JP2008195681A - Gene transfer agent, nucleic acid complex and gene transfer material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a gene transfer agent having such temperature responsibility that the gene transfer agent is hydrophilic at lower temperatures than a prescribed temperature (T) lower than the temperature of a living body and is hydrophobic at temperatures higher than the prescribed temperature (T). <P>SOLUTION: This gene transfer agent comprising a branched chain-having polymer material is characterized in that the polymer is hydrophilic at lower temperatures than a prescribed temperature (T) lower than the temperature of a living body and is hydrophobic at temperatures higher than the prescribed temperature (T). The polymer material is obtained by living-polymerizing a vinylic monomer in the presence of an iniferter comprising preferably a compound having three or more N,N-dialkyl-dithiocarbamoylmethyl molecular groups in the same molecule under irradiation of light to produce a homopolymer comprising the branched polymer and then block-polymerizing N-isopropylacrylamide or its derivative to the obtained homopolymer. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a gene introduction agent, and a nucleic acid complex and a gene introduction material using the gene introduction agent.

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

As a non-viral vector for transporting DNA into cells, a cationic star polymer is described in WO2004 / 092388.
WO2004 / 092388

  In the above-mentioned WO 2004/092388, the nucleic acid complex of a nucleic acid and a gene introduction agent is dispersed in an aqueous solution, and thus cannot be placed at a certain site in a living body for a long time.

  The present invention eliminates the above-mentioned conventional problems, and has a temperature responsiveness 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. It is an object of the present invention to provide an introduced gene introduction agent, a nucleic acid complex using the gene introduction agent, and a gene introduction material.

The gene introduction agent of the present invention is a gene introduction agent comprising a polymer material having a branched chain, wherein the polymer material is hydrophilic at a temperature lower than a predetermined temperature (T) lower than a living body temperature, It is characterized by being hydrophobic at a temperature higher than the temperature (T).
The polymer referred to in the present invention includes oligomers such as monomer dimers.

  The polymer material is preferably a polymer having a copolymer block of at least a cationic monomer and N-isopropylacrylamide or a derivative thereof, and includes a cationic monomer, a nonionic monomer, and N-isopropylacrylamide or a derivative thereof. A copolymer may also be used.

  The molecular weight of the cationic polymer block is preferably 2,000 to 500,000, and the molecular weight of the nonionic polymer block is preferably 2,000 to 500,000.

  The molecular weight of this polymer (block copolymer) is preferably 3,000 to 600,000.

  The predetermined temperature (T) is preferably a temperature between 25 and 35 ° C.

  This block copolymer uses a compound having three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule as an iniferter, and a branched monomer obtained by subjecting a vinyl monomer as a cationic monomer to light irradiation living polymerization. It is preferably a mold polymer.

  The compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule has a benzene ring as a nucleus and three or more N, N-dialkyl-dithio as a branched chain in this nucleus. Those having a carbamylmethyl molecular group bonded thereto are preferred.

  The vinyl monomer is one selected from the group consisting of 3-N, N-dimethylaminopropylacrylamide, 2-N, N-dimethylaminoethyl methacrylate, N, N-dimethylacrylamide, and methoxyethyl (meth) acrylate. The above is preferable.

The nucleic acid complex of the present invention comprises a complex of the gene introduction agent of the present invention and a nucleic acid.
The gene transfer material of the present invention is obtained by supporting this nucleic acid complex on a base material.

  Since the gene introduction agent of the present invention is hydrophilic and water-soluble at a temperature lower than the above-mentioned predetermined temperature, it is dissolved in water and complexed with a nucleic acid and administered to a living body or carried on a substrate ( For example, it is applied), and then is set to a temperature higher than a predetermined temperature, so that it becomes hydrophobic (water insoluble) and adheres to a living body or a substrate.

  When administered to a living body, the gene introduction agent becomes water-insoluble due to the body temperature of the living body, so that the nucleic acid complex is left in place for a long period of time. Since this gene introduction agent does not use an organic solvent, it can be applied to a substrate made of a hybrid material.

  The gene introduction agent of the present invention is a polymer having a branched chain 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). Made of material.

  As the above-mentioned polymer material, a compound having three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule is used as an iniferter, and at least a cationic monomer and N-isopropylacrylamide or a derivative thereof are used as light. A branched polymer subjected to irradiation living polymerization is preferred.

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

  As a compound having three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule, three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups are bonded as a branched chain to the benzene ring. These are preferred, and specific examples are as follows. That is, as a 3-branched chain, 1,3,5-tri (bromomethyl) benzene and sodium N, N-dithiocarbamate (sodium N, N-dithiocarbamate) can be obtained by addition reaction in ethanol. , 3,5-tri (N, N-dithiocarbamylmethyl) benzene, and four branched chains include 1,2,4,5-tetrakis (bromomethyl) benzene and sodium N, N-dithiocarbamylate ( 1,2,4,5-tetrakis (N, N-dithiocarbamylmethyl) benzene obtained by addition reaction with sodium N, N-dithiocarbamate) in ethanol. Hexakis (N, N-dithio) obtained by addition reaction of bromomethyl) benzene and sodium N, N-dithiocarbamate in ethanol Rubamirumechiru) is a benzene.

  The above iniferter is almost insoluble in polar solvents such as alcohol, but is easily soluble in nonpolar solvents. The nonpolar solvent is preferably a hydrocarbon, an alkyl halide or a halogenated alkylene, particularly benzene, toluene, chloroform or methylene chloride, and particularly preferably toluene.

As the cationic monomer to be polymerized to this iniferter, vinyl monomers such as vinyl monomers, acrylic acid derivatives, styrene derivatives and the like are particularly suitable. Specifically, 3-N, N-dimethylaminopropylacrylamide CH ₂ ═CHCONHC _At least one selected from the group consisting of ₃ H ₆ N (CH ₃ ) ₂ , 2-N, N-dimethylaminoethyl methacrylate, N, N-dimethylacrylamide and methoxyethyl (meth) acrylate is preferred.

  In one embodiment of the present invention, an iniferter is first polymerized with a cationic monomer to obtain a cationic homopolymer, and N-isopropylacrylamide or a derivative thereof is block copolymerized therewith.

  In order to react the iniferter with the cationic monomer, a raw material solution containing the iniferter and the monomer is prepared, and this is irradiated with light to generate a reaction product in which the monomer is bound to the iniferter.

  The concentration of the 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.

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

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

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

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

  The molecular weight of the cationic homopolymer depends on the number of branched chains, but is preferably about 2,000 to 500,000, particularly 2,000 to 150,000, and particularly 2,000 to 100,000.

  N-isopropylacrylamide or a derivative thereof is block copolymerized with the cationic homopolymer made of the branched polymer thus produced to obtain a target polymer material. The polymer chain of N-isopropylacrylamide or a derivative thereof has a temperature dependency that becomes hydrophilic at a low temperature and hydrophobic at a high temperature, so that the polymer material has the temperature responsiveness.

To block copolymerize N-isopropylacrylamide or a derivative thereof, the branched polymer synthesized as described above is dissolved in a solvent such as methanol, mixed with N-isopropylacrylamide or a derivative thereof, What is necessary is just to superpose | polymerize by irradiation. The concentration of the branched polymer 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 preferably about 0.3 to 30% by weight. It is. 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 another aspect of the present invention, N-isopropylacrylamide or a derivative thereof is first polymerized with respect to the iniferter to form a homopolymer, and then a cationic monomer such as the vinyl monomer is blocked in the homopolymer. Polymerization agents may be obtained by polymerization.

  In yet another embodiment of the present invention, a cationic monomer, N-isopropylacrylamide or a derivative thereof, and a nonionic monomer are used as monomers. The order of polymerization with respect to the iniferter is arbitrary. That is, the arrangement order of the cationic block, the block of N-isopropylacrylamide or a derivative thereof, and the nonionic polymer block constituting one branched chain is arbitrary.

  As the nonionic monomer, polyethylene glycol (meth) acrylate, polyethylene glycol alkyl ester (meth) acrylate, polyvinylpyrrolidone, or the like can be used. The molecular weight of the nonionic polymer block is preferably 2,000 to 500,000.

  The molecular weight of the block copolymer (polymer material) is preferably 3,000 to 600,000, particularly preferably 3,000 to 150,000.

  The gene introduction agent made of this polymer material is insoluble in water at a temperature higher than about 30 ° C. and water-soluble at a temperature lower than about 30 ° C.

  Therefore, when an aqueous solution of a gene introduction agent complexed with nucleic acid (concentration is preferably about 3 to 150 mg / mL) at a temperature lower than about 30 ° C., for example, about 10 to 25 ° C. is administered to the living body, the temperature rises depending on the body temperature of the living body. Warm and water insoluble. Therefore, the gene introduction agent combined with the nucleic acid is placed at a specific site administered in the living body for a long time, and the nucleic acid is locally supplied around the site for a long time.

  Also, a water-insoluble nucleic acid-carrying polymer is prepared by adhering an aqueous solution of a gene transfer agent complexed with this nucleic acid to a substrate by coating or the like, raising the temperature to a temperature higher than 30 ° C., and drying as necessary. A coating or the like is formed.

  The gene introduction agent (vector) composed of the cationic polymer thus produced surrounds the nucleic acid as a nucleic acid-containing complex, whereby the inactivation and degradation of the nucleic acid by the enzyme in the living body can be suppressed.

  In order to combine a vector made of a cationic polymer and a nucleic acid, the nucleic acid may be added and mixed at room temperature to a dispersion having a concentration of about 1 to 1000 μg / mL. It is preferable to add an excessive amount of a cationic polymer to the nucleic acid and to complex the cationic polymer in a saturated state with respect to the nucleic acid as a nucleic acid-containing complex.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Example 1
i) Synthesis of iniferter According to the following reaction formula, 1,2,4,5-tetrakis (NN-diethyldithiocarbamylmethyl) benzene was synthesized as follows.

  1.0 g of 1,2,4,5-tetrakis (bromomethylbenzene) and 4.0 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, dried under reduced pressure, dissolved in 200 mL of chloroform, and extracted and separated by adding 150 mL of water to remove sodium bromide. After repeating this operation three times, the chloroform layer was dried over magnesium sulfate for 24 hours, filtered, added with n-hexane, recrystallized and purified, and white 1,2,4,5-tetrasakis ( Acicular crystals of (N, N-diethyldithiocarbamylmethyl) benzene were obtained (yield 90%). High-performance liquid chromatography confirmed that the raw material peak disappeared and the purified product was a single substance.

The measurement result of ¹ H NMR (in CDCl ₃ ) is δ1.26-1.31 ppm (t, 24H, CH ₂ CH ₃ ), δ 3.69-3.77 ppm (q, 8H, N (CH ₂ CH ₃ ) ₂ ), Δ 3.99-4.07 ppm (q, 8H, N (CH ₂ CH ₃ ) ₂ ), δ 4.57 ppm (s, 8H, Ar—CH ₂ ), δ 7.49 ppm (s, 2H, Ar—H) It became.

ii) Synthesis of cationic homopolymer comprising 4-branched star polymer by photopolymerization According to the following reaction formula, 1,2,4,5-tetrakis [(N, N-diethyldithiocarbamyl) ) A cationic homopolymer comprising 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-tetrasakis (N, N-diethyldithiocarbamylmethyl) benzene synthesized by i) above was dissolved in 20 mL of chloroform, and 3-N, N-dimethylaminopropylacrylamide was dissolved. (3-N, N-DMAPAAm) 5.0 g was added and mixed, and the total amount was adjusted to 50 mL with chloroform. After purging with high-purity nitrogen gas for 5 minutes with vigorous stirring in a quartz cell, ultraviolet light was irradiated for 20 minutes with a 200 W high-pressure mercury lamp. The irradiation intensity was adjusted to 1 mW / cm ² (250 nm) using an illuminometer (UVR-1, TOPCON, Tokyo, Japan). The polymerization solution is concentrated with an evaporator, purified by reprecipitation of the polymer with diethyl ether, dissolved in a small amount of water, filtered through a 0.2 μm filter, and lyophilized to form a 4-branched star homopolymer pDMAPAAm. A cationic polymer was obtained (polymerization rate 40%). The molecular weight was determined to be 16,000 by GPC.

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) Synthesis of polymer material (4-branched pDMAPAAm-b-pNIPAM) by block copolymerization of N-isopropylacrylamide with a cationic homopolymer According to the following reaction formula, 1,2,4,5- Tetrakis [(N, N-diethyldithiocarbamyl) -poly (N-isopropylacrylamide) -block-poly (3-N, N-dimethylaminopropylacrylamide) -methyl] benzene (hereinafter referred to as pDMAPAAm-b-pNIPAM) Was synthesized.

  That is, the whole amount of the 4-branched pDMAPAAm homopolymer synthesized in the above ii) was dissolved in 20 mL of methanol, and 0.1 g of N-isopropylacrylamide (NIPAM) was mixed to adjust the total amount to 60 mL with methanol. Polymerization material pDMAPAAm-b-pNIPAM consisting of a block polymer of 4-branched pDMAPAAm and poly N-isopropylacrylamide (pNIPAM) after light irradiation polymerization under the same conditions as in ii) and purification in methanol / diethyl ether system Got. The molecular weight was determined to be 19,000 by GPC.

<Preparation of treatment composition for imparting gene transfer activity to material surface>
The preparation was performed in a clean bench controlled at an ambient temperature of 20 to 25 ° C. All the solutions such as the buffer solution and physiological saline used for the preparation here were also adjusted to 20 to 25 ° C. As the DNA, a plasmid (Promega, pGL3 control vector) encoding firefly luciferase was used.

  Using DNA as a TE buffer solution having a concentration of 0.033 μg / μL, the 4-branched pDMAPAAm-b-NIPAM block polymer is dissolved in physiological saline, and the concentrations thereof are 0.05 μg / μL, 0.15 μg / μL, 0.25 μg. / ΜL, 0.50 μg / μL or 0.75 μg / μL. 900 μL of the DNA solution and 600 μL each of the 4-branched pDMAPAAm-b-NIPAM block polymer solution of each concentration were mixed to prepare a treatment composition that imparts gene transfer activity to the material surface of the present invention.

<Evaluation of gene transfer activity on material surface>
200 μL each of the treatment agent composition for imparting gene transfer activity was added to each well of the 24-well culture plate on the surface of the material of the present invention, and the plate was gently shaken to extend the whole and incubated at 37 ° C. for 30 minutes. .
COS-1 cells cultured in a 100 mm dish were treated with trypsin, diluted with complete medium, and the number of cells was adjusted to 6 × 10 ⁴ cells / mL with a Birkerchilk blood count plate to obtain a cell suspension.

  Each well of the 24-well culture plate prepared above was rinsed once with PBS heated to 37 ° C., and 1 mL of the cell suspension heated to 37 ° C. was added thereto and cultured as it was for 48 hours. After 48 hours of culturing, the medium was removed, and after washing twice with PBS, 200 μL of a cell lysing agent was added to each well and allowed to stand at 4 ° C. for 30 minutes to obtain a sample for evaluation of gene transfer activity.

  FIG. 1 shows the results of evaluation of gene transfer activity by luciferase assay. For firefly luciferase activity, Promega's assay kit was used, and correction was performed at the protein concentration. Protein quantification was performed with Bradford reagent from BioRad.

<Comparative example>
The 4-branched pDMAPAAm homopolymer was dissolved in physiological saline to obtain a solution having a concentration of 0.75 μg / μL. It was mixed with a TE buffer solution of DNA having a concentration of 0.033 μg / μL, coated onto a 24-well culture plate in the same manner as in the Examples, and the gene transfer activity was evaluated in the same manner. The results are shown in FIG.
From FIG. 1, it was confirmed that a composition capable of imparting gene introduction activity to the material surface was obtained according to the present invention.

It is a graph which shows the result of an Example and a comparative example.

Claims (13)

In a gene introduction agent comprising a polymer material having a branched chain,
The gene introduction agent, wherein the polymer material 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).   2. The gene introduction agent according to claim 1, wherein the polymer material is a polymer having a copolymer block of at least a cationic monomer and N-isopropylacrylamide or a derivative thereof.   The gene introduction agent according to claim 2, wherein the molecular weight of the cationic polymer block is 2,000 to 500,000.   4. The gene introduction agent according to claim 2, wherein the polymer material is a block copolymer of a cationic monomer, a nonionic monomer, and N-isopropylacrylamide or a derivative thereof.   The gene introduction agent according to claim 4, wherein the molecular weight of the nonionic polymer block is 2,000 to 500,000.   The gene introduction agent according to any one of claims 2 to 5, wherein the polymer material has a molecular weight of 3,000 to 600,000.   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.   8. The block copolymer according to claim 2, wherein the block copolymer is an iniferter having a compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule. A gene transfer agent, which is a branched polymer obtained by light-irradiating living polymerization of a functional monomer and N-isopropylacrylamide or a derivative thereof.   9. The compound according to claim 8, wherein the compound having three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule has a benzene ring as a nucleus and three or more N, N- A gene transfer agent characterized in that a dialkyl-dithiocarbamylmethyl molecular group is bound thereto.   The gene introduction agent according to claim 8 or 9, wherein the cationic monomer is a vinyl monomer.   11. The vinyl monomer according to claim 10, wherein the vinyl monomer is selected from the group consisting of 3-N, N-dimethylaminopropylacrylamide, 2-N, N-dimethylaminoethyl methacrylate, N, N-dimethylacrylamide and methoxyethyl (meth) acrylate. A gene introduction agent characterized by comprising at least one kind.   A nucleic acid complex comprising the complex of the gene introduction agent according to any one of claims 1 to 11 and a nucleic acid.   A gene transfer material obtained by supporting the nucleic acid complex of claim 11 on a substrate.

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