JP2010136632A - Method for preparing nucleic acid complex and nucleic acid complex - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for preparing a nucleic acid complex capable of efficiently complexing a nucleic acid with a low-molecular weight and low pKa compound comprising a polymer of 2-N, N-dimethylaminoethyl methacrylate and/or its derivatives which is used as a gene transfer agent, and a nucleic acid complex which is prepared by the method and has low-molecular weight and excellent gene transfer efficiency. <P>SOLUTION: There is disclosed the method for preparing the nucleic acid complex by complexing the nucleic acid with the gene transfer agent by mixing a solution containing the gene transfer agent with a solution containing a nucleic acid, wherein the gene transfer agent comprises a polymer having a branched chain, and the branched chain comprises at least one polymer block comprising 2-N, N-dimethylaminoethyl methacrylate and/or its derivatives, and a lower alcohol exists in the mixed system. There is also disclosed a nucleic acid complex prepared by the method for preparing the nucleic acid complex. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for preparing a nucleic acid complex and a nucleic acid complex prepared by the method for preparing the nucleic acid 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. As vectors for the introduction of foreign DNA, many viruses, including retroviruses, adenoviruses or herpes viruses, have been modified to carry therapeutic genes and are used in human clinical trials for gene therapy. However, the risk of infection and immune response remains.

  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).

Non-patent document 1 below reports a study on a gene introduction agent comprising a homopolymer of 2-N, N-dimethylaminoethyl methacrylate.
The background of the research reported in this non-patent document 1 is as follows.
That is, regarding the aggregating power of DNA, a cationic polymer having a high molecular weight and a high pKa is stronger. With such a polymer, DNA can be aggregated more compactly to form fine particles of a high-density nucleic acid complex. it can.
On the other hand, excessive aggregation results in poor release efficiency of DNA in cells (so-called overcondensed state), resulting in poor gene expression efficiency.

  Therefore, research using a low pKa cationic polymer has been studied for a long time. By forming nucleic acid complex microparticles with these polymers that are not fully cationized under neutral conditions, there is room for proton binding capacity in acidic endosomes after endocytosis, and the proton sponge effect. It is expected that it can be enhanced. In addition, since the cohesive force is low, it is expected that DNA is easily released in the cell.

2-N, N-dimethylaminoethyl methacrylate is a low pKa cationic monomer having a pKa of about 7, and Non-Patent Document 1 reports a study on a low pKa cationic polymer using the same.
WO2004 / 092388 JP2007-70579 Japanese Patent Application No. 2008-205256 Wetering P., Cherng, JY, Talsma, H., Crommelin, DJ, Hennink, WE, (1998), J Control Release 53, 145-153

  The gene introduction agent in which the polymer chain extends radially from the benzene ring described in Patent Documents 1 and 2 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 polyplex particles was increased and the gene transfer activity was improved, but there were problems listed in (1) and (2) below.

(1) In order to agglomerate the DNA into small pieces, a device for increasing the charge density has been devised. On the other hand, the finely agglomerated fine particles once exist stably without being dissociated (so-called over). Therefore, it becomes difficult to release DNA inside the cell. Unreleased DNA is naturally not transcribed into mRNA, leading to protein expression.
(2) Generally, cell culture and gene introduction are performed under neutral conditions. Therefore, a monomer having a high pKa is selected as a raw material for a cationic polymer having a sufficiently high ionic strength even under neutral conditions. However, a cationic polymer composed of such a high pKa monomer, under neutral conditions, has almost 100% of its side chain nitrogen atoms cationized, and after incorporation into the cell, it is acidic within the endosome. Even so, it lacks capacity to attract protons. For this reason, the above-mentioned proton sponge phenomenon cannot be obtained.

  In the case of the thermosensitive-cationic block copolymer described in Patent Document 3, the introduction of the thermosensitive polymer block portion may reduce the above problems (1) and (2). In the thermosensitive-cationic block copolymer, the cationic polymer block and the thermosensitive polymer block are essential, and there is a portion that is not involved in the inclusion of nucleic acid, and the efficiency is poor. In addition, it is necessary to perform polymerization in two stages using two types of monomers, which makes the synthesis complicated and expensive. Further, in the nucleic acid complex, the culture substrate is also an essential component because it is insolubilized by the strong cohesive force of the polymer block of N-isopropylacrylamide that is not compatible with nucleic acid and deposited on the substrate surface. That is, it cannot be applied to the usage as an intangible drug, and there is a problem that it cannot be administered by injecting into the blood vessel, subcutaneously or abdominal cavity, or ingested.

  The homopolymer of 2-N, N-dimethylaminoethyl methacrylate reported in Non-Patent Document 1 is a category of cationic polymer, but its pKa is about 7 and is actually a cell culture environment. It has almost no charge under sexual conditions. For this reason, the cohesive strength of nucleic acid is low, and it does not function as a gene introduction agent unless it is an ultra high molecular weight body of 300,000 or more. However, high molecular weight substances are disadvantageous in terms of degradation, metabolism, excretion, etc., are unsuitable for in vivo use, and are difficult to put into practical use as pharmaceuticals. Cytotoxicity also appears strongly in high molecular weight products.

  The present invention solves the above-mentioned conventional problems, uses a low molecular weight substance composed of a low pKa 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof as a gene introduction agent, and efficiently uses a nucleic acid for this. It is an object of the present invention to provide a method for preparing a nucleic acid complex to be complexed and a nucleic acid complex prepared by this method and having a low molecular weight and excellent gene transfer efficiency.

  The method for preparing a nucleic acid complex of the present invention (Claim 1) is a method for preparing a nucleic acid complex in which a nucleic acid is complexed with a gene introduction agent by mixing the gene introduction agent and the nucleic acid in a solution. The introduction agent comprises a polymer material having a branched chain, and the branched chain contains at least a polymer block of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof, It is characterized by the presence of a lower alcohol.

  The method for preparing a nucleic acid complex according to claim 2 is characterized in that, in claim 1, the lower alcohol is an alcohol having 1 to 5 carbon atoms.

  The method for preparing a nucleic acid complex of claim 3 is characterized in that, in claim 2, the lower alcohol is ethanol.

  The method for preparing a nucleic acid complex according to claim 4 is the method according to any one of claims 1 to 3, wherein the amount of the lower alcohol present in the mixed system is based on the amount of the gene introduction agent in the mixed system. 300 to 300,000% by weight.

  The method for preparing a nucleic acid complex according to claim 5 is the compound according to any one of claims 1 to 4, wherein the polymer material has three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule. Is a branched polymer obtained by light-irradiating living polymerized with at least 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof.

  The method for preparing a nucleic acid complex according to claim 6 is the method according to claim 5, wherein the compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule has a benzene ring as a nucleus and branches to this nucleus. It is characterized in that three or more N, N-dialkyl-dithiocarbamylmethyl groups are bonded as a chain.

  The method for preparing a nucleic acid complex according to claim 7 is the method according to any one of claims 1 to 6, wherein the branched chain consists only of a polymer block of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof. It is characterized by this.

  The method for preparing a nucleic acid complex according to claim 8 is the method according to any one of claims 1 to 7, wherein the molecular weight of the polymer block of the 2-N, N-dimethylaminoethyl methacrylate and / or derivative thereof is from 5,000 to It is characterized by being 100,000.

  The method for preparing a nucleic acid complex according to claim 9 is characterized in that, in any one of claims 1 to 8, the gene introduction agent has a molecular weight of 10,000 to 150,000.

  The nucleic acid complex of the present invention (invention 10) is prepared by the method for preparing a nucleic acid complex according to any one of claims 1 to 9.

  According to the method for preparing a nucleic acid complex of the present invention (Claim 1), a polymer material containing a polymer block of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof in a branched chain is used as a gene introduction agent. Furthermore, since the lower alcohol exists in the mixed system containing the gene introduction agent and the nucleic acid, the nucleic acid can be efficiently combined with the gene introduction agent.

  This is because, when a solution of a gene introduction agent having a polymer comprising 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof, which is a low pKa polymer, in a branched chain is mixed with a lower alcohol, the polymer main chain is mixed. And the dimethylamino group of the side chain dissociate, or the association between the polymer molecules dissociates, and as a result, the polymer has sufficient basicity to include the nucleic acid. It is guessed that this is due to

  As described above, in the present invention, a polymer comprising 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof used as a gene introduction agent is improved in basicity in the presence of a lower alcohol and includes a nucleic acid. However, the inherently low pKa and the higher pKa polymer have less nucleic acid inclusion force, so that the nucleic acid release is superior to Patent Documents 1 and 2.

  Moreover, since it is more efficient than that of Patent Document 3 in which a temperature-sensitive polymer block is separately introduced in addition to the basic polymer block, and can be synthesized by one polymerization reaction using one kind of monomer, Easy to synthesize and low cost.

  Moreover, when a gene introduction agent using 2-N, N-dimethylaminoethyl methacrylate and / or its derivative and a nucleic acid are combined in the presence of a lower alcohol, a low molecular weight of about 10,000 to 30,000 is sufficient. Since it becomes a nucleic acid complex having a high ζ-potential, there is no problem such as the cytotoxicity of the ultrahigh molecular weight substance reported in Non-Patent Document 1, and it is excellent in adaptability for in vivo use.

  The lower alcohol used in the present invention is preferably an alcohol having 1 to 5 carbon atoms (Claim 2), and particularly preferably ethanol (Claim 3).

  Further, the amount of the lower alcohol present in the mixed system is preferably 300 to 300,000% by weight with respect to the amount of the gene introduction agent in the mixed system (claim 4). When the amount of the lower alcohol is within the above range, the polymer block composed of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof has sufficient basicity to complex the nucleic acid.

  In the present invention, the polymer material uses an iniferter as a compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule, and includes at least 2-N, N-dimethylaminoethyl methacrylate and / or A branched polymer obtained by photoirradiation living polymerization of the derivative is preferable (Claim 5), and the compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule is a benzene ring. It is preferable that three or more N, N-dialkyl-dithiocarbamylmethyl groups are bonded as a branched chain to the nucleus.

  The branched chain preferably comprises only a polymer block of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof (Claim 7).

  In the present invention, the molecular weight of the polymer block of the 2-N, N-dimethylaminoethyl methacrylate and / or derivative thereof is preferably 5,000 to 100,000 (Claim 8), and the molecular weight of the gene introduction agent is It is preferable that it is 10,000-150,000 (Claim 9).

  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 gene introduction agent and the nucleic acid can be more efficiently suppressed while suppressing high molecular weight. Can be combined to obtain a nucleic acid complex having a low molecular weight and excellent gene transfer efficiency.

  The nucleic acid complex of the present invention (Claim 10) is prepared by the above-described method for preparing a nucleic acid complex, and is capable of reducing cytotoxicity by lowering the molecular weight, resulting in gene transfer efficiency suitable for biological use. It is excellent.

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

  The method for preparing a nucleic acid complex of the present invention is a method for preparing a nucleic acid complex in which a nucleic acid is mixed with a gene introduction agent by mixing the gene introduction agent and the nucleic acid in a solution, and the gene introduction agent is branched. A polymer material having a chain, wherein the branched chain contains a polymer block of at least 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof, and a lower alcohol is present in the mixed system. It is characterized by this.

  As the polymer material, 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 a derivative thereof is added thereto. What consists of the polymerized branched polymer is suitable.

  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 a derivative solution containing the derivative thereof is prepared and irradiated 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 derivatives thereof 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.

200-400 nm is suitable for the wavelength of the light to irradiate, for example, a low pressure mercury lamp, a high pressure mercury lamp, etc. can be used. The irradiation time of the light depends on the irradiation intensity is preferably about 1 to 200 minutes, about 1 minute to 360 minutes with a low irradiation intensity of about 1μW ^/ cm ² ~10mW ^/ cm 2 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, any irradiation light source may be used as long as it includes light having a wavelength of 240 to 400 nm. For example, a low-pressure mercury lamp or a high-pressure mercury lamp 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 comprising 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof in the gene introduction agent to be obtained is preferably about 5,000 to 100,000, particularly preferably about 10,000 to 80,000, Moreover, it is preferable that the polymer block which consists of 2-N, N- dimethylamino ethyl methacrylate and / or its derivative in a branched chain consists of about 5-150 monomer units. If the molecular weight of the polymer block comprising 2-N, N-dimethylaminoethyl methacrylate and / or its derivative is too small, the nucleic acid cannot be sufficiently aggregated even when a lower alcohol is used in combination, and is too large. And the nucleic acid complex is disadvantageous because of its high molecular weight.

  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 having only a polymer block composed of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof is about 10,000 to 150,000, although it depends on the number of branched chains. About 20,000 to 100,000, especially about 30,000 to 80,000 is preferable. 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 ζ-potential of about +20 mV to +50 mV when complexed with a nucleic acid even when the molecular weight is a low molecular weight within the above range.

  In the method for preparing a nucleic acid complex of the present invention, the gene introduction agent synthesized as described above and a nucleic acid are mixed together in a system in which a lower alcohol is present.

  The lower alcohol is preferably an alcohol having 1 to 5 carbon atoms, such as methanol, ethanol, propanol, butanol, pentanol, etc. Among them, ethanol is particularly preferable. Alcohols with more than the above range do not dissociate the pseudo bond between the polymer main chain and the dimethylamino group on the side chain of the above-mentioned gene introduction agent, or dissociate the polymers, so that they can be combined with nucleic acids. I can't do it. In addition, such an alcohol may not be mixed uniformly with other solvents such as water present in the mixed system. In addition, these lower alcohols may be used individually by 1 type, and 2 or more types may be mixed and used for them.

  In order to complex the vector comprising the gene introduction agent and the nucleic acid, a lower alcohol is added at room temperature to the dispersion having a concentration of about 1 to 1000 μg / ml, and then the nucleic acid is added and mixed. Good. It is preferable that an excessive amount of a gene introduction agent is added to the nucleic acid, and the gene introduction agent is saturated with respect to the nucleic acid to form a nucleic acid complex.

  The amount of the lower alcohol present in the mixed system containing the gene introduction agent and the nucleic acid is about 300 to 300,000% by weight, particularly 500 to 200,000, based on the amount of the gene introduction agent in the mixture system. It is preferable that it is about weight%. If the amount of the lower alcohol is too small, the polymer chain composed of 2-N, N-dimethylaminoethyl methacrylate and / or its derivative, which is a gene introduction agent, will not be sufficiently cationic, so that the nucleic acid and 2-N, N- It becomes difficult to complex with a gene introduction agent having a branched chain composed of a polymer block of dimethylaminoethyl methacrylate and / or a derivative thereof. Moreover, when there is too much quantity of a lower alcohol, the cytotoxicity of alcohol will appear strongly.

  This mixed system is practically an aqueous solution containing the gene introduction agent, nucleic acid, lower alcohol, buffer system component, and inorganic salts, and the concentration of the nucleic acid is about 10 μg / mL to 100 μg / mL. The weight ratio of water and lower alcohol is preferably about water / alcohol = 95/5 to 30/70.

  Thus, if a lower alcohol is present in the mixed system, the nucleic acid can be introduced even if it is a gene transfer agent having a branched chain composed of a polymer block of 2-N, N-dimethylaminoethyl methacrylate and / or its derivative, which is a low pKa monomer. Can be included.

  As another embodiment of the present invention, nucleic acid may be mixed at room temperature with a dispersion of a gene introduction agent, and then this mixture and lower alcohol may be mixed. Alcohol may be mixed, and then this mixed solution and the gene introduction agent dispersion may be mixed.

  However, by mixing a dispersion of the gene introduction agent and a lower alcohol, the pseudo bond between the branched main chain of the gene introduction agent and the dimethylamino group of the side chain is dissociated, or between the polymers Therefore, it is preferable to mix a dispersion of a gene introduction agent and a lower alcohol, and then mix this mixture and a nucleic acid.

  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.

  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.

  Hereinafter, the present invention will be described more specifically with reference to examples.

i) Synthesis of iniferter 1,2,4,5-tetrakis (N, N-diethyldithiocarbamylmethyl) benzene as an iniferter was synthesized according to the following reaction formula.

  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 liters of methanol, stirred for 30 minutes, and then 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 δ1.26-1.31 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) It became.

ii) Polymerization of 2-N, N-dimethylaminoethyl methacrylate to iniferter According to the following reaction formula, 1,2,4,5-tetrakis [(N, N-diethyldithiocarbamyl) poly (2-N, N- Dimethylaminoethyl methacrylate) -methyl] benzene (hereinafter sometimes referred to as pDMAEMAA) was synthesized.

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 high-purity nitrogen gas for 10 minutes with vigorous stirring in a 3 mm thick soft glass cell, irradiation with mixed ultraviolet rays having a wavelength of 250 nm to 400 nm was performed for 180 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. This polymerization solution is concentrated with an evaporator, the polymer is reprecipitated with n-hexane, purified by repeating reprecipitation three times with chloroform / n-hexane, and dissolved in a small amount of benzene after evaporating n-hexane. The solution was filtered through a 0.2 μm filter and freeze-dried to obtain a 4-branched star homopolymer pDMAEMAA. The number average molecular weight using polyethylene glycol as a standard substance was measured by GPC to be 32,000 (Mw / Mn = 1.5).

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, it became 2H, O-CH ₂₎ and.

iii) Preparation of nucleic acid complex (Example 1)
32 μg of the branched star polymer synthesized in the above ii) was collected and 30 μL of TE buffer (10 mM)
Tri · HCL, 1 mM EDTA). 30 μL of ethanol was mixed here. Plasmid DNA (pGL3 control) encoding Promega firefly luciferase was dissolved in TE buffer and adjusted to a concentration of 0.033 μg / μL. 60 μL of the cationic polymer solution was added to 90 μL of this branched star polymer solution and gently mixed at room temperature. The amount of ethanol in this mixed system is about 1,000% by weight with respect to the branched star polymer, and the weight ratio of water / alcohol is 50/50. The number of positive charges per unit weight in the branched star polymer was calculated from the molecular weight 157 of the monomer unit in the polymer, and was assumed to be 100% dissociated even under neutral conditions. The number of negative charges per unit weight in DNA was calculated from the number of base pairs in the sequence map and the average molecular weight 660 of nucleobases. Thereby, the mixing ratio of the positive charge and the negative charge in the nucleic acid complex is such that the number of positive charges is 20 times the number of negative charges.

iv) Preparation of nucleic acid complex (Comparative Example 1)
32 μg of branched star polymer was dissolved in 60 μL of TE buffer. The DNA solution was prepared in the same manner as iii). A branched star polymer and a DNA solution were mixed and maintained at room temperature as in iii) to prepare a nucleic acid complex.

v) Measurement of ζ-potential The nucleic acid complex prepared in iii) and iv) was diluted 10-fold with physiological saline, and the ζ-potential was measured. A Sysmex Zetasizer Nano-ZS and a disposable 1 mL capacity cell were used. The cell temperature was set at 25 ° C. The various coefficients of the particles were calculated with the values of polystyrene particles.

  In Example 1, a ζ-potential of 28.0 mV was observed. On the other hand, in Comparative Example 1, no ζ-potential was observed, suggesting that charged fine particles were not formed.

vi) Confirmation of complexation of DNA and gene introduction agent by ethidium bromide Ethidium bromide is known as a compound that emits strong fluorescence when intercalated into DNA. Usually, it is used to visualize bands by electrophoresis of DNA, and if the DNA is in a free state, strong fluorescence is observed by mixing ethidium bromide. If the gene introduction agent and the nucleic acid form a nucleic acid complex due to the aggregation of the gene introduction agent, the binding between the DNA molecule and ethidium bromide is limited, and fluorescence is not observed. This principle is used to evaluate whether the DNA is condensed. All operations were performed at room temperature.

  The nucleic acid complex prepared in iii) and iv) was transferred to a 96-well microplate. To this, 40 μL of an aqueous ethidium bromide solution having a concentration of 20 μg / mL was added and allowed to stand for 10 minutes. After 10 minutes, the fluorescence intensity was measured at an excitation wavelength of 560 nm and a fluorescence wavelength of 620 nm using a fluorescence microplate reader (Dia-Latron, CT9000D). The fluorescence intensity is 100% of the solution obtained by adding 40 μL of 20 μg / mL aqueous ethidium bromide solution to a solution obtained by mixing 90 μL of 0.033 μg / μL DNA solution and 60 μL of TE buffer, and 20 μg to 150 μL of TE buffer. The fluorescence intensity of a solution obtained by adding 40 μL of an aqueous ethidium bromide solution at a concentration of / mL was calculated as 0%.

  As a result, in Comparative Example 1, it was found that there was no difference from the fluorescence intensity of the solution of DNA alone, and the DNA was not aggregated. In Example 1, the relative value was about 30%, indicating that DNA was aggregated and the intercalation of ethidium bromide was limited. This result was confirmed to be consistent with the result of ζ-potential of v).

vii) Gene transfer activity COS-1 derived from African green monkey kidney cells was used as cells, and a pGL3 control vector was used as 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 nucleic acid complex prepared in the above iii) or iv) was used as a gene vector. 25 μL of this gene vector was added to 200 μL of OPTI-MEM at room temperature and incubated for 30 minutes. Cultured cells were washed with room temperature PBS and transfected. Forty-eight hours after transfection, 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.

  As a result, in Example 1, gene transfer activity was expressed, and the activity was about 50 to 80% higher than that of commercially available Lipofectamine 2000. On the other hand, in Comparative Example 1, it was considered that the activity was almost the same as that of the system using Nacked DNA, the nucleic acid complex was not formed, and the gene introduction activity was not exhibited.

Claims (10)

A method for preparing a nucleic acid complex in which a nucleic acid is complexed with a gene introduction agent by mixing the gene introduction agent and the nucleic acid in a solution,
The gene introduction agent comprises a polymer material having a branched chain,
The branched chain contains at least a polymer block of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof;
A method for preparing a nucleic acid complex, wherein a lower alcohol is present in the mixed system.   The method for preparing a nucleic acid complex according to claim 1, wherein the lower alcohol is an alcohol having 1 to 5 carbon atoms.   3. The method for preparing a nucleic acid complex according to claim 2, wherein the lower alcohol is ethanol.   The amount of the lower alcohol present in the mixed system according to any one of claims 1 to 3 is 300 to 300,000% by weight with respect to the amount of the gene introduction agent in the mixed system. A method for preparing a nucleic acid complex.   5. The polymer material according to claim 1, wherein the polymer material includes a compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule as an iniferter, and includes at least 2-N, N -A method for preparing a nucleic acid complex comprising a branched polymer obtained by light-irradiating living polymerization of dimethylaminoethyl methacrylate and / or a derivative thereof.   6. The compound according to claim 5, wherein the compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule has a benzene ring as a nucleus and three or more N, N-dialkyls as branched chains in the nucleus. -A method for preparing a nucleic acid complex, wherein a dithiocarbamylmethyl group is bonded.   The method for preparing a nucleic acid complex according to any one of claims 1 to 6, wherein the branched chain consists only of a polymer block of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof.   The nucleic acid complex according to any one of claims 1 to 7, wherein the molecular weight of the polymer block of the 2-N, N-dimethylaminoethyl methacrylate and / or derivative thereof is 5,000 to 100,000. Body preparation method.   The method for preparing a nucleic acid complex according to any one of claims 1 to 8, wherein the gene introduction agent has a molecular weight of 10,000 to 150,000.   A nucleic acid complex prepared by the method for preparing a nucleic acid complex according to any one of claims 1 to 9.