KR100718077B1 - Mannosylated chitosan derivative and gene delivery system using thereof - Google Patents

Abstract

The present invention relates to a chitosan derivative modified as a mannose group as a gene carrier. The present invention also relates to a complex of the gene carrier and DNA, and a gene therapy agent comprising the complex.

The gene transporter of the present invention is a non-viral gene transporter that overcomes the disadvantages of the existing viral gene transporter, and can exhibit high cell expression and high gene expression rate.

Gene Carriers, Chitosan, Mannose Receptors

Description

Mannosylated chitosan derivative and gene delivery system using

1 is a ¹ H-NMR spectrum of a chitosan derivative (MC) having a mannose group.

FIG. 2 is an electrophoretic image of MC / DNA (pGL3-Control) complexes according to DNA and various charge ratios ('K' in the figure means '1,000').

Figure 3 is an electrophoresis image of salping the protective ability from the DNase I of MC100,000 / DNA complex at charge ratio 5.

FIG. 4 is an electrophoresis photograph showing the dissociation of DNA from MC100,000 / DNA complex at charge ratio 5. FIG.

5 is a photograph of an atomic force microscope of the MC100,000 / DNA complex at charge ratio 5;

FIG. 6 is a graph of cytotoxicity of NCTC 3749 cells of chitosan and MC ('K' in the figure means '1,000').

7 is a graph showing the gene expression efficiency of Raw 264.7 cells of MC100,000 / DNA (pGL3-Control) complex at charge ratio 5 ('K' means '1,000' in the figure).

8 is a graph illustrating the ability of IL-12 and INF-γ to be produced by the MC100,000 / DNA (pmIL-12) complex from dendritic cells ('K' in the figure means '1,000').

9 is a graph showing the ability of IL-12 and INF-γ after treating MC100,000 / DNA (pmIL-12) complexes from tumor tissues.

10 is a graph illustrating changes in tumor volume of the control and treatment groups by date.

Figure 11 is a graph confirming the degree of apoptosis after treatment with MC100,000 / DNA (pmIL-12) complex by Western blot.

12 is a photograph confirming the degree of apoptosis through TUNEL analysis.

Figure 13 is a graph confirming the degree of cell division cycle inhibition by Western blot.

Figure 14 is a photograph showing the activity of MMP-2 and MMP-9 through gelatin zymography assay.

Figure 15 is a photograph taken by immunostaining FGF-2.

The present invention relates to chitosan derivatives as new gene carriers.

Gene therapy involves the injection of new genes or genetic material created by DNA recombination into a patient's cell to correct genetic defects or add new functions to the cell to cause cancer, infectious diseases, and It is a method of preventing or treating all genetic defects such as autoimmune diseases. Recently, the concept of gene therapy has been expanded and applied to the treatment of acquired diseases such as AIDS caused by human immunodeficiency virus (HIV) infection as well as congenital genetic diseases transmitted to offspring.

These gene therapy methods include direct injection of proteins, drug therapy to help or inhibit the expression of specific genes in the body, and injection of DNA with genetic information into the body to express the injected gene. However, direct protein injection and drug therapy have the risk of causing an immune response to foreign substances, the need for continuous infusion and expensive production costs. To overcome these problems with protein drugs, the idea was to use genes. DNA has the ability to produce proteins in vivo. Therefore, the basic concept of gene therapy is to inject DNA in vivo in the method of injecting the existing protein directly, so that the injected DNA continuously produces the protein.

There are some issues that need to be addressed before this new level of treatment can be used as a common and effective treatment in many medical institutions. Once you have a gene with a genetic code that can produce some specific protein. Next, there must be a gene expression system called a plasmid that can express the gene. Finally, there is a need for a carrier that can safely transport an expression containing a specific gene to a desired location. Since DNA has a problem of being easy to be hydrolyzed in vivo and having low efficiency of entering into cells, many studies on gene delivery systems have been actively conducted to solve these problems and to apply gene therapy.

Until recently, viral vectors with high transfection efficiency have been used as gene carriers in clinical trials. However, this is not only complicated in the manufacturing process, but also limited in safety issues such as immunogenicity, infectivity, inflammation, non-specific DNA insertion and the size of the acceptable DNA is limited to apply to humans. Therefore, non-viral vectors are currently in the spotlight as substitutes for viral vectors.

Nonviral vectors have the advantage of being able to be repeatedly administered with a minimum of immune response, capable of specific delivery to specific cells, being stable for storage, and easy to produce large quantities. It is mainly used as a positive liposome-based liposome-based N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride { N- [1- (2,3 -dioleyloxy) propyl] -N, N, N -trimethylammonium chloride (DOTMA)}, alkylammonium, cationic cholesterol derivatives, gramicidin, etc. Although it is reported to enter the cell membrane, there are problems such as complicated manufacturing process, low inclusion rate of DNA having a large molecular weight, decrease in transfection activity by serum components on in vivo ( in vivo ) , particle size and the like. In addition, lipofectin, which is currently used most, is also a cytotoxic problem.

Recently, cationic polymers in non-viral vectors have attracted much attention because they can form complexes with negatively charged DNA and ionic bonds. These include poly-L-lysine (PLL), poly (4-hydroxy-L-proline ester), polyethyleneimine (PEI), poly [α- (4-aminobutyl) -L-glycolic acid], polyamidoamine Dendrimers, poly [N, N '-(dimethylamino) ethyl] methacrylate (PDMAEMA), which compress DNA to form nanoparticles, protecting DNA from enzymatic degradation, and rapidly penetrating into cells to endo Help get out of the moth. However, most non-viral gene carriers have lower immunorejection response and cytotoxicity than viral gene carriers, but the gene expression rate is relatively low. This is the most important problem.

Chitosan is also a material that offers ample potential as a nonviral gene carrier because of its biocompatibility, biodegradability, low toxicity and high cationicity. However, due to the low transfection efficiency, there are many difficulties in practical applications. For efficient gene expression, there are several steps to be taken. For effective penetration into cells, complexes formed by binding to DNA must be condensed down to a size of 150 nm, prevent DNA degradation from various enzymes, enter the cell, and the complex must exit the endosomes well, and the cell nucleus It must enter and dissociate the DNA from the carrier. (M. Koeping-Hoeggaerd et. Al. (2001) "Chitosan as a nonviral gene delivery system.Structure-property relationships and characteristics compared with polyethylenimine in vitro and after lung administration in vivo" Gene Therapy 8: 1108-1121)

The present inventors, such as the non-toxic and excellent biocompatibility but low transfection efficiency, complement the shortcomings of the chitosan, and succeeded in the development of a new gene carrier that can increase the gene expression rate by inducing the rapid release from the endosomes patent It has been filed (Korean Patent Application 10-2004-22301). The gene carrier uses a chitosan derivative in which imidazoling is introduced into chitosan. However, the gene carrier was completely cell specific.

It is an object of the present invention to provide a non-viral gene carrier that can overcome the disadvantages of existing viral gene carriers, which can exhibit cell specificity and high efficiency of gene expression.

The present invention relates to a chitosan derivative modified as a mannose group as a gene carrier. The present invention also relates to a complex of the gene carrier and DNA, and a gene therapy agent comprising the complex.

More specifically, the present invention relates to a polymer compound comprising the following general formula (1) as a main repeating unit.

Wherein n> x and n is an integer of 57 or more.

Preferably, the polymer compound has a molecular weight of 10,000 or more, up to about 1,000,000 can be used.

In the present invention, the compound is a polymer (hereinafter referred to as polymer compound or MC of the present invention) in which a mannose group is bonded to chitosan.

Another aspect of the present invention relates to a gene carrier comprising the polymer compound.

Moreover, this invention relates to the complex of the high molecular compound of this invention and DNA. The complex is an ionic bond product of the polymer compound and DNA. Hereinafter, the carrier and the complex of the present invention will be described in more detail.

The gene delivery system of the present invention enhances gene delivery efficiency by introducing a mannose group capable of specifically binding to a cell having a mannose receptor in chitosan, a positive polysaccharide that is nontoxic and excellent in biocompatibility as a natural polymer. It is a cationic high molecular compound prepared to induce endocytosis.

The cell may be any cell in vivo as long as the cell has a mannose receptor. Regarding immunotherapy using genes, for example, antigen presenting cells (APC) may be mentioned. Known cells with mannose receptors are macrophage cells and dendritic cells.

When the polymer compound of the present invention as described above is mixed with DNA in an aqueous solution, a cooperative bond between electrostatically complementary chains is formed between the polymer compound of the present invention, which is a cationic polymer, and DNA, which is anionic polymer, and eventually, It will form a complex. In this case, the polymer compound / DNA charge ratio of the present invention is preferably about 5 to about 20.

The DNA can be any gene. Regarding cancer treatment using genes, there may be mentioned, for example, cytokine genes such as the interleukin-12 gene.

The goal of cytokine gene therapy for cancer treatment is to produce an immune response to the systemic body that is specific to cancer tissues and can prevent cancer metastasis, even though the therapeutic effect lasts for a long time. Among many kinds of cytokines, interleukin-12 is known to be the most effective substance for inducing anticancer immunity. Interleukin-12 induces the division of natural killer cells (NK) or T cells, enhances cytolytic activity, interferon-γ (INF-γ) and some tumor necrosis factor (TNF-α). It is known to increase the immune response of the Th1-type by inhibiting tumor growth, metastasis, angiogenesis, and induce apoptosis.

Interleukin-12 is produced mainly by antigen presenting cells (APCs), such as dendritic cells (DCs) or macrophages (macrophages), and exhibits a pleiotropic effect on immune effector cells. In particular, dendritic cells are the only antigen-presenting cells capable of inducing a primary immune response, and are known to induce immune memory. Dendritic cells are also known to be essential for inducing cell-mediated immune responses against cancer cells because of their ability to stimulate cytotoxic T lymphocytes, which is why they are an important focus for the development of vaccines and immunotherapeutic agents for the treatment of cancer. It is a cell. Interestingly, immature DCs (immature DC) have a large number of mannose receptors to facilitate the endocytosis of antigens that expose the mannose sugar surface. For this reason, dendritic cells with mannose receptors are good targets for cationic polymers to which mannose is introduced as gene carriers.

Given the anticancer effects of interleukin-12 and the ability of dendritic cells to induce systemic immunity, sustained secretion of interleukin-12 from dendritic cells is much more effective in combination with the ability of dendritic cells to recognize and label tumor antigens. It is thought to induce an immune response. In recent years, cancer vaccines using dendritic cells have been developed to differentiate the cells isolated from the blood cells of the patient into dendritic cells, which are able to release antigens derived from cancer cells or immunoregulatory cytokines such as interleukin-12. Injecting genes into genes and then administering them back into the patient's body is being studied. However, ex vivo treatment of dendritic cells is time-consuming and expensive, and may present a risk of infection to the patient. In addition, considering the toxicity and rapid in vivo degradation, it is expected that the delivery method using a gene delivery system is more effective than the injection method of the protein itself in vivo.

Until recently, viral vectors with high transfection efficiency have been used as gene carriers in clinical trials. However, this is not only complicated in the manufacturing process, but also limited in safety issues such as immunogenicity, infectivity, inflammation, non-specific DNA insertion and the size of the acceptable DNA is limited to apply to humans. Therefore, nonviral vectors are currently in the spotlight as substitutes for viral vectors. Nonviral vectors have the advantage of being able to be repeatedly administered with a minimum of immune response, capable of specific delivery to specific cells, being stable for storage, and easy to produce large quantities. Among them, cationic polymers are attracting much attention because they can form complexes through negatively charged DNA and ionic bonds.

Among cationic polymers, in particular, chitosan, by itself, is a material that shows sufficient potential as a non-viral gene carrier due to its biocompatibility, biodegradability, low toxicity and high cationicity. The present invention has greatly improved gene transfer efficiency by imparting cell specificity thereto. In addition, chitosan itself is known as an immune stimulating substance that increases antigen recognition, and therefore, it is particularly suitable for use as a gene carrier for delivering cytokines.

Another aspect of the present invention is a gene therapy agent comprising the complex as an active ingredient. The therapeutic target may be any disease that can be treated by delivering genes to cells. Examples of such treatments include genetic disease treatment using gene transfer, immune disease treatment using gene transfer, cancer treatment using gene transfer, cancer immunotherapy treatment of cancer using immunological approach, among others. Can be mentioned.

Specific examples of the complex applicable to the treatment of cancer immunity include a complex of the polymer compound of the present invention and a cytokine gene, particularly an interleukin-12 gene, wherein the target cell is a dendritic cell having a mannose receptor ( Antigen presenting cells (APC), such as DC) and macrophage. In this case, the complex of the present invention enters target cells through mannose receptors, promotes the production of interleukin-12 from antigen-presenting cells, and thus exhibits anticancer immune effects. In other words, the interleukin-12 gene is specifically delivered to antigen-labeled cells around cancer tissues, thereby making it possible to treat cancer immunologically.

Hereinafter, the present invention will be described in detail with reference to Examples, which are merely illustrative of the present invention and do not limit the scope of the present invention.

pGL3 -Control

The brand name was pGL3-Control and the size was 5.3kb, purchased from Promega (Madison, WI, USA), and used to express strong luc + ( luciferase ) using the SV40 promoter.

pmIL -12

The CMV IE promotor was used to express p35 and p40 through the internal ribosome entry site. The product name was pUMVC3-mIL12, size 6.2kb, and the product was purchased from Aldevron (Fargo, ND, USA).

Example 1 Preparation of Chitosan Derivatives (MC) Containing Mannose

A water-soluble chitosan (WSC) of 18,000, 50,000, 100,000 was used, mixed with mannopyranosylphenylisothiocynate dissolved in DMSO and reacted at room temperature for 24 hours, precipitated with isopropanol, washed with water several times, and centrifuged, and the precipitate was dried in a vacuum oven. The degree of introduction of mannose in chitosan was confirmed by NMR measurement. The bound mannose accounts for 8.6, 4.6, and 5.8 mol% of the total, and is named MC18,000, MC50,000, and MC100,000, respectively. 1 shows a ¹ H-NMR spectrum of MC100,000.

Example 2 Complex Formation of MC and DNA

To calculate the charge ratio, DNA was calculated as one charge per molecular weight of 330, MC was calculated as one charge per repeat unit of chitosan including mannose. DNA was allowed to fluoresce using ethidium bromide. MC and DNA (luciferase marker gene (pGL3-Control)] (0.1 μg) prepared in Example 1 were spontaneously complexed for 30 minutes at various charge ratios in 10 mM phosphate buffer (PBS, pH 7.4). The formation of the complex was then confirmed by electrophoresis for 100 V for 40 minutes on a 1% agarose gel using Tris-Acetate (TAE) running buffer. The results are shown in FIG. Regardless of the molecular weight of chitosan, the charge ratio seems to form a complete complex when MC (N) / DNA (P) is 3 or more.

Experimental Example 1: Inhibition of DNA Decomposition from Deoxyribonuclease (DNase I) of MC / DNA Complex and Dissociation of DNA from Complex>

  DNase I was added to the complex formed in Example 2, 2 units per 0.1 g of DNA was reacted at 37 ° C. for 30 minutes, and the results are shown in FIG. 3. In addition, by treating the complex formed in Example 2 with sodium dodecyl sulfate (SDS) it was confirmed that the DNA dissociated from the complex is not degraded, but in the original form, the results are shown in FIG. According to the chitosan molecular weight, there was a similar tendency, and in the case of MC100,000, it was confirmed that the DNA remained undecomposed from the charge ratio 5 which completely formed the complex, thereby confirming that MC plays a role of protecting DNA from enzymes. In addition, it was confirmed that even when the DNA was forcibly dissociated from the complex using SDS, the original DNA form was maintained intact, indicating that the DNA was safely protected without losing its function in the complex.

Experimental Example 2: Particle Size and Shape of MC / DNA Complex

  MC / DNA was prepared at various charge ratios as in the preparation method, and measured by atomic force microscopy, and the results are shown in FIG. 5. In the case of MC100,000 / DNA complex, when the charge ratio was increased to 5 or more, agglomeration between particles was observed and the particle size was greatly increased. The experiment was fixed by. When the charge ratio was 5, the average particle size was 136.7 ± 33.5 nm, and as shown in FIG. This fits well with the requirement that the particle size be less than 150 nm for rapid entry into the cell.

If you look at the morphology using AFM, etc., when the charge ratio exceeds 20, the size increases and tends to aggregate a lot. Especially in animal experiments, the amount of DNA added is much higher than the amount used in cell experiments.As the charge ratio increases, the amount of polymer added increases as the amount of ionic bond increases, thereby increasing the complex concentration. There is a lot of difficulty in making it. This is because it shows an easy clustering phenomenon. Therefore, the efficiency of the cell experiment was good to increase the charge ratio, but when applied to animal experiments, it was difficult when the charge ratio was over 20. Therefore, the MC / DNA charge ratio is preferably about 5 to 20.

Experimental Example 3: Cytotoxicity Test of MC

NCTC 3749 cell lines (a type of macrophage cell line) were each dispensed in 96 well-plates at a concentration of 4 × 10 ⁴ per well and grown for 24 hours using RPMI 1640 medium. Chitosan and MC prepared in Example 1 was treated by concentration for 24 hours Cell titer 96 Aqueous daily solution cell division test [CellTiter 96  Aqueous One Solution Cell Proliferation Assay (Promega, Madison, USA)] was used to determine the cytotoxicity. Cytotoxicity results are shown in FIG. 6. MC has been shown to be more cytotoxic than chitosan (WSC) itself, proving that MC is a biocompatible material.

Experimental Example 4: Transfection Efficiency of MC / DNA Complex

Raw 264.7 cells, one of the macrophage lines containing mannose receptors, were dispensed in 24 well-plates at 3 x 10 ⁵ cells per well for 18 hours in DMEM medium containing 10% fetal bovine serum (FBS). Grow at 37 ° C. under 5% carbon dioxide. Then, in the same manner as in Example 2 to form a complex with luciferase (luciferase) marker gene (pGL3-Control) and put in each well with a medium without FBS. After incubation at 37 ° C. and 5% carbon dioxide for 4 hours, the medium is removed and placed in DMEM medium containing fresh 10% FBS and incubated at 37 ° C. and 5% carbon dioxide for 48 hours. After removing the medium, 100 μl of cell lysis buffer was added and placed at -20 ° C for 24 hours. After one day, it is dissolved at room temperature and then transferred to an e-tube in each well and centrifuged for 15 minutes at 10000 rpm to obtain only the supernatant. The obtained supernatant was measured for activity using a luciferase assay kit. In addition, in order to determine how much the transfection efficiency of MC decreases with increasing the concentration of mannose, various concentrations of mannose were first treated with cells 15 minutes before the transfection experiment. (In the figure, RLU stands for 'relative light unit') The results are shown in FIG. As can be seen, MC showed much better gene expression compared to chitosan itself, and as the concentration of mannose increases, chitosan itself is not significantly affected by gene expression, whereas MC has consistently improved transfection efficiency. It was confirmed that the decrease, it was confirmed that the receptor-mediated gene delivery by mannose to some extent. Similar results were obtained when the molecular weight of chitosan was varied, and the gene transfer efficiency was the highest in the case of MC100,000, which was higher than the molecular weight of chitosan. Proceeded.

Next, the interleukin-12 gene, which is not a reporter gene, but an actual therapeutic gene, was extracted from rats and transferred to cells differentiated into dendritic cells to determine whether similar results were obtained. The results are shown in FIG. When the gene was delivered using chitosan or MC compared to the case of delivering only the interleukin-12 gene, it was confirmed that the generation degree of interleukin-12 was much higher, and the cell experiment obtained by delivering the luciferase gene, rather than the chitosan itself. It was confirmed that MC is much more effective in generating interleukin-12 from dendritic cells, and thus, it was confirmed that interferon-γ was also more effectively induced by MC.

Example 5 Tumor Induction and Anticancer Immunotherapy Using MC / pmIL-12 Complex

In order to cause cancer in rats, hairs of 8-week-old BALB / c mice were removed, and 2 x 10 ⁶ cells of CT-26 cells (colon cancer cell lines of mice) were injected into the back of mice through subcutaneous injection. Tumor volume was calculated using the formula v = 0.5 × a × b ² , where a and b in turn are the shortest and longest diameter of the tumor. Treatment of the tumor began about a week after the tumor size reached 100-120 mm ³ . MC / pmIL-12 complex was prepared in the same manner as in Example 2 charge ratio 5, the amount of treated DNA was 50 μg per mouse, a total of four tumor sites for 3 weeks every 3-4 days for 2 weeks Injected topically. The control group was injected with normal saline. Tumor size was measured twice a week for up to 21 days. All treatments and controls were sacrificed on day 21 and tumors were harvested for Western blot, immunostaining, and ELISA measurements.

Experimental Example 5-1: Cytokine Secretion and Tumor Growth Inhibitory Effect by MC / pmIL-12 Complex>

The extracted tumor was frozen in liquid nitrogen and dissolved, homogenized using a lysis buffer, centrifuged at 5000 rpm for 5 minutes, and then the supernatant was removed from the ELISA kit (R & D System, Minneapolis (MN) was used to quantify mIL-12 p70 and mINF-γ. The results are shown in FIG. Compared with the control group, the treatment group treated with MC / pmIL-12 complex was found to be much more effective in inducing mIL-12 p70 and mINF-γ. In addition, as shown in Example 3 as a result of measuring the volume of the tumor (FIG. 10), it can be seen that the growth of the tumor is significantly delayed until 13 days in the treated group compared to the control group, which is MC / pmIL-12 complex This may be due to the effects of cytokines induced on.

Experimental Example 5-2: Induction of Apoptosis of Tumor Tissue by MC / pmIL-12 Complex>

As in Experiment 5, Western blot was used to confirm whether the effect of delaying tumor growth was caused by apoptosis of tumor tissues, and the factors involved in apoptosis, Bad, Bid, Apaf-1 and cancer activator Bcl. The expression level of -xL was examined. As shown in FIG. 11, compared to the control group, the treatment group showed a significant increase in Bad, Bid, and Apaf-1, and conversely, Bcl-xL decreased, and apoptosis was induced by MC / pmIL-12 complex in tumor tissue. Was found to be induced. In addition, in the case of dark brown staining of the apoptosis site through the TUNEL assay (terminal deoxy-U nick end labeling assay), the treatment group was able to confirm that the apoptosis occurred in a much larger area than the control group. .

Experimental Example 5-3: Inhibitory Effect of Cell Division Cycle of Tumor Tissue by MC / pmIL-12 Complex>

In order to confirm whether the inhibition of tumor tissue growth is related to the inhibition of the cell division cycle, the expression level of the cell division cycle inhibitors p21, p27, and p53 was confirmed by Western blot, and the results are shown in FIG. 13. In the same context as the results of Experiment 6, p21, p27, and p53 were significantly increased in the treated group compared to the control group, and cyclin D1 was found to decrease, and thus the cells shifted from the G1 phase to the S phase. The cleavage cycle was confirmed to be inhibited by the administration of the MC / pmIL-12 complex.

Experimental Example 5-4: Angiogenesis Inhibitory Effect by MC / pmIL-12 Complex>

  Considering that new neovascularization is a very important factor in the growth of cancer, gelatin zymography assay was performed to determine the degree of inhibition of angiogenesis after treatment of MC / pmIL-12 complex. Shown in MMP-2 and MMP-9 activities, which are involved in cancer growth, metastasis, and blood vessel formation, were significantly lower in the treatment group than in the control group. Especially, MMP-2 showed little activity in the treatment group. It was confirmed that the production inhibitory effect. In addition, the MC / pmIL-12 complex was significantly reduced in FGF-2 expression in the treated group when stained with angiogenesis factor FGF-2 (Fibroblast growth factor) dark brown as shown in FIG. 15. It was confirmed again that there was some inhibitory effect on angiogenesis due to the treatment of.

Compared to chitosan without mannose group, MC having a mannose group introduced therein was superior in gene transfer to cells having a mannose receptor, and MC / pmIL-12 was applied to a mouse subcutaneously induced colon cancer. Injecting the complex effectively induces interleukin-12 and interferon-γ, thereby slowing the growth rate of cancer, inhibiting angiogenesis, inhibiting the progress of the cell division cycle, and eventually inducing tissue apoptosis.

The present invention is a non-viral gene carrier that overcomes the shortcomings of the existing viral gene carriers, and provides a gene carrier that can be expressed cell-specific and high efficiency gene expression, and also provides a gene therapy agent comprising the same.

In particular, there have been no studies on the delivery of interleukin-12 genes through direct receptor mediation to immune cells around cancerous tissues in vivo. Significant growth is expected in treatment.

Claims (8)

A polymeric compound comprising the following formula as a major repeating unit:

Wherein n> x and n is an integer of 57 or more. The polymer compound according to claim 1, wherein the molecular weight is 10,000 to 1,000,000. Gene delivery system comprising the polymer compound of claim 1. A complex of the polymer compound of claim 1 with DNA. The composite of claim 4 wherein the polymer compound / DNA charge ratio is 5-20. Gene therapy comprising the complex of claim 4 as an active ingredient. The gene therapy agent according to claim 6, wherein the subject to be treated using the gene is a genetic disease, an immune disease or a cancer. The gene therapy agent according to claim 6, wherein the DNA of the complex is an interleukin-12 gene, and the therapy agent is for immunological treatment of cancer.

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