JP2009275002A - Gene transfer agent, nucleic acid complex and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a gene transfer agent and a nucleic acid complex, excellent in gene transfer efficiency. <P>SOLUTION: This gene transfer agent comprises a cationic branched type polymer becoming hydrophilic at a temperature lower than a prescribed temperature (T) which is lower than a living body temperature, and hydrophobic at a higher temperature than the prescribed temperature (T), and the cationic branched type polymer is characterized by using a compound having ≥3 N,N-disubstituted dithiocarbamylmethyl groups in the same molecule as an iniferter, performing the photo-irradiated living polymerization of N-isopropylacrylamide or its derivative to form its polymer block chains on the iniferter, and further performing the photo-irradiated living polymerization of a cationic monomer to form cationic polymer block chains. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

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

  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

I. In these Patent Documents 1 and 2, the branched chain consists of 3-N, N-dimethylaminopropylacrylamide polymerized by light irradiation, and 3 to 6 branched chains are provided for the aromatic ring.

  Since this branched chain captures and stores DNA, it is expected that the greater the number of branched chains, the higher the capacity for storing DNA. However, as a result of various studies, it was confirmed that a gene introduction agent having 6 branched chains does not necessarily exhibit a higher DNA occluding ability than a gene introduction agent having 4 branched chains. This is considered to be because the degree of freedom of movement of the branched chain is reduced particularly on the base end side of the branched chain due to electrical repulsion between the branched chains.

  That is, as in Patent Documents 1 and 2, in the case of a star polymer in which 3-N, N-dimethylaminopropyl acrylamide is polymerized directly on an aromatic ring to form a branched chain, on the base end side of the branched chain In this case, since the distance between the branched chains is short, it is considered that the movement of the branched chain is likely to be limited by the electric repulsive force (repulsive force between cations). That is, this branched chain can move around the aromatic ring as a rotation center, but when the branched chains repel each other on the branch chain base end side, there is a range in which the branch chain can move in the base end side rotation direction. Narrow. In other words, the degree of freedom of movement of the branched chain is reduced. If the degree of freedom of movement of the branched chain is small, the ability of the branched chain to embed DNA may be reduced.

  As described above, when the number of branched chains is increased, the degree of freedom of movement of the branched chains is limited. Therefore, even if the number of branched chains is increased, the DNA storage capacity does not increase immediately.

II. 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).

III. The present invention provides a gene introduction agent having a temperature responsiveness that is hydrophilic at a temperature lower than a predetermined temperature (T) lower than a living body temperature and is hydrophobic at a temperature higher than the predetermined temperature (T). It is an object of the present invention to provide a nucleic acid complex used, which is considered to be capable of improving gene transfer efficiency, a nucleic acid complex using the gene transfer agent, and a method for producing the same. To do.

  The gene transfer agent of the present invention (Claim 1) is a cation that 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 cationic branched polymer comprises a compound having three or more N, N-disubstituted-dithiocarbamylmethyl groups in the same molecule as an iniferter. N-isopropylacrylamide or a derivative thereof is light-irradiated living polymerized to form a polymer block chain, and then a cationic monomer is light-irradiated living polymerized to form a cationic polymer block chain. And

  The gene transfer agent according to claim 2 is the compound 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 and a branch chain in the nucleus. Three or more of the N, N-disubstituted-dithiocarbamylmethyl groups are bonded.

  The gene introduction agent according to claim 3 is characterized in that, in claim 1 or 2, the N, N-disubstituted-dithiocarbamylmethyl group is an N, N-dialkyl-dithiocarbamylmethyl group.

  The gene introduction agent 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- It is at least one selected from the group consisting of N, N-dimethylaminostyrene and 4-aminostyrene.

  The gene introduction agent 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.

  The gene transfer agent 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 1,000,000.

  The gene introduction agent 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 nucleic acid complex of the present invention (invention 8) is obtained by combining the gene introduction agent according to any one of claims 1 to 7 and a nucleic acid.

  The method for producing a nucleic acid complex according to the present invention (invention 9) includes the step of bringing the cationic branched polymer according to any one of claims 1 to 7 and a nucleic acid at a temperature higher than the predetermined temperature (T). A method for producing a nucleic acid complex, comprising producing a nucleic acid complex by mixing.

  The method for producing a nucleic acid complex according to claim 10 is characterized in that, in claim 9, 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. And

  The gene introduction agent of the present invention is a star-type polymer having an aromatic ring as a nucleus, and a cationic branch in which a polymer block of N-isopropylacrylamide or a derivative thereof is introduced into the inner shell and a cationic polymer block is introduced into the outer layer Type polymer.

  N-isopropylacrylamide or a derivative thereof introduced into the cationic branched polymer is hydrophilic at a temperature lower than a predetermined temperature (T) lower than the living body temperature, but is higher than the predetermined temperature (T). It is a temperature sensitive monomer that undergoes phase transition and becomes hydrophobic when placed in a high temperature environment. That is, the gene introduction agent of the present invention is obtained by polymerizing N-isopropylacrylamide or a derivative thereof as a temperature sensitive monomer on an aromatic ring serving as a nucleus and then polymerizing a cationic monomer. Therefore, when this gene introduction agent is in a state higher than the predetermined temperature (T), the proximal end side of the branched chain of the star polymer is composed of a hydrophobic polymer block chain, and the distal end side thereof is a cation. It is composed of a functional polymer block chain.

  In this gene introduction agent, the cationic polymer block chain captures and incorporates an anionic nucleic acid. Further, this gene introduction agent has an attractive force or a cohesive force between the hydrophobic polymer block chains constituting the proximal end side of the branched chain in an aqueous solution having a temperature higher than the predetermined temperature (T). A plurality of star polymers are aggregated or aggregated in such a manner that their central portions (aromatic rings and branched chain base ends) overlap each other (hereinafter this phenomenon is sometimes referred to as “intermolecular aggregation phenomenon”). )), And is considered to constitute a dendrimer-like star polymer composite. In this star polymer complex, n × m branch chains are radially extended from the center, where n is the branch chain of one star polymer and m is the number of aggregated star polymers. The number of branched chains is extremely large.

In addition, since the terminal side of each branch chain of the star polymer complex is cationic and repels each other, each branch chain radiates from the center of the star polymer complex with a distance between each other. Distract in the direction.
Thus, the radially elongated star-type polymer complex has a significantly higher ability to embed DNA than a single star-type polymer present alone.

  Thus, according to the gene introduction agent of the present invention, a nucleic acid complex having excellent gene introduction efficiency can be produced.

  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 1,000,000 (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).

  When the gene introduction agent and the nucleic acid are mixed in an aqueous solution so that the gene introduction agent of the present invention is combined with the nucleic acid at a temperature lower than the predetermined temperature (T), the polymer block of N-isopropylacrylamide (or a derivative thereof) It becomes hydrophilic and is extended in an aqueous solution, and a nucleic acid complex is formed in a state where each branched chain is freely extended. In such a nucleic acid complex, the effect of improving the gene introduction activity based on the specific dendrimer-like steric structure of the above-mentioned star polymer complex cannot be obtained.

  Therefore, in the method for producing a nucleic acid complex of the present invention, the nucleic acid complex is produced by mixing the cationic branched polymer and the nucleic acid at a temperature higher than the predetermined temperature (T) (claim 9). In this case, 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 10).

  Embodiments of a gene introduction agent of the present invention, a nucleic acid complex using the same, and a method for producing the same will be described in detail below.

[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, as an iniferter, a compound having three or more N, N-disubstituted-dithiocarbamylmethyl groups in the same molecule, and N-isopropylacrylamide or a derivative thereof is subjected to light irradiation living polymerization. Is a branched polymer in which a cationic monomer is further photoirradiated by living polymerization to form a cationic polymer block chain after the polymer block chain is formed, and an aromatic ring is used as a nucleus, and then is radially extended. It is a 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. N-isopropylacrylamide or a derivative thereof is subjected to light irradiation living polymerization to form a polymer block chain of N-isopropylacrylamide or a derivative thereof, and then a cationic monomer is subjected to light irradiation living polymerization to form a cationic polymer block. A branched chain formed of a block copolymer of which a chain is formed and the base side is a polymer block of N-isopropylacrylamide or a derivative thereof (hereinafter sometimes referred to as “temperature-sensitive polymer block”) and the tip side is a cationic 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.

  In order to react an iniferter with N-isopropylacrylamide or a derivative thereof, a raw material solution containing the iniferter and N-isopropylacrylamide or a derivative thereof is prepared, and light irradiation is performed on the iniferter, whereby N-isopropylacrylamide is added to the iniferter. Or the reaction product which the derivative | guide_body couple | bonded is produced | generated. Two or more of N-isopropylacrylamide and its derivatives may be used. 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 N-isopropylacrylamide or a derivative thereof in this 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.

  This light irradiation produces the target branched polymer in the reaction solution. Therefore, N-isopropylacrylamide or its derivative homopolymer (hereinafter referred to as “temperature-sensitive homopolymer”), which is purified as necessary and comprises a branched polymer. May be referred to as “.”).

  A cationic monomer is block-copolymerized by light irradiation living polymerization with respect to the temperature-sensitive homopolymer made of the branched polymer thus produced to obtain a target cationic branched polymer.

As the cationic monomer to be polymerized to this temperature-sensitive homopolymer, vinyl monomers such as acrylic acid derivatives and styrene derivatives are suitable, such as 3-N, N-dimethylaminopropylacrylamide, 2-N, N-dimethylamino. Ethyl methacrylate, 4-N, N-dimethylaminostyrene, 4-aminostyrene and the like can be mentioned. Particularly, since it has excellent hydrolysis resistance, 3-N, N-dimethylaminopropylacrylamide CH ₂ = CHCONHC ₃ H Acrylic monomers such as ₆ N (CH ₃ ) ₂ and 2-N, 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.

In order to block copolymerize a cationic monomer to a temperature-sensitive homopolymer by light irradiation living polymerization, the temperature-sensitive homopolymer synthesized as described above is dissolved in a solvent such as methanol, and the cationic monomer is mixed therewith. Then, polymerization may be performed by irradiation with light. The concentration of the temperature-sensitive homopolymer in the solution when starting the polymerization reaction is preferably about 0.01 to 10% by weight, and the concentration of the cationic monomer is preferably about 0.3 to 30% by weight. . 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, N-isopropylacrylamide or a derivative thereof is first photoirradiated and living polymerized with respect to the iniferter, and then a cationic monomer is photoirradiated and living block copolymerized. A branched copolymer (block copolymer) having a branched chain composed of a block copolymer chain composed of a cationic polymer block 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 1,000,000, particularly preferably 3,000 to 150,000. If the molecular weight of the cationic branched polymer is outside this range, it is difficult to form a nucleic acid complex. In particular, if the molecular weight of the cationic branched polymer is too small, the molecular design of a branched copolymer suitable for the present invention is not possible. When the molecular weight of the cationic branched polymer is too large, it is not preferable because the metabolic property and biodegradability tend to be inferior.

  The molecular weight of each of the thermosensitive polymer block and the cationic polymer block in the cationic branched polymer also varies depending on the type of monomer constituting each polymer block, and cannot be generally stated. The molecular weight of the warm polymer block is 1,000 to 10,000, especially 30,000 to 8,000, and the molecular weight of the cationic polymer block is 5,000 to 100,000, especially 15,000 to 80,000. Preferably there is.

  If the molecular weight of the temperature-sensitive polymer block is too small, it is stable in an aqueous solution even at a predetermined temperature (T) lower than the living body temperature, for example, at a temperature of 30 ° C. or higher, within the preferred molecular weight range of the above-mentioned cationic branched polymer Therefore, if the intermolecular aggregation phenomenon described above does not occur and is too large, the affinity of the cationic branched polymer to water is too low, and it is not preferable because it is emulsified in water to form macro particles.

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

  Since the gene introduction agent of the present invention can be produced by light irradiation living polymerization of N-isopropylacrylamide or a derivative thereof to iniferter, and then light irradiation living polymerization of a cationic monomer, the synthesis is easy and rapid. This is also very advantageous in terms of a large degree of freedom in molecular design, such as introduction of different polymer blocks or modification of functional groups.

[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.

  Here, even when the concentration of the cationic branched polymer in the aqueous solution of the cationic branched polymer is less than 0.05 mg / mL, 50 mg / mL However, 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 intermolecular aggregation phenomenon described above is obtained. The aggregation effect cannot be obtained, and the object of the present invention cannot be achieved. In order to sufficiently obtain the intermolecular aggregation phenomenon of the cationic branched polymer, the temperature at the time of mixing the cationic branched polymer and the nucleic acid should be higher than the predetermined temperature (T) by several degrees C. Is preferred. However, it cannot be denied that the solubility of the polymer itself may change, adversely affect the intermolecular aggregation phenomenon, and unexpected phenomena such as salt precipitation and phase separation may occur when cooling to a temperature that acts on cells. Therefore, it is preferable that the temperature at the time of mixing is 10 ° C. or higher than the 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. The aqueous solution here means a solution containing water as a main solvent component, and contains Tris / EDTA buffer, NaCl, ethanol, etc. well-known to those skilled in the art within a range not interfering with the intermolecular aggregation phenomenon. Is also possible.

  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 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. did. 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 4-branched star-type N-isopropylacrylamide homopolymer 188.6 mg of 1,2,4,5-tetrasakis (N, N-diethyldithiocarbamylmethyl) benzene synthesized by i) was added to 20 mL of toluene. It melt | dissolved, N-isopropylacrylamide 1.0g was added and mixed, and the whole quantity was adjusted to 50 mL with toluene. The solution was purged with high-purity nitrogen gas (G1 grade, flow rate 2 L / min) for 10 minutes while stirring vigorously in a 3 mm thick soft glass cell, and then a 300 W short arc xenon lamp (manufactured by Asahi Spectroscopic Co., Ltd., MAX-301). The mixture was irradiated with mixed ultraviolet rays having a wavelength of 250 nm to 400 nm for 20 minutes. 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, and the ether is evaporated, followed by vacuum drying to give a 4-branched star type N -An isopropylacrylamide homopolymer was obtained (polymerization rate 13%).
The molecular weight of this product was measured to be 5,000 (Mw / Mn = 1.18) by GPC.

iii) Synthesis of 4-branched star N-isopropylacrylamide / cationic block copolymer
1.0 g of the N-isopropylacrylamide homopolymer synthesized in ii) and 7.9 g of 3-N, N-dimethylaminopropylacrylamide were dissolved in about 30 mL of toluene. This solution was purged with high purity nitrogen gas (G1 grade, flow rate 2 L / min) for 10 minutes while stirring vigorously in a glass container, and then light was irradiated in the same manner as in ii) except that the light irradiation time was 40 minutes. Performs irradiation polymerization, reprecipitates three times in chloroform / diethyl ether system, evaporates the ether, dissolves in a small amount of water, filters through a 0.2 μm filter, freeze-drys, and then four-branch star type N-isopropylacrylamide / cationic block copolymer was obtained (polymerization rate 28%).

  The molecular weight of this product was determined to be 73,000 (Mw / Mn = 1.86) by GPC.

  From the above, a temperature-sensitive polymer block of N-isopropylacrylamide (molecular weight 5,000) and a cationic polymer block of 3-N, N-dimethylaminopropylacrylamide (molecular weight 68,000) are radially formed with benzene as the central core. It was confirmed that the introduced 4-branched block polymer was synthesized.

[Comparative Example 1]
iv) Synthesis of 4-branched star-type cationic homopolymer 45.6 mg of 1,2,4,5-tetrasakis (N, N-diethyldithiocarbamylmethyl) benzene synthesized in i) of Example 1 was added to 30 mL of toluene. Then, 7.9 g of 3-N, N-dimethylaminopropylacrylamide was added and mixed, and the total amount was adjusted to 50 mL with toluene. This solution was purged with high purity nitrogen gas (G1 grade, flow rate was 2 L / min) for 10 minutes while vigorously stirring in a glass container, and then the light irradiation time was changed to 40 minutes. The photo-irradiation polymerization was carried out by the above method, followed by reprecipitation treatment three times with a chloroform / diethyl ether system, followed by vacuum drying.

The total amount of the polymer component was dissolved in about 30 mL of methanol, and 3.4 g of 3-N, N-dimethylaminopropylacrylamide was added and mixed to make the total amount 50 mL with methanol. After purging again with high purity nitrogen gas (G1 grade, flow rate is 2 L / min) for 10 minutes with vigorous stirring in a glass container, the solution was concentrated after 15 minutes of light irradiation, and polymerized with diethyl ether The components were separated. The separated polymer component was reprecipitated 6 times with chloroform / diethyl ether system, then ether was evaporated, dissolved in a small amount of water, filtered through a 0.2 μm filter, freeze-dried, and then branched into four types. A star-type cationic homopolymer was obtained (polymerization rate: 34%).
The molecular weight of this product was measured to be 71,000 (Mw / Mn = 1.31) by GPC.

[Analysis of polymers]
The 4-branched star type N-isopropylacrylamide / cationic block copolymer synthesized in iii) of Example 1 was dissolved in heavy water at room temperature and became emulsion when the temperature was adjusted to 40 ° C. ^{When 1} H NMR is measured at 40 ° C., the NMR signal derived from N-isopropylacrylamide confirmed in the measurement at room temperature is decreased, and the polymer block of N-isopropylacrylamide becomes a cationic polymer block by heating. It was suggested that it was shielded. It was considered that micelles surrounding the N-isopropylacrylamide polymer block were formed together with the phenomenon of cloudiness.

  The measurement result of the particle size with a static light scattering device (MALVERN ZETA-SIZER NANO) is 6.9 nm for the N-isopropylacrylamide / cationic block copolymer synthesized in Example iii) at 20 ° C., Comparative Example 1. The iv) 4-branched cationic homopolymer having a molecular weight of 71,000 had a particle diameter of 7.3 nm. This result was confirmed to be consistent with the GPC measurement result. On the other hand, when N-isopropylacrylamide / cationic block copolymer was heated to 40 ° C. and the particle size was measured, the maximum value was 13.1 nm. This suggests that the N-isopropylacrylamide / cationic block copolymer forms micelles by associating a plurality of molecules in water at 40 ° C., that is, a dendrimer-like 3D radial structure. It was done.

[Verification of solubility / concentration balance]
The 4-branched star N-isopropylacrylamide / cationic block copolymer synthesized in iii) of Example 1 was dissolved in water to a concentration of 50 mg / mL. When heated to 40 ° C., the solution became cloudy (emulsified) and could not be filtered with a 0.2 μm filter.

  In Example 1, ii), the chain length of the N-isopropylacrylamide polymer block could be freely extended by lowering the iniferter concentration to ½ amount and / or extending the polymerization time. After introduction of the cationic polymer block (Mn is about 45,000-60,000) in iii) of Example 1, the water solubility of this 4-branched star N-isopropylacrylamide / cationic block copolymer As a result of confirmation of (40 ° C.), the N-isopropylacrylamide polymer block was a homogeneous aqueous solution with a block chain length of 10,000 or less in molecular weight. Confirmed that dendrimer-like 3D structure is self-assembled by the association of N-isopropylacrylamide polymer block and electrostatic repulsion of cationic polymer chain in aqueous solution, and structural superiority is expressed over gene transfer activity It was done.

[Gene transfer activity]
In vitro cellular gene transfer experiments were performed.
COS-1 cells were used as the cells, and pGL3 control vector was used as the 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 N-isopropylacrylamide / cationic block copolymer synthesized in iii) of Example 1 was used as a gene introduction agent, and the number of positive charges per unit weight in the gene introduction agent was N-isopropyl by GPC. It calculated | required by calculating from the molecular weight 156 of each molecular weight of an acrylamide block and a cationic block, and the monomer unit of a 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. 1 (Example 1).

  Further, the gene introduction activity was examined in the same manner as described above except that the cationic homopolymer composed of the 4-branched star polymer synthesized in iv) of Comparative Example 1 was used as a gene introduction agent. (Comparative Example 1).

  Further, the gene introduction activity was examined in the same manner as described above except that the temperature was adjusted at 15 ° C. when mixing the aqueous solution of the gene introduction agent with the Tris / EDTA solution of DNA and OPTI-MEM, and the results are shown in FIG. (Comparative Example 2).

  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 (10)

A gene transfer agent comprising a cationic branched polymer that is hydrophilic at a temperature lower than a predetermined temperature (T) lower than a living body temperature and is hydrophobic at a temperature higher than the predetermined temperature (T). ,
The cationic branched polymer uses a compound having three or more N, N-disubstituted-dithiocarbamylmethyl groups in the same molecule as an iniferter, and N-isopropylacrylamide or a derivative thereof is subjected to light irradiation living polymerization. Then, after the formation of the polymer block chain, a cationic polymer block chain is formed by further photoirradiating living polymerization of a cationic monomer.   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 gene transfer agent, wherein a di-substituted dithiocarbamylmethyl group is bonded.   3. The gene introduction agent 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 gene introduction agent characterized by being at least one selected from the group consisting of aminostyrene.   The gene introduction agent 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 gene introduction agent according to any one of claims 1 to 5, wherein the molecular weight of the cationic branched polymer is 3,000 to 1,000,000.   The gene introduction agent according to any one of claims 1 to 6, wherein the predetermined temperature (T) is a temperature of 25 to 35 ° C.   A nucleic acid complex obtained by combining the gene introduction agent according to any one of claims 1 to 7 and a nucleic acid.   A nucleic acid complex produced by mixing the cationic branched polymer according to any one of claims 1 to 7 and a nucleic acid at a temperature higher than the predetermined temperature (T). A method for producing a composite.   10. The method for producing a nucleic acid complex according to claim 9, wherein the cationic branched polymer is mixed with nucleic acid as an aqueous cationic branched polymer solution having a concentration of 0.05 to 50 mg / mL.

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