the nucleic acid.

  When the nucleic acid complex gene introduction agent is attached to a substrate, the substrate is preferably a sheet. The thickness of the sheet-like substrate is preferably about 0.05 to 10 mm, and the size of the sheet surface is 1 to 20 mm on one side and 1 to 20 mm on the other side in the case of a square, In the case of an ellipse, the diameter is preferably about 1 to 20 mm. As a material for the base material, a synthetic resin such as polyolefin, polystyrene, polyester, polyamide, polyurethane, silicone resin, or fluororesin is suitable. This substrate may be porous.

The adhesion amount of the nucleic acid complex gene introduction agent to the substrate is preferably about 0.001 to 10 mg per 1 cm ^{2 of the} substrate surface.

  A gene transfer material comprising a substrate carrying a nucleic acid complex gene transfer agent is placed on a sheet-like substrate so as to surround a subcutaneous tissue, a myocardial tissue, a diseased tissue, or a diseased blood vessel, or is applied to a film of a covered stent. Therefore, it is used in such a manner that it is placed in the living body or is attached to the outer surface of the living body using an adhesive tape.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples unless it exceeds the gist.

i) Synthesis of Iniferter As an iniferter, 1,2,4,5-tetrakis (N, N-diethyldithiocarbamylmethyl) benzene was synthesized as follows.

  5.0 g of 1,2,4,5-tetrakis (bromomethylbenzene) and 34.0 g of sodium N, N-diethyldithiocarbamate were added to 100 mL of ethanol, and the mixture was stirred at room temperature for 4 days in the dark. The precipitate was filtered, poured into 3 L of methanol, stirred for 30 minutes and filtered. This operation was repeated a total of 4 times. After the precipitate was dissolved in 200 mL of toluene, 100 mL of methanol was added and heated to 50 ° C., stored in a refrigerator for 15 hours for recrystallization, and the crystals were separated by filtration and washed with a large amount of methanol. The crystals were dried at room temperature under reduced pressure to obtain white needle-like crystals of 1,2,4,5-tetrakis (N, N-diethyldithiocarbamylmethyl) benzene (yield 90%). High-performance liquid chromatography confirmed that the raw material peak disappeared and the purified product was a single substance.

The measurement result of ¹ H-NMR (in CDCl ₃ ) is as follows: δ 1.26-1.31 ppm (t, 24H, CH ₂ CH ₃ ), δ 3.69-3.77 ppm (q, 8H, N (CH ₂ CH _{3 2} ), δ 3.99-4.07 ppm (q, 8H, N (CH ₂ CH ₃ ) ₂ ), δ 4.57 ppm (s, 8H, Ar—CH ₂ ), δ 7.49 ppm (s, 2H, Ar— H).

ii) Synthesis of a thermosensitive cationic homopolymer comprising a 4-branched star polymer by photopolymerization 1,2,4,5 using 2-N, N-dimethylaminoethyl methacrylate as a thermosensitive cationic monomer A thermosensitive cationic homopolymer comprising tetrakis [(N, N-diethyldithiocarbamyl) poly (2-N, N-dimethylaminoethyl methacrylate) -methyl] benzene (hereinafter sometimes referred to as pDMAEMAA). Polymer synthesis was performed.

Namely, 45.6 mg of 1,2,4,5-tetrakis (N, N-diethyldithiocarbamylmethyl) benzene synthesized by i) above was dissolved in 20 mL of toluene, and 2-N, N-dimethylaminoethyl methacrylate was dissolved. 8.5 g was added and mixed, and the total amount was adjusted to 50 mL with toluene. After purging with high-purity nitrogen gas for 10 minutes with vigorous stirring in a 3 mm thick soft glass cell, mixed ultraviolet rays of 250 nm to 400 nm were irradiated for 60 minutes with a 300 W short arc xenon lamp (manufactured by Asahi Spectroscopic Co., Ltd., MAX-301). The irradiation intensity was adjusted to 2.5 mW / cm ² by attaching UVD-C405 (detection wavelength range of 320 nm to 470 nm) to UIT-150 manufactured by USHIO. The polymerization solution is concentrated with an evaporator, the polymer is reprecipitated with n-hexane, purified by repeating reprecipitation three times with a chloroform / n-hexane system, and n-hexane is evaporated and dissolved in a small amount of benzene. The solution was filtered through a 0.2 μm filter and then freeze-dried to obtain a temperature-sensitive cationic homopolymer composed of the target 4-branched star polymer (pDMAEMAA). The number average molecular weight of polyethylene glycol of this thermosensitive cationic homopolymer as a standard substance was measured by GPC as 11,000 (Mw / Mn = 1.4), and 2-N, introduced by this reaction It can be seen that the molecular weight of the N-dimethylaminoethyl methacrylate polymer block is about 10,000 from the difference between the molecular weight of the gene introduction agent and the molecular weight of the iniferter (723). This temperature-sensitive cationic homopolymer is used as the gene introduction agent of Example 1.

The measurement result of ¹ H-NMR (in CD ₃ OD) is δ0.8-1.2 ppm (br, 3H, —CH ₂ —CH ₃ —), δ 1.6-2.0 ppm (br, 2H, —CH). _{2 -CH 3 -), δ2.2-2.4ppm (} br, 6H, N-CH 3), δ2.5-2.7ppm (br, 2H, CH 2 -N), δ4.0-4.2ppm (br, 2H, O-CH 2) was.

iii) Synthesis of a non-thermosensitive cationic homopolymer comprising a 4-branched star polymer by photopolymerization 1,2,4,5 using 3-N, N-dimethylaminopropylacrylamide as a cationic vinyl monomer Non-thermosensitive cationic property comprising tetrakis [(N, N-diethyldithiocarbamyl) poly (3-N, N-dimethylaminopropylacrylamide) -methyl] benzene (hereinafter sometimes referred to as pDMAPAAm). A homopolymer was synthesized.

That is, 45.6 mg of 1,2,4,5-tetrakis (N, N-diethyldithiocarbamylmethyl) benzene synthesized by i) above was dissolved in 20 mL of toluene, and 3-N, N-dimethylaminopropylacrylamide was dissolved. 3.4 g of (3-N, N-DMAPAAm) was added and mixed, and the total amount was adjusted to 50 mL with toluene. After purging with high-purity nitrogen gas for 5 minutes while vigorously stirring in a 3 mm thick soft glass cell, a mixed ultraviolet ray of 250 nm to 400 nm was irradiated for 15 minutes with a 300 W short arc xenon lamp (manufactured by Asahi Spectroscopic Co., Ltd., MAX-301). The irradiation intensity was adjusted to 2.5 mW / cm ² by attaching UVD-C405 (detection wavelength range of 320 nm to 470 nm) to UIT-150 manufactured by USHIO. The polymerization solution is concentrated with an evaporator, the polymer is reprecipitated with diethyl ether, purified by repeating reprecipitation three times with a chloroform / diethyl ether system, ether is evaporated, dissolved in a small amount of water, 0.2 μm After filtration through a filter, the product was freeze-dried to obtain a non-thermosensitive cationic homopolymer composed of the target 4-branched star polymer (pDMAPAAm) (polymerization rate: 32%). The number average molecular weight of this non-thermosensitive cationic homopolymer was measured by GPC as 13,000 (Mw / Mn = 1.3). This non-thermosensitive cationic homopolymer is used as the gene introduction agent of Comparative Example 1.

The measurement result of ¹ H NMR (in D ₂ O) is as follows: δ1.5-1.8 ppm (br, 2H, —CH ₂ CH ₂ CH ₂ —), δ 2.1-2.2 ppm (br, 6H, N— _{CH 3), δ2.2-2.4ppm (br,} 2H, CH 2 -N), δ3.0-3.4ppm (br, 2H, NH-CH 2), δ7.4-7.8ppm (br, 1H, -NH-).

iv) Measurement of cloud point of gene introduction agent of Example 1 and Comparative Example 1 Temperature-sensitive cationic homopolymer which is a gene introduction agent of Example 1, and non-thermosensitive cation which is a gene introduction agent of Comparative Example 1 An aqueous solution of a hydrophilic homopolymer was prepared, and the temperature at which the solution became cloudy (cloud point) was measured by raising the temperature of this aqueous solution.

  That is, 3% by weight aqueous solutions of each gene transfer agent were prepared, and the absorbance of light having a wavelength of 660 nm in the temperature range of 20 ° C. to 40 ° C. of these aqueous solutions was measured. From this measurement result, the thermosensitive cationic homopolymer which is the gene introduction agent of Example 1 has LCST (Lower Critical Solution Temperature) around 32 ° C., and is hydrophobic and uncharged in a cell culture environment. The polymer was found to be a thermosensitive polymer that suspends in water. On the other hand, the non-thermosensitive cationic homopolymer, which is the gene introduction agent of Comparative Example 1, showed 100% transmittance without turbidity in the range of 20 ° C to 40 ° C. It turns out that it doesn't change.

v) Evaluation of gene transfer activity Gene transfer activity was evaluated according to the following procedures (1) to (3).
(1) Preparation of polymer solution of gene introduction agent and preparation of DNA solution COS-1 derived from African green monkey kidney cells was used for cells, and pGL3 control vector was used for DNA. The number of COS-1 cells was adjusted to 40,000 cells / mL, seeded in a 24 Well culture dish, and gene transfer was performed after 24 hours of culture. The temperature-sensitive cationic homopolymer that is the gene introduction agent of Example 1 prepared in the above ii) and the non-temperature-sensitive cationic homopolymer that is the gene introduction agent of Comparative Example 1 prepared in the above iii) Each was dissolved in physiological saline to obtain a polymer solution having a concentration (polymer / saline) of 8 μg / 60 μL. DNA was dissolved in TE buffer, and the concentration (DNA / buffer) was adjusted to 3 μg / 90 μL.

(2) Evaluation of gene introduction activity in medium containing serum The gene introduction activity of the gene introduction agents of Example 1 and Comparative Example 1 in a medium containing serum was evaluated.

  Each polymer solution prepared in (1) above and the DNA solution were mixed at room temperature (25 ° C.), and then the solution was heated to 37 ° C. to form a nucleic acid complex. 25 μL of this nucleic acid complex was added to complete medium (DMEM + 10% FCS + antibiotics) containing 1 mL of serum and incubated for 30 minutes. About 1 mL of the nucleic acid complex complete medium solution was brought into contact with the cultured cells washed with PBS at 37 ° C., and cultured for 48 hours. After 48 hours, gene transfer activity was evaluated by luciferase assay (Promega, assay kit reagent). Correction was performed at protein concentration, and protein quantification was performed with Bradford reagent from BioRad.

(3) Evaluation of gene transfer activity in medium not containing serum The gene transfer activity of the gene transfer agents of Example 1 and Comparative Example 1 in a medium not containing serum was evaluated.

  Each polymer solution prepared in (1) above and the DNA solution were mixed at room temperature (25 ° C.), and then the solution was heated to 37 ° C. to form a nucleic acid complex. 25 μL of this nucleic acid complex was added to 200 μL of serum-free medium (OPTI-MEM) and incubated for 30 minutes. About 200 μL of the OPTI-MEM solution of this nucleic acid complex was added to cultured cells washed with PBS at 37 ° C., and after transfection for 3 hours, 1 mL of complete medium was added, followed by further incubation for 45 hours. After 45 hours, gene transfer activity was evaluated by luciferase assay (Promega, assay kit reagent). Correction was performed at protein concentration, and protein quantification was performed with Bradford reagent from BioRad.

  The evaluation results of (2) and (3) are shown in FIG.

[Discussion]
The non-thermosensitive cationic homopolymer that is the gene introduction agent of Comparative Example 1 is excellent in gene introduction activity in a medium not containing serum, but the gene introduction activity was significantly reduced in a medium containing serum. On the other hand, the temperature-sensitive cationic homopolymer that is the gene introduction agent of Example 1 had a slightly lower gene introduction activity than the gene introduction agent of Comparative Example 1 in a medium not containing serum. This is because the difference in pKa between 2-N, N-dimethylaminoethyl methacrylate (pKa = 7) used as a monomer and 3-N, N-dimethylaminopropylacrylamide (pKa = 10) is the difference in gene transfer activity. It is thought that appeared. The low molecular weight of 2-N, N-dimethylaminoethyl methacrylate (pDMAEMAA), which is a thermosensitive cationic monomer, has a low DNA aggregation ability at room temperature (about 25 ° C.), and becomes warm when heated. If the hydrophobic aggregation effect is not added, DNA cannot be sufficiently aggregated. That is, it can be seen that in a medium that does not contain serum, the gene transfer agent having a high pKa cationic polymer has a higher DNA aggregating ability.

  On the other hand, in the medium containing serum, the gene introduction agent of Example 1 shows much superior gene introduction activity. This is because the charge of the nucleic acid complex between the gene introduction agent and the nucleic acid of Example 1 is decreased by heating the solution, and the anionic protein contained in the serum becomes difficult to aggregate, or the nucleic acid complex is hydrophobic. This is thought to be due to the action of the cells in a state where the affinity with the cells was increased due to the adsorption of proteins and lipids having both affinity in the serum. On the other hand, since the gene introduction agent of Comparative Example 1 does not become hydrophobic even when heated, the nucleic acid complex aggregates anionic proteins in the serum and the nucleic acid is less likely to be taken into the cells. Therefore, the gene transfer activity is considered to have decreased.

  The gene introduction agent of Example 1 shows similar activity in each of a medium containing serum and a medium not containing serum. That is, the gene introduction agent of the present invention can efficiently introduce a gene even in a medium that does not give stress to cells by containing serum.

Claims (13)

A gene introduction method for introducing a gene into a cell by contacting the cell with a nucleic acid complex obtained by combining a gene introduction agent and a nucleic acid,
The gene introduction agent is a thermosensitive polymer whose physical properties change at a predetermined temperature (T),
A gene introduction method comprising contacting the nucleic acid complex and cells at a temperature equal to or higher than the predetermined temperature (T) in a medium containing serum.   2. The gene introduction method according to claim 1, wherein the temperature-sensitive polymer exhibits a cationic property at a temperature lower than a predetermined temperature (T) and exhibits a hydrophobic property at a temperature higher than the predetermined temperature (T). .   3. The nucleic acid complex according to claim 1, wherein the nucleic acid complex is prepared by mixing a solution containing a gene introduction agent and a nucleic acid at a temperature lower than a predetermined temperature (T), and then heating the solution to a temperature equal to or higher than a predetermined temperature (T). A gene transfer method characterized by being obtained by combining a gene transfer agent and a nucleic acid.   The temperature-sensitive polymer according to any one of claims 1 to 3, wherein the thermosensitive polymer comprises a branched polymer having a branched chain, and the branched chain includes at least 2-N, N-dimethylaminoethyl methacrylate and / or Alternatively, a gene introduction method comprising a polymer block of a derivative thereof.   5. The branched polymer according to claim 4, wherein the branched polymer comprises a compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule as an iniferter, and at least 2-N, N-dimethylaminoethyl methacrylate. And / or a derivative polymerization method of light-induced living polymerization.   6. The gene introduction method according to claim 4 or 5, wherein the branched chain comprises only a polymer block of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof.   The molecular weight of the polymer block of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof contained in the branched polymer according to any one of claims 4 to 6 is 5,000 to 100,000. A gene introduction method characterized by:   The gene introduction method according to any one of claims 1 to 7, wherein the gene introduction agent has a molecular weight of 10,000 to 150,000.   The gene introduction method according to any one of claims 1 to 8, wherein the predetermined temperature (T) is 30 to 34 ° C.   In any 1 item | term of the Claims 3 thru | or 9, after mixing the solution containing the said gene introduction agent and a nucleic acid at 0-30 degreeC, this solution is heated at 32-100 degreeC, A gene introduction method comprising combining with a nucleic acid.   The gene introduction method according to any one of claims 1 to 10, wherein the nucleic acid complex and cells are contacted at 34 to 45 ° C. A method for preparing a nucleic acid complex in which a nucleic acid and a gene introduction agent are combined by mixing a solution containing the gene introduction agent and the nucleic acid,
The gene introduction agent uses a compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule as an iniferter, and contains at least 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof. It consists of a branched polymer that is polymerized by light irradiation living,
A mixing step of mixing the gene introduction agent and the nucleic acid at a temperature of the solution lower than a predetermined temperature (T);
After the mixing step, the solution is heated to a predetermined temperature (T) or higher, then serum is added to the solution to combine the gene introduction agent and the nucleic acid,
A method for preparing a nucleic acid complex having   A nucleic acid complex prepared by the method for preparing a nucleic acid complex according to claim 12.

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