JP2009275001A - Method for producing nucleic acid complex - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid complex excellent in gene transfer efficiency. <P>SOLUTION: This method for producing the nucleic acid complex is provided by compounding the nucleic acid with a gene transfer agent comprising a cationic branched type polymer obtained by using an aromatic ring as a core and having a plurality of cationic branched chains radially extended from the core, and becoming hydrophilic at a lower temperature than a prescribed temperature (T) which is lower than a living body temperature, and hydrophobic at a higher temperature than the prescribed temperature (T). The nucleic acid complex is also provided by producing the cationic branched type polymer by using a compound having ≥3 N,N-disubstituted dithiocarbamylmethyl groups in the same molecule as an iniferter, forming cationic block chains by performing the photo-irradiated living polymerization of a cationic monomer and then further performing the photo-irradiated living polymerization of N-isopropylacrylamide or its derivative to form its polymer blocks, and mixing the cationic branched type polymer with the nucleic acid at a higher temperature than the prescribed temperature (T). <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a nucleic acid complex in which a gene introduction agent and a nucleic acid are complexed.

  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.

Patent Documents 1 and 2 describe cationic star polymers as non-viral vectors for transporting DNA into cells.
WO2004 / 092388 JP2007-070579

  In Patent Documents 1 and 2, since the nucleic acid complex of the nucleic acid and the gene introduction agent is dispersed in an aqueous solution, it cannot be placed at a certain site in the living body for a long time.

  In order to solve such disadvantages, the present applicant uses a compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule as an iniferter, and performs photoirradiation living polymerization of a cationic monomer thereon, Next, a gene transfer agent comprising a branched polymer obtained by light-irradiating living polymerization of N-isopropylacrylamide was proposed (Japanese Patent Application No. 2007-34889, hereinafter referred to as “prior application”).

  The gene transfer agent of the prior application is hydrophilic at a temperature lower than a predetermined temperature (T) lower than the living body temperature, and is hydrophobic at a temperature higher than the predetermined temperature (T).

  The present invention discloses a temperature responsiveness disclosed in this prior application that is hydrophilic at a temperature lower than a predetermined temperature (T) lower than the living body temperature and becomes hydrophobic at a temperature higher than the predetermined temperature (T). It is an object of the present invention to provide a method for producing a nucleic acid complex using a gene transfer agent having the above-mentioned, wherein the gene transfer efficiency is further improved.

  The method for producing a nucleic acid complex of the present invention (Claim 1) is a cationic branched polymer having a plurality of cationic branched chains, each having an aromatic ring as a nucleus and extending radially therefrom. A nucleic acid is combined with a gene introduction agent comprising a cationic branched polymer that is hydrophilic at a temperature lower than a predetermined temperature (T) and is hydrophobic at a temperature higher than the predetermined temperature (T). A method for producing a nucleic acid complex, wherein a compound having three or more N, N-disubstituted-dithiocarbamylmethyl groups in the same molecule is used as an iniferter, and a cationic monomer is irradiated with light by living polymerization. After forming a cationic polymer block chain, N-isopropylacrylamide or its derivative is further subjected to light irradiation living polymerization to form the polymer block chain. To produce a down of branched polymer, characterized in that to produce a nucleic acid complex by mixing the cationic branched polymer and nucleic acids by the temperature higher than a predetermined temperature (T).

  The method for producing a nucleic acid complex according to claim 2 is the method according to claim 1, wherein the compound having three or more N, N-disubstituted-dithiocarbamylmethyl groups in the same molecule has a benzene ring as a nucleus, It is characterized in that three or more N, N-disubstituted-dithiocarbamylmethyl groups are bonded as a branched chain.

  The method for producing a nucleic acid complex according to claim 3 is the method according to claim 1 or 2, wherein the N, N-disubstituted-dithiocarbamylmethyl group is an N, N-dialkyl-dithiocarbamylmethyl group. And

  The method for producing a nucleic acid complex according to claim 4 is the method according to any one of claims 1 to 3, wherein the cationic monomer is 3-N, N-dimethylaminopropylacrylamide, 2-N, N-dimethylaminoethyl methacrylate. , 4-N, N-dimethylaminostyrene and at least one selected from the group consisting of 4-aminostyrene.

  The method for producing a nucleic acid complex according to claim 5 is characterized in that, in any one of claims 1 to 4, the molecular weight of the polymer block of N-isopropylacrylamide or a derivative thereof is 1,000 to 10,000. To do.

  The method for producing a nucleic acid complex according to claim 6 is characterized in that, in any one of claims 1 to 5, the molecular weight of the cationic branched polymer is 3,000 to 600,000.

  The method for producing a nucleic acid complex according to claim 7 is characterized in that, in any one of claims 1 to 6, the predetermined temperature (T) is a temperature between 25 and 35 ° C.

  The method for producing a nucleic acid complex according to claim 8 is the method according to any one of claims 1 to 7, wherein the cationic branched polymer is used as an aqueous cationic branched polymer solution having a concentration of 0.05 to 50 mg / mL. It is characterized by mixing with nucleic acid.

  According to the present invention, a nucleic acid complex excellent in gene transfer efficiency can be produced.

  That is, in the prior application, the temperature-responsive gene introduction agent and the nucleic acid are mixed at a temperature lower than the predetermined temperature (T). It has been found that a nucleic acid complex excellent in gene transfer efficiency can be obtained by mixing and introducing a gene transfer agent and a nucleic acid at a temperature higher than (T).

The reason is presumed as follows.
The gene introduction agent used in the present invention is a star-type polymer having benzene as a core, and a cationic branch in which a cationic polymer block is introduced into the inner shell and a polymer block of N-isopropylacrylamide or a derivative thereof is introduced into the outer layer. Type polymer.

  When this gene introduction agent is placed in a temperature environment higher than the predetermined temperature (T), the poly (N-isopropylacrylamide (or a derivative thereof)) block undergoes phase transition and aggregates in a hydrophobic bond. Single molecule micelles surrounding the (N-isopropylacrylamide (or derivative)) block are formed. On the other hand, the cationic polymer blocks on the inner shell side electrostatically repel ions. As a result, this star-shaped polymer becomes a self-cyclized 3D structure, and it is possible to condense and hold nucleic acids with high density by this complicated 3D structure (hereinafter, this phenomenon is referred to as “self It may be referred to as “cyclization phenomenon”.), Which shows high gene transfer activity.

  In contrast, when the gene introduction agent and the nucleic acid are mixed in an aqueous solution so that the gene introduction agent used in the present invention is complexed with the nucleic acid at a temperature lower than the predetermined temperature (T), hydrophilic poly (N-- The isopropylacrylamide (or derivative thereof) block is extended in an aqueous solution, and a nucleic acid complex is formed with each branched chain extended freely. In such a nucleic acid complex, the effect of improving gene introduction activity based on a specific three-dimensional structure by self-cyclization of a star polymer like the nucleic acid complex obtained by the method of the present invention cannot be obtained.

  In the present invention, a compound having three or more N, N-disubstituted-dithiocarbamylmethyl groups in the same molecule as an iniferter has a benzene ring as a nucleus and three or more N, N-disubstituted-dithiocarbamylmethyl group is preferably bonded (Claim 2), and this N, N-disubstituted-dithiocarbamylmethyl group is N, N-dialkyl-dithiocarbamylmethyl group. (Claim 3).

  In addition, as cationic monomers for photoirradiation living polymerization to iniferter, 3-N, N-dimethylaminopropylacrylamide, 2-N, N-dimethylaminoethyl methacrylate, 4-N, N-dimethylaminostyrene and 4-amino are used. At least one selected from the group consisting of styrene is preferred (Claim 4).

  The molecular weight of the polymer block of N-isopropylacrylamide or a derivative thereof introduced into the cationic branched polymer is preferably 1,000 to 10,000 (Claim 5). The molecular weight is preferably 3,000 to 600,000 (Claim 6).

  Further, the predetermined temperature (T) lower than the living body temperature at which the cationic branched polymer becomes hydrophilic or hydrophobic is preferably a temperature between 25 and 35 ° C. (Claim 7).

  In the present invention, it is preferable to mix the cationic branched polymer, which is a gene introduction agent, with the nucleic acid as an aqueous solution having a concentration of 0.05 to 50 mg / mL (claim 8).

  Hereinafter, embodiments of the method for producing a nucleic acid complex of the present invention will be described in detail.

  The method for producing a nucleic acid complex of the present invention is a cationic branched polymer having a plurality of cationic branched chains that are radially extended from an aromatic ring as a nucleus, and has a predetermined temperature lower than the living body temperature. A nucleic acid complex obtained by combining a nucleic acid and a gene introduction agent comprising a cationic branched polymer that is hydrophilic at a temperature lower than (T) and hydrophobic at a temperature higher than the predetermined temperature (T) In which a compound having three or more N, N-disubstituted-dithiocarbamylmethyl groups in the same molecule is used as an iniferter, and a cationic monomer is subjected to light irradiation living polymerization to the cationic polymer block. After forming a chain, further, N-isopropylacrylamide or a derivative thereof is subjected to light irradiation living polymerization to form a polymer block chain, thereby forming the cationic branched type. To produce a combined, characterized in that to produce a nucleic acid complex by mixing the cationic branched polymer and nucleic acids by the temperature higher than a predetermined temperature (T).

[Gene transfer agent]
<Cationic branched polymer>
First, the cationic branched polymer constituting the gene introduction agent according to the present invention and the production method thereof will be described.

  This cationic branched polymer uses a compound having three or more N, N-disubstituted-dithiocarbamylmethyl groups in the same molecule as an iniferter, and a cationic monomer is subjected to light irradiation living polymerization to the cationic polymer. A branched polymer in which N-isopropylacrylamide or its derivative is further light-irradiated and polymerized to form the polymer block chain after forming the block chain, and the aromatic ring is the nucleus, and then is radially extended. The cationic branched polymer having a plurality of cationic branched chains.

  This cationic branched polymer is hydrophilic at a temperature lower than a predetermined temperature (T) lower than the living body temperature by having a polymer block chain of temperature-sensitive N-isopropylacrylamide or a derivative thereof, It is hydrophobic at a temperature higher than the predetermined temperature (T). The predetermined temperature (T) lower than the living body temperature is preferably 25 to 35 ° C., for example, around 30 ° C., and when the polymer block chain of N-isopropylacrylamide is introduced, the predetermined temperature (T) is usually The temperature (T) is about 30 ° C.

  The cationic branched polymer according to the present invention is an iniferter comprising a compound having three or more N, N-disubstituted-dithiocarbamylmethyl groups, preferably N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule. And a cationic monomer is light-irradiated living polymerized to form a cationic polymer block chain, and then N-isopropylacrylamide or a derivative thereof is light-irradiated living polymerized to form a polymer block of N-isopropylacrylamide or a derivative thereof. A branched chain formed of a block copolymer in which a chain is formed, the base end side is a cationic polymer block, and the front end side is a polymer block of N-isopropylacrylamide or a derivative thereof (hereinafter sometimes referred to as “temperature-sensitive polymer block”). It is manufactured by forming.

  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-disubstituted-dithiocarbamylmethyl groups in the same molecule as an iniferter, the N, N-disubstituted-dithiocarbamylmethyl group, preferably N, N A group in which 3 or more dialkyl-dithiocarbamylmethyl groups are bonded as a branched chain is preferable, and specific examples are as follows. 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-dialkyl-dithiocarbamylmethyl group is preferably an alkyl group having 2 to 18 carbon atoms such as an ethyl group, but is not limited to an alkyl group, It may be an aromatic hydrocarbon group such as a phenyl group. That is, not only N, N-dialkyl-dithiocarbamylmethyl group but also N, N-diaryldithiocarbamylmethyl group and the like substituted with aliphatic hydrocarbon group and / or aromatic hydrocarbon group. An N-disubstituted dithiocarbamylmethyl group can achieve the object.

  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.

As the cationic monomer to be polymerized to this iniferter, vinyl monomers such as acrylic acid derivatives and styrene derivatives are preferable, and 3-N, N-dimethylaminopropylacrylamide, 2-N, N-dimethylaminoethyl methacrylate, 4 -N, N-dimethylaminostyrene, 4-aminostyrene and the like are mentioned, but since 3-H, N-dimethylaminopropylacrylamide CH ₂ = CHCONHC ₃ H ₆ N (CH _{3) 2, 2-N,} acrylic monomers such as N- dimethylaminoethyl methacrylate are preferred. A cationic monomer may be used individually by 1 type, and 2 or more types may be mixed and used for it.

  To react the iniferter with the cationic monomer, prepare a raw material solution containing the iniferter and the cationic monomer, and irradiate it with light to generate a reaction product in which the cationic monomer is bound to the iniferter. Let 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 the cationic monomer in the raw material solution is preferably 0.5M or more, for example, 0.5 to 2.5M. The concentration of the iniferter is preferably about 0.1 to 100 mM.

The wavelength of the irradiated light is preferably 300 to 400 nm. The irradiation time of the light depends on the irradiation intensity, about 1 to 60 minutes are preferred, about 1 to 30 minutes at a low irradiation intensity of about 1μW ^/ cm ² ~10mW ^/ cm 2 is particularly preferred.

  Since the target branched polymer is produced in the reaction solution by this light irradiation, the cationic homopolymer comprising the branched polymer is obtained by purification as necessary.

  N-isopropylacrylamide or a derivative thereof is block-copolymerized by light irradiation living polymerization to the cationic homopolymer made of the branched polymer thus produced to obtain a target cationic branched polymer.

In order to block copolymerize N-isopropylacrylamide or its derivative by light irradiation living polymerization, the cationic branched polymer (homopolymer) synthesized as described above is dissolved in a solvent such as methanol, and N- Isopropyl acrylamide or a derivative thereof may be mixed and polymerized by irradiation with light. Two or more of N-isopropylacrylamide and its derivatives may be used. The concentration of the cationic homopolymer in the solution when starting the polymerization reaction is preferably about 0.01 to 10% by weight, and the concentration of N-isopropylacrylamide or a derivative thereof is about 0.3 to 30% by weight. Is preferred. The light irradiation conditions are preferably a light wavelength of 250 to 400 nm, an irradiation time of 1 to 150 minutes, and an irradiation intensity of about 100 to 10,000 μW / cm ² .

  In this way, the cationic monomer is first photoirradiated and living polymerized with respect to the iniferter, and then N-isopropylacrylamide or its derivative is photoirradiated and living block copolymerized. A branched copolymer (block copolymer) having a branched chain composed of block copolymer chains composed of warm polymer blocks can be obtained.

  In the present invention, the molecular weight of the cationic branched polymer comprising the block copolymer varies depending on the iniferter, the cationic monomer, and the type of N-isopropylacrylamide or a derivative thereof, and cannot be generally described. 3,000 to 600,000, particularly preferably 3,000 to 150,000. If the molecular weight of the cationic branched polymer is too small, the molecular design of the branched copolymer suitable for the present invention cannot be sufficiently performed. If the molecular weight of the cationic branched polymer is too large, the Degradability tends to be inferior, which is not preferable.

  Also, the molecular weight of each of the cationic polymer block and the temperature-sensitive polymer block in the cationic branched polymer varies depending on the type of monomer constituting each polymer block, and cannot be generally described. The molecular weight of the functional polymer block is 5,000 to 1,500,000, particularly 15,000 to 150,000, and the molecular weight of the temperature-sensitive polymer block is 1,000 to 10,000, especially 3,000 to 8, 000 is preferred.

  If the molecular weight of the cationic polymer block is too small, the gene transfer activity tends to be inferior, and if it is too large, the molecular weight of the cationic branched polymer increases undesirably.

  In addition, if the molecular weight of the thermosensitive polymer block is too small, it will be stable in an aqueous solution even at a predetermined temperature (T) lower than the living body temperature, for example, 30 ° C. or higher, and the above-mentioned self-cyclization phenomenon will not occur. If it is too large, the cationic branched polymer is emulsified to form macro particles, which is not preferable.

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

  The present inventors have previously reported that gene transfer activity can be enhanced by photocrosslinking such a cationic branched polymer, but this product has a pale yellowish brown coloration. On the other hand, the gene introduction agent (cationic branched polymer) according to the present invention is colorless without coloring to the light yellowish brown color seen in the photocrosslinked product. This is considered because there is little or no damage to the amino group etc. of the side chain of the cationic polymer block by photocrosslinking. Since the aqueous solution of a gene introduction agent that acts on a gene or a cell as a gene introduction agent is more colorless than a colored solution, the gene introduction agent of the present invention is like this. Also excellent in that there is no coloring.

[Production of nucleic acid complex]
Inactivation of nucleic acids by enzymes in the body by enclosing the nucleic acid as a nucleic acid-containing complex using the gene transfer agent (vector) according to the present invention comprising the cationic branched polymer obtained as described above. , Decomposition can be suppressed.

  In the present invention, the gene introduction agent comprising the above-mentioned cationic branched polymer is preferably an aqueous solution having a concentration of 0.05 to 50 mg / mL, and the predetermined temperature (T) is applied to the aqueous solution of the gene introduction agent. By mixing the nucleic acid at a higher temperature, the gene introduction agent and the nucleic acid are combined. At this time, it is preferable that the gene introduction agent is mixed in an excess amount with respect to the nucleic acid, and the gene introduction agent is saturated with respect to the nucleic acid to be complexed as a nucleic acid-containing complex. The aqueous gene introduction agent solution described above means a solution containing water as a main solvent component, and can contain Tris / EDTA buffer, NaCl, ethanol, and the like well known to those skilled in the art.

  Here, even when the concentration of the cationic branched polymer in the aqueous solution of the cationic branched polymer in the aqueous solution of the cationic branched polymer is less than 0.05 mg / mL, 50 mg / mL Even if it exceeds, a nucleic acid complex suitable for finally acting on a living cell cannot be formed. In particular, when the concentration of the cationic branched polymer is high, when the mixing temperature of the cationic branched polymer and the nucleic acid is raised above the predetermined temperature (T), the frequency of aggregation between molecules increases and emulsification occurs. As a result, macro particles are formed, which is not preferable. A more preferable cationic branched polymer concentration in the aqueous cationic branched polymer solution when mixing the cationic branched polymer and the nucleic acid is 0.1 to 5.0 mg / mL.

  In addition, if the temperature at the time of mixing the cationic branched polymer aqueous solution and the nucleic acid is lower than the predetermined temperature (T), the cationic branched polymer becomes hydrophilic, and the nucleic acid of the nucleic acid due to the self-cyclization phenomenon described above. The aggregation effect cannot be obtained, and the object of the present invention cannot be achieved. In order to sufficiently obtain the self-cyclization phenomenon of the cationic branched polymer, the temperature at the time of mixing the cationic branched polymer and the nucleic acid should be higher by several degrees C. than the above-mentioned predetermined temperature (T). preferable. However, if this temperature is too high, the solubility of the polymer itself will change, adversely affecting the self-cyclization phenomenon, and unexpected phenomena such as salt precipitation and phase separation when cooling to a temperature that acts on cells Therefore, the temperature during mixing is preferably 10 ° C. or higher than the above-mentioned predetermined temperature (T). For example, when the predetermined temperature (T) is 30 ° C., it is preferable to mix and complex the cationic branched polymer and the nucleic acid at 32-37 ° C.

  More specifically, the cationic branched polymer and the nucleic acid are mixed by preparing an aqueous cationic branched polymer solution having a predetermined concentration as described above, separately preparing an aqueous nucleic acid solution, and mixing both solutions. Is performed by mixing the two solutions at the temperature.

  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 complex of the present invention is preferably about 50 to 400 nm. If it is smaller than this, the action of the enzyme may reach the nucleic acid inside the nucleic acid 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, the DNA 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 complex 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 complex as an active ingredient further comprises a pharmaceutically acceptable carrier (osmotic pressure regulator, stabilizer, preservative, solubilizer, pH adjuster, thickener, etc.) if necessary. It is possible to mix. Any known carrier can be used.

  Moreover, the medicine which contains this nucleic acid complex as an active ingredient includes those containing two or more kinds of nucleic acid-containing complexes having 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 administration method and the specific site to be treated. However, the dosage should be sufficient to produce a therapeutic response.

  This nucleic acid 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.) .

  In addition, an aqueous solution of the nucleic acid complex is attached to a substrate by coating or the like, and dried as necessary to form a coating of a polymer carrying nucleic acids.

  When the nucleic acid complex is attached to a substrate, a sheet is preferable as the substrate. 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 to the substrate is preferably about 0.001 to 10 mg per 1 cm ^{2 of the} substrate surface.

  Gene transfer material consisting of a substrate carrying a nucleic acid complex can be applied by placing a sheet-like substrate surrounding the subcutaneous tissue, myocardial tissue, diseased tissue, or diseased blood vessel, or by applying it to a covered stent film. It is used in such a manner that it is placed in a living body or 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.

[Example 1]
i) Synthesis of Iniferter 1,2,4,5-Tetrakis (N, N-diethyldithiocarbamylmethyl) benzene as an iniferter 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 stirred at room temperature for 4 days under light shielding. The precipitate was filtered, poured into 3 liters 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 cationic homopolymer comprising a 4-branched star polymer by photopolymerization Using 1,3-N, N-dimethylaminopropylacrylamide as a cationic monomer, 1,2,4,5-tetrakis [(N, A cationic homopolymer composed of N-diethyldithiocarbamyl) poly (3-N, N-dimethylaminopropylacrylamide) -methyl] benzene (hereinafter sometimes referred to as pDMAPAAm) 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. 7.9 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 with vigorous stirring in a 3 mm thick soft glass cell, irradiation with mixed ultraviolet light having a wavelength of 250 nm to 400 nm was performed for 40 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 repeated reprecipitation three times with chloroform / diethyl ether system, ether is evaporated and dissolved in a small amount of water, 0.2 μm The solution was filtered through a filter and freeze-dried to obtain a cationic homopolymer consisting of a 4-branched star homopolymer pDMAPAAm (polymerization rate: 32%). The molecular weight of this cationic homopolymer was measured by GPC as 58,000 (Mw / Mn = 1.31).

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-).

iii) Block Copolymerization of N-Isopropylacrylamide into Cationic Homopolymer 3.0 g of the 4-branched cationic homopolymer of molecular weight 58,000 synthesized in ii) above was dissolved in about 30 mL of methanol, and N- Isopropyl acrylamide 1.0g was added and melt | dissolved, and the whole quantity was 50 mL with methanol. After purging high purity nitrogen gas for 10 minutes while vigorously stirring this liquid in a glass container, polymerization was performed by light irradiation in the same manner as in the above ii) except that the light irradiation time was changed to 10 minutes, and thereafter Concentrate and reprecipitate with diethyl ether, collect the polymer component, perform reprecipitation treatment three times with chloroform / diethyl ether system, tetrakis {N, N-diethyldithiocarbamyl-poly (N-isopropylacrylamide)- Block-poly (3-N, N-dimethylaminopropylacrylamide) -methyl} benzene was obtained (polymerization rate 20%).
The molecular weight was determined to be 61,000 (Mw / Mn = 1.68) by GPC.

  From the above, a 4-branched star block copolymer (4) in which a polymer block of 3-N, N-dimethylaminopropylacrylamide (molecular weight 58,000) and a polymer block of N-isopropylacrylamide (molecular weight 3,000) were introduced (4 It was confirmed that a branched star-type cationic / thermosensitive block copolymer) was synthesized.

  In NMR in heavy water, white turbidity occurred in the tube by heating, and the phenomenon of a decrease or disappearance of the peak signal derived from the polymer block of N-isopropylacrylamide was confirmed. Although it was possible to extend the polymer block chain length of N-isopropylacrylamide by extending the polymerization time, the polymer block chain length of 3-N, N-dimethylaminopropylacrylamide had a molecular weight of 58,000. In the case, the solubility in water (30 ° C. or higher) was lowered from the region where the polymer block chain length of N-isopropylacrylamide exceeded the molecular weight of 10,000, and there was a tendency for suspension emulsification.

iv) Analysis of block copolymers
A 100 mg / mL aqueous solution of the 4-branched star-type cationic / thermosensitive block copolymer synthesized in iii) was prepared under a temperature condition of 20 ° C. When this solution was heated to 35 ° C., the solution became turbid, and it was confirmed that the polymer was sensitive to temperature and the N-isopropylacrylamide block chain became hydrophobic and became turbid. When the solution was diluted with water at 35 ° C., the solution became a colorless and transparent solution with about 20-fold dilution (5 mg / mL concentration), and filtration with a 0.2 μm syringe filter became possible.

  As a result of measuring the particle diameter with a static light scattering apparatus (MALVERN ZETA-SIZER NANO), the cationic homopolymer had a particle diameter of 6.3 nm and the cationic / thermosensitive block copolymer had a value of 6.7 nm. Here, not only the molecular weight (particle diameter) of the cationic / thermosensitive block copolymer is not significantly different from the value calculated by GPC, but also no significant change such as double or several times is confirmed. It was thought that the self-cycling phenomenon occurred in the water / water-sensitive block copolymer, in which a plurality of molecules do not associate in water and form micelles in the same molecule, that is, the leading end of the polymer associates. .

v) Gene transfer activity In vitro cell gene transfer experiments were performed.
COS-1 cells were used as cells, and pGL3 control vector was used as DNA. It seed | inoculated to the 24Well culture dish by the seeding density 6 * 10 < ⁴ > piece / mL, and gene introduction was performed 24 hours after culture | cultivation. The 4-branched star-type cationic / N-isopropylacrylamide block copolymer synthesized in the above iii) is used as a gene introduction agent, and the number of positive charges per unit weight in the gene introduction agent is determined by the GPC cationic block and N- The molecular weight was calculated from each molecular weight of the isopropylacrylamide block and the molecular weight 156 of the monomer unit of the cationic polymer. The number of negative charges per unit weight in DNA was calculated from the number of base pairs according to the sequence map and the average molecular weight 660 of nucleobases.

  This gene transfer agent aqueous solution (concentration of 1 mg / mL) was heated to 37 ° C. and stirred, and further mixed with a Tris / EDTA solution of DNA and OPTI-MEM adjusted to 37 ° C. and incubated for 30 minutes. . The mixing ratio was adjusted so that the number of charges was 20 times the number of negative charges, and the solution was adjusted so that 0.5 μg of DNA was administered to each well, and added to the cultured cells. After 3 hours, the OPTI-MEM solution was removed, washed with PBS, and then added with a complete medium and cultured. After 48 hours of culturing, 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 results are shown in FIG.

[Comparative Example 1]
The gene transfer activity was examined according to v) of Example 1 except that the cationic homopolymer composed of the 4-branched star polymer synthesized in Example ii) was used as the gene transfer agent. Is shown in FIG.

[Comparative Example 2]
According to v) of Example 1 except that the temperature was adjusted at 28 ° C. in mixing of the aqueous solution of the gene introduction agent with the Tris / EDTA solution of DNA and OPTI-MEM in v) of Example 1. The gene transfer activity was examined, and the results are shown in FIG.

  As shown in FIG. 1, according to the present invention, a single-molecule micelle three-dimensional structure can be obtained simply by introducing an N-isopropylacrylamide block having no charge and no ionic interaction with DNA and setting the mixing temperature with DNA to 30 ° C. or higher. It can be seen that a significant advantage is expressed and the gene transfer activity is enhanced.

It is a graph which shows the evaluation result of the gene transfer activity in Example 1 and Comparative Examples 1 and 2.

Claims (8)

A cationic branched polymer having a plurality of cationic branched chains, each having an aromatic ring as a nucleus and radially extending therefrom, and is hydrophilic at a temperature lower than a predetermined temperature (T) lower than a living body temperature. A method for producing a nucleic acid complex obtained by combining a nucleic acid and a gene introduction agent comprising a cationic branched polymer that is hydrophobic at a temperature higher than the predetermined temperature (T),
A compound having three or more N, N-disubstituted-dithiocarbamylmethyl groups in the same molecule is used as an iniferter, and a cationic monomer is photoirradiated by living polymerization to form a cationic polymer block chain. , N-isopropylacrylamide or a derivative thereof is subjected to light irradiation living polymerization to form the polymer block chain to produce the cationic branched polymer,
A method for producing a nucleic acid complex, wherein the cationic branched polymer and a nucleic acid are mixed at a temperature higher than the predetermined temperature (T) to form a nucleic acid complex.   The compound having three or more N, N-disubstituted-dithiocarbamylmethyl groups in the same molecule as defined in claim 1 having a benzene ring as a nucleus and three or more N, N- A method for producing a nucleic acid complex, wherein a di-substituted dithiocarbamylmethyl group is bonded.   3. The method for producing a nucleic acid complex according to claim 1, wherein the N, N-disubstituted-dithiocarbamylmethyl group is an N, N-dialkyl-dithiocarbamylmethyl group.   4. The cationic monomer according to claim 1, wherein the cationic monomer is 3-N, N-dimethylaminopropylacrylamide, 2-N, N-dimethylaminoethyl methacrylate, 4-N, N-dimethylaminostyrene, and 4. -A method for producing a nucleic acid complex, which is at least one selected from the group consisting of aminostyrenes.   The method for producing a nucleic acid complex according to any one of claims 1 to 4, wherein the molecular weight of the polymer block of N-isopropylacrylamide or a derivative thereof is 1,000 to 10,000.   The method for producing a nucleic acid complex according to any one of claims 1 to 5, wherein the molecular weight of the cationic branched polymer is 3,000 to 600,000.   The method for producing a nucleic acid complex according to any one of claims 1 to 6, wherein the predetermined temperature (T) is a temperature between 25 and 35 ° C.   The nucleic acid complex according to any one of claims 1 to 7, wherein the cationic branched polymer is mixed with nucleic acid as a cationic branched polymer aqueous solution having a concentration of 0.05 to 50 mg / mL. Manufacturing method.

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