JP2011072257A - Gene transfer agent, method for producing the same, and nucleic acid complex - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a gene transfer agent capable of more efficiently transferring a gene into a cell in a medium containing blood serum, a method for producing the gene transfer agent, and a nucleic acid complex in which the gene transfer agent is combined with a nucleic acid. <P>SOLUTION: There is disclosed a gene transfer agent comprising a polymer material having a branched-chain, wherein the branched-chain is a polymer in which at least 2-N, N-dimethylaminoethylmethacrylate and/or its derivatives are polymerized, and a part of constituent units derived from 2-N, N-dimethylaminoethylmethacrylate and/or its derivatives of the polymer is hydrolyzed. There is disclosed a nucleic acid complex comprising the gene transfer agent and a nucleic acid. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a gene introduction agent comprising a polymer material having a branched chain, a method for producing the gene introduction agent, and a nucleic acid complex in which the gene introduction agent and a nucleic acid are complexed.

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

  Synthetic polymer vectors and cationic lipid vectors have been researched and developed as gene transfer technologies to replace virus vectors that have problems with safety, quality stability, and manufacturing costs. For example, 2-N, N-dimethylaminoethyl methacrylate A gene introduction agent containing a cationic polymer composed of a cationic vinyl monomer has been reported (Non-patent Document 1).

  The present inventors have proposed that a synthetic polymer vector for transporting DNA into a cell is a branched structure vector in which an aromatic ring such as benzene is used as a nucleus and a cationic polymer chain radially extends to condense DNA at high density. Have found that small nucleic acid complex fine particles can be formed and genes can be efficiently introduced into cells, and patent applications have been filed earlier (Patent Documents 1 and 2).

  The present applicants have also introduced a gene comprising a thermosensitive cationic polymer that exhibits a cationic property at a temperature lower than a predetermined temperature (T) but is hydrophobic at a temperature higher than the predetermined temperature (T). Using an introducing agent, a solution containing the gene introducing agent and the nucleic acid is mixed at a temperature lower than a predetermined temperature (T), and then the solution is heated to a predetermined temperature (T) or more to combine the gene introducing agent and the nucleic acid. And a nucleic acid complex was obtained by bringing the nucleic acid complex and cells into contact with each other at a predetermined temperature (T) or higher in a medium containing serum (patent). Reference 3).

WO2004 / 092388 JP 2007-70579 A Japanese Patent Application No. 2009-126718

Smedt, S., Demeester, J., Hennik, W., (2000), Cationic polymer based gene delivery system, J Pharmaceutical research 17,113-126

  The gene introduction agent in which the polymer chain extends radially from the benzene ring described in Patent Documents 1 and 2 can be arranged with a higher charge density due to its structure compared to a linear polymer composed of the same monomer unit. It is. Therefore, a complex with a nucleic acid such as DNA or RNA can be more strongly aggregated, and fine polyplex particles having a smaller particle diameter can be formed. For this reason, the cell membrane permeability of the polyplex particles is increased and the gene transfer activity is improved, but there are the following problems.

  When a gene is introduced into cells, a medium containing serum is more suitable as a medium for culturing cells than a medium not containing serum. This is because a medium containing no serum has a large stress on cells due to lack of nutrients, and adversely affects cells after culturing. Stem cells such as iPS cells in which control of cell differentiation is particularly important This is because it is not suitable for the introduction of genes into.

  However, the gene introduction agent containing the above-mentioned cationic polymer does not show high gene introduction activity in a medium containing serum. This is because the gene introduction agent containing the above-mentioned cationic polymer has a high zeta potential even under a neutral condition of about pH 7, and can agglutinate nucleic acids. This is thought to be because anionic proteins and the like are also adsorbed and aggregated to form macro particles, which are difficult to be taken up into cells and DNA is not easily recognized by transporters.

  As a gene introduction method for avoiding this problem, the present applicants have introduced a thermosensitive cationic polymer that exhibits a cationic property at a temperature lower than a predetermined temperature (T) and exhibits a hydrophobic property at a temperature higher than the predetermined temperature (T). The method described in Patent Document 3 used as an agent was proposed.

  In the gene introduction method of Patent Document 3, since the gene introduction agent is cationic when the temperature is lower than a predetermined temperature (T), the gene introduction agent and the nucleic acid can be mixed efficiently, and at a temperature higher than the predetermined temperature (T). Since the gene introduction agent changes to hydrophobic, that is, nonionic, the gene introduction agent and the nucleic acid are combined at a predetermined temperature (T) or higher, and then the nucleic acid complex and the cell are brought into contact with each other. As a result, it was possible to prevent the nucleic acid complex from being complexed with the anionic substance in the serum, and as a result, the gene transfer efficiency was improved.

However, this gene introduction method has the following problems (1) to (4).
(1) Since the gene introduction agent changes to hydrophobic by setting the temperature to a predetermined temperature (T) or higher, and the nucleic acid complex consisting of the gene introduction agent and the nucleic acid also changes to hydrophobic, the anion in the serum It is less susceptible to the effects of sex proteins, but is likely to aggregate with hydrophobic substances in serum.
(2) When the gene introduction agent changes to hydrophobicity, it may be affected by components that solubilize hydrophobic substances such as cholesterol and albumin contained in serum and ionic lipids.
(3) Since the polystyrene petri dish generally used for cell culture is hydrophobic with a water contact angle of about 70 °, the hydrophobic gene introduction agent is easily adsorbed to the hydrophobic polystyrene petri dish.
(4) Nucleic acid complexes composed of gene transfer agents that have changed to hydrophobicity tend to aggregate in an aqueous solution. By utilizing the property that these nucleic acid-nucleic acid complexes are hydrophobically aggregated, the nucleic acid complex, such as in muscle, may be localized, or a portion where it is preferable that the nucleic acid complex is preferably localized. It is possible to introduce a gene into the blood vessel, but when this nucleic acid complex is administered into a living blood vessel, it is not preferable because it aggregates in the blood vessel.

The present invention has been made in view of the above-described conventional problems, and provides a gene introduction agent capable of more efficiently introducing genes into cells in a medium containing serum, and a method for producing the gene introduction agent. The purpose is to do.
Another object of the present invention is to provide a complex of this gene introduction agent and a nucleic acid.

  The gene transfer agent of the present invention (Claim 1) is a gene transfer agent comprising a polymer material having a branched chain, and the branched chain polymerizes at least 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof. A part of the structural unit derived from 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof of the polymer is a polymer chain that is hydrolyzed. Is.

  The gene introduction agent according to claim 2 is characterized in that, in claim 1, LCST (Lower Critical Solution Temperature) of the gene introduction agent is 40 to 60 ° C.

  The gene introduction agent according to claim 3 is the gene introduction agent according to claim 1 or 2, wherein the gene introduction agent is a compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule, and at least the above-mentioned It is obtained by hydrolyzing a branched polymer obtained by photoirradiation living polymerization of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof.

  The gene introduction agent according to claim 4 is characterized in that, in claim 3, the hydrolysis is performed by dialysis of the branched polymer.

  The gene introduction agent according to claim 5 is the gene transfer agent according to claim 3 or 4, wherein the compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule has a benzene ring as a nucleus and a branched chain in the nucleus. And three or more N, N-dialkyldithiocarbamylmethyl groups bonded to each other.

  The gene introduction agent according to claim 6 is the polymer according to any one of claims 1 to 5, wherein the polymer obtained by polymerizing at least 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof is 2-N. , N-dimethylaminoethyl methacrylate and / or a polymer composed solely of a derivative thereof.

  The gene introduction agent according to claim 7 is characterized in that, in any one of claims 1 to 6, a molecular weight per one of the branched chains is 300 to 60,000.

  The gene introduction agent according to claim 8 is the molecular weight of the structural unit derived from 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof contained in the gene introduction agent according to any one of claims 1 to 7. , 1,200 to 290,000.

  The gene transfer agent according to claim 9 is characterized in that, in any one of claims 1 to 8, the molecular weight of the gene transfer agent is 2,000 to 300,000.

  The method for producing a gene introduction agent according to the present invention (Claim 10) is a method for producing the gene introduction agent according to any one of Claims 1 to 9, wherein the gene introduction agent is radially extended from the aromatic ring to the aromatic ring. A branched polymer production process for producing a branched polymer by introducing a plurality of branched chains, and 2 contained in the branched chain of the branched polymer by hydrolyzing the obtained branched polymer. A hydrolysis step of hydrolyzing a part of the structural unit derived from -N, N-dimethylaminoethyl methacrylate and / or a derivative thereof.

  The method for producing a gene introduction agent according to claim 11 is characterized in that, in claim 10, the hydrolysis step is a step of dialysis of the branched polymer.

  The nucleic acid complex of the present invention (Claim 12) is obtained by combining the gene introduction agent according to any one of Claims 1 to 9 and a nucleic acid.

  In the present invention, 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof (hereinafter, 2-N, N-dimethylaminoethyl methacrylate and / or a derivative may be simply abbreviated as “DMAEM”). ) Based on the novel finding found by the present inventors that the polymer has temperature sensitivity and the side chain portion of the polymer is hydrolyzable. In the gene introduction agent of the present invention, as a branched chain, at least a part of a structural unit derived from DMAEM of a polymer obtained by polymerizing DMAEM (hereinafter sometimes referred to as “DMAEM unit”) is hydrolyzed. Therefore, even when a gene is introduced into cells in a medium containing serum, the gene can be efficiently introduced.

  That is, as described in Patent Document 3, a branched chain composed of a polymer chain having DMAEM as a polymerization component and having a structural unit derived from DMAEM removes the amine solvated to the branched chain by heating. Solvation causes the charge of the branched chain to be as close to zero as possible, and the entire gene transfer agent becomes hydrophobic. In the gene transfer agent of the present invention, the DMAEM unit of such a branched chain Since a part is hydrolyzed, the gene introduction agent as a whole becomes hydrophilic due to the effect of the anionic functional group generated by the hydrolysis. Thereby, the gene introduction agent of the present invention exhibits high gene introduction activity without adsorbing anionic proteins, serum lipids, and the like even in a medium containing serum.

  Especially in the gene introduction agent of this invention, it is preferable that LCST (Lower Critical Solution Temperature) is 40-60 degreeC (Claim 2). When the LCST of the gene introduction agent is 40 to 60 ° C., the gene introduction agent is partially hydrophobic under the conditions of 35 to 37 ° C., which is the optimum temperature for culturing cells. Shows hydrophilicity and does not cause aggregation between gene introduction agents. As a result, the nucleic acid complex consisting of this gene transfer agent is less likely to cause aggregation of serum hydrophobic substances and nucleic acid complexes, and solubilizes hydrophobic substances such as cholesterol and albumin contained in serum. In addition, it is difficult to be affected by components to be absorbed, and further, adsorption to a hydrophobic polystyrene petri dish is prevented.

  In this specification, LCST (Lower Critical Solution Temperature) is a temperature (cloud point) at which an aqueous solution containing a gene introduction agent changes from a transparent solution to a cloudy suspension when the temperature of the aqueous solution containing the gene introduction agent is increased. And the temperature at which the gene transfer agent changes from hydrophilic to hydrophobic and exhibits phase separation behavior. In other words, when the LCST of the gene introduction agent of the present invention is 40 to 60 ° C., this gene introduction agent exhibits hydrophilicity at 35 to 37 ° C., which is a temperature for introducing a gene into cells, and therefore is hydrophobic in serum. The problem of agglomeration with the active substance is less likely to occur.

  The gene transfer agent according to the present invention is a branched polymer obtained by using a compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule as an iniferter and subjecting at least the DMAEM to light irradiation living polymerization. It is preferable that the branched polymer is hydrolyzed (claim 3), and this hydrolysis is particularly preferably carried out by dialysis of the branched polymer ( Claim 4).

  The compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule has a benzene ring as a nucleus and three or more N, N-dialkyldithiocarbamylmethyl groups as branched chains in this nucleus. Are preferably bonded (Claim 5), and the branched chain is preferably a polymer chain composed only of the DMAEM (Claim 6). When the branched chain is a polymer chain composed only of DMAEM, a part of this polymer chain is hydrolyzed to have both a hydrophobic part and a hydrophilic part. Can get to.

  Moreover, it is preferable that the molecular weight per said branched chain is 300-60,000 (Claim 7). When the molecular weight per branched chain is within this range, a gene introduction agent capable of efficiently introducing a gene can be obtained while suppressing the increase in the molecular weight of the gene introduction agent. The total molecular weight of the DMAEM units contained in the gene introduction agent is preferably 1,200 to 290,000 (claim 8). When the molecular weight of the DMAEM unit is within the above range, a gene introduction agent having excellent hydrophilicity can be obtained. Furthermore, the molecular weight of the gene introduction agent is preferably 2,000 to 300,000 (claim 9). If the molecular weight of the gene introduction agent is within the above range, the gene introduction agent and the nucleic acid can be more efficiently combined while suppressing high molecular weight, and the gene has a low molecular weight and excellent gene introduction efficiency. An introducer can be obtained.

  The method for producing a gene introduction agent of the present invention comprises a branched polymer production process for introducing a plurality of branched chains into an aromatic ring, and a DMAEM contained in the branched chain by hydrolyzing the branched polymer. A hydrolysis step of hydrolyzing a part (claim 10), and this hydrolysis step is preferably a step of dialysis of the branched polymer (claim 11).

  The nucleic acid complex of the present invention is a nucleic acid complex formed by complexing the gene introduction agent of the present invention and a nucleic acid (claim 12).

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

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

[Gene transfer agent]
The gene introduction agent of the present invention is a gene introduction agent comprising a polymer material having a branched chain, wherein the branched chain is obtained by polymerizing at least 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof (DMAEM). A polymer, wherein a part of the DMAEM unit of the polymer is a hydrolyzed polymer chain.

  As described above, the gene introduction agent of the present invention has a branched chain composed of a polymer chain containing a structural unit derived from DMAEM, and a part of the DMAEM unit in the branched chain is hydrolyzed. Because of the anionic functional group generated by this hydrolysis, the entire gene transfer agent shows hydrophilicity, hardly adsorbs anionic proteins and lipids in serum, and even in a medium containing serum, efficiency Gene can be introduced automatically.

  The method for producing such a gene introduction agent of the present invention is not particularly limited, but a branched polymer for producing a branched polymer by introducing a plurality of branched chains radially extending from the aromatic ring into the aromatic ring. The gene introduction agent of the present invention comprising a coalescence production step and a hydrolysis step of hydrolyzing a part of the DMAEM unit contained in the branched chain of the branched polymer by hydrolyzing the obtained branched polymer It is preferable to manufacture by this manufacturing method.

  Hereinafter, the gene introduction agent of the present invention will be described in accordance with the production procedure when the gene introduction agent of the present invention is produced, but the method of producing the gene introduction agent of the present invention is limited to the following methods. It is not something.

<Branched polymer production process>
The branched polymer used in the present invention is preferably a compound in which a compound having three or more N, N-disubstituted dithiocarbamylmethyl groups in the same molecule is used as an iniferter, and at least DMAEM is subjected to light irradiation living polymerization on the iniferter. .

  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 an aromatic 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 compound in which three or more dialkyldithiocarbamylmethyl 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-dialkyldithiocarbamylmethyl 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 group. That is, not only N, N-dialkyldithiocarbamylmethyl group but also N, N-substituted by aliphatic hydrocarbon group and / or aromatic hydrocarbon group including N, N-diaryldithiocarbamylmethyl group and the like. A di-substituted dithiocarbamylmethyl group can achieve the object.

  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 an alkylene halide, and particularly preferably benzene, toluene, chloroform or methylene chloride, especially toluene.

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

  The concentration of DMAEM in the raw material solution is preferably 0.5 M or more, for example, 0.5 M to 2.5 M, and the concentration of iniferter is preferably about 1 to 20 mM.

The wavelength of the irradiated light is preferably 250 to 400 nm. For example, a short arc xenon lamp, a low pressure mercury lamp, a high pressure mercury lamp, or the like can be used. The irradiation time of the light depends on the irradiation intensity, about 1 to 90 minutes are preferred, about 1 to 60 minutes at a low irradiation intensity of about 1μW ^/ cm ² ~10mW ^/ cm 2 is particularly preferred.

  By this light irradiation, the target branched polymer is produced in the reaction solution. Therefore, by purifying as necessary, a polymer chain composed of DMAEM units is introduced into the branched chain portion, and the end of the branched chain is N, A homopolymer which is an N-disubstituted dithiocarbamylmethyl group is obtained.

  The molecular weight per branched chain of this branched polymer is preferably about 300 to 60,000, particularly about 3,000 to 30,000. This molecular weight can be adjusted by controlling the light irradiation time. That is, by extending the reaction time, the polymerization reaction can be advanced to obtain a branched polymer having a large molecular weight.

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

  The branched chain of the branched polymer according to the present invention is preferably a homopolymer composed of only one monomer having the above-mentioned DMAEM as a monomer, but one or more monomers different from DMAEM and DMAEM are introduced. It may be a block copolymer or a random copolymer.

As other monomers in this case, vinyl monomers such as acrylic acid derivatives and styrene derivatives are suitable. Specifically, N, N-dimethylacrylamide, methoxyethyl (meth) acrylate, 3-N, N- Dimethylaminopropylacrylamide CH ₂ = CHCONHC ₃ H ₆ N (CH ₃ ) ₂ , 4-N, N-dimethylaminostyrene, and at least one vinyl monomer selected from the group consisting of 4-aminostyrene derivatives In particular, cationic vinyl monomers such as 3-N, N-dimethylaminopropylacrylamide CH ₂ ═CHCONHC ₃ H ₆ N (CH ₃ ) ₂ are preferable. These vinyl monomers may be used alone or in combination of two or more.

  In order to react an iniferter with a monomer different from DMAME and DMAEM, a raw material solution containing monomers other than the iniferter, DMAEM, and DMAEM is prepared in the same manner as in the case of reacting the iniferter with DMAEM. Is irradiated with light to obtain a random copolymer in which monomers other than DMAEM and DMAEM are bonded to the iniferter.

In addition, to the above iniferter, first, DMAEM is block-polymerized to form a homopolymer, and then 3-N, N-dimethylaminopropylacrylamide is block-polymerized to the homopolymer, and the base end side of the branched chain is The block polymer of DMAEM may be a branched chain composed of 3-N, N-dimethylaminopropylacrylamide block polymer on the tip side of the branched chain. Thus, when making a branched chain into a block copolymer of two or more types of monomers, the order of polymerization with respect to the iniferter is arbitrary.
In either case, the end of the branched chain is an N, N-disubstituted dithiocarbamylmethyl group.

<Hydrolysis step>
By hydrolyzing the branched polymer obtained in the branched polymer production process, a part of the DMAEM unit constituting the branched chain of the branched polymer is hydrolyzed.

  The branched polymer can be hydrolyzed by a general method under alkaline conditions, but is preferably performed by dialysis of the branched polymer. For example, it is preferable to prepare an aqueous solution of about 1 to 20% by weight of the branched polymer, transfer the prepared solution to a dialysis cellulose tube, and dialyze it with desalted water as a dialysis solution. The preferred temperature for dialysis in this way is about 20 to 30 ° C., and the dialysis time varies depending on the degree of hydrophilicity of the target gene introduction agent, but usually, from the dissolution of the branched polymer. It is preferably about 0.5 to 30 hours, particularly about 1 to 20 hours when counted. The target gene introduction agent can be obtained by freeze-drying the obtained polymer after elapse of a predetermined dialysis time.

As a gene introduction agent of the present invention that can be obtained by such a production method, LCST (Lower Critical Solution Temperature) is preferably about 40 to 60 ° C, particularly about 40 to 50 ° C. If the LCST is less than the above lower limit, the gene introduction agent exhibits hydrophobicity in a cell culture environment, and thus the nucleic acid complex comprising the gene introduction agent and the nucleic acid or between the nucleic acid complex and the serum is contained. Gene transfer efficiency decreases due to aggregation of hydrophobic substances. In addition, the nucleic acid complex may be affected by a hydrophobic substance-solubilizing component in the serum, or it may cause problems such as being easily adsorbed on a polystyrene dish. In addition, when the LCST exceeds the above upper limit, the gene introduction agent aggregates in an ion-binding manner within and / or between the molecules of the gene introduction agent, or the nucleic acid complex with the gene introduction agent is an anionic polymer. It becomes difficult to form. If the hydrolysis is excessively performed, the number of anionic sites in the side chain increases, and the gene introduction agent that is originally a cationic polymer is changed to anionic, and the LCST tends to exceed the above upper limit.
In addition, the measuring method of LCST is as describing in the term of the below-mentioned Example.

  As described above, the molecular weight per branched chain of the branched polymer subjected to hydrolysis is preferably about 300 to 60,000, particularly about 1,000 to 30,000. The total molecular weight of the DMAEM units contained in the agent is preferably about 1,200 to 290,000, particularly preferably about 10,000 to 150,000, although it depends on the number of branched chains. In this case, the total molecular weight of the DMAEM units contained in the gene introduction agent is the total molecular weight of the unhydrolyzed DMAEM units and the hydrolyzed DMAEM units.

  In the DMAEM unit contained in the gene introduction agent of the present invention, the ratio of hydrolyzed DMAEM is not particularly limited as long as it satisfies the above-mentioned LCST. That is, in the present invention, it is preferable to hydrolyze the aforementioned branched polymer so as to satisfy the above-mentioned LCST. The degree of hydrolysis can be easily controlled by adjusting the dialysis time in the above-described hydrolysis by dialysis, that is, the longer the dialysis time, the higher the hydrolysis rate, and thus the higher the LCST, A highly hydrophilic gene transfer agent can be obtained.

  Further, the molecular weight of the gene introduction agent of the present invention is preferably about 2,000 to 300,000, particularly preferably about 10,000 to 150,000.

[Nucleic acid complex]
By enclosing the nucleic acid as a nucleic acid-containing complex by the gene introduction agent (vector) of the present invention, it is possible to suppress inactivation and degradation of the nucleic acid by an enzyme in the living body.

  In order to combine the gene introduction agent and the nucleic acid, the nucleic acid may be added to and mixed with a solution of the gene introduction agent having a concentration of about 0.1 to 1000 μg / mL. It is preferable to add an excessive amount of the gene introduction agent to the nucleic acid and to saturate the gene introduction agent with respect to the nucleic acid so that the gene introduction agent and the nucleic acid are combined.

  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. Such a series of genes is well known to those skilled in the art.

  The particle size of the nucleic acid complex is preferably about 50 to 400 nm. This particle size is measured by, for example, a dynamic light scattering method using a laser. If the particle size 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, 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 complex of the present invention can be administered to a living body by any method besides being used for a culture test.

  Intravenous or arterial injection is particularly preferable as a method of administration to a living body, but intramuscular, adipose tissue, subcutaneous, intradermal, intralymphatic, intralymphatic, body cavity (pericardial cavity, thoracic cavity, abdominal cavity, brain) In addition to administration into the spinal cavity and the like, it is also possible to administer directly into the diseased tissue.

  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 method of administration, 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.) .

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples unless it exceeds the gist.

i) Synthesis of Iniferter 1,2,4,5-Tetrakis (N, N-diethyldithiocarbamylmethyl) benzene as an iniferter was synthesized as follows.

  5.0 g of 1,2,4,5-tetrakis (bromomethylbenzene) and 34.0 g of sodium N, N-diethyldithiocarbamate were added to 100 mL of ethanol and stirred at room temperature for 4 days under light shielding. The precipitate was filtered, poured into 3 L of methanol, stirred for 30 minutes, and then filtered. This operation was repeated a total of 4 times. After the precipitate was dissolved in 200 mL of toluene, 100 mL of methanol was added and heated to 50 ° C., stored in a refrigerator for 15 hours for recrystallization, and the crystals were separated by filtration and washed with a large amount of methanol. The crystals were dried at room temperature under reduced pressure to obtain white needle-like crystals of 1,2,4,5-tetrakis (N, N-diethyldithiocarbamylmethyl) benzene (yield 90%). High-performance liquid chromatography confirmed that the raw material peak disappeared and the purified product was a single substance.

The measurement result of ¹ H-NMR (in CDCl ₃ ) is as follows: δ 1.26-1.31 ppm (t, 24H, CH ₂ CH ₃ ), δ 3.69-3.77 ppm (q, 8H, N (CH ₂ CH _{3 2} ), δ 3.99-4.07 ppm (q, 8H, N (CH ₂ CH ₃ ) ₂ ), δ 4.57 ppm (s, 8H, Ar—CH ₂ ), δ 7.49 ppm (s, 2H, Ar— H).

ii) Synthesis of a thermosensitive cationic homopolymer comprising a 4-branched star polymer by photopolymerization 1,2,4,5-tetrakis [(N , N-diethyldithiocarbamyl) poly (2-N, N-dimethylaminoethyl methacrylate) -methyl] benzene (hereinafter sometimes referred to as pDMAEMAA) was synthesized. It was.

Namely, 45.6 mg of 1,2,4,5-tetrakis (N, N-diethyldithiocarbamylmethyl) benzene synthesized by i) above was dissolved in 20 mL of toluene, and 2-N, N-dimethylaminoethyl methacrylate was dissolved. 8.5 g was added and mixed, and the total amount was adjusted to 50 mL with toluene. After purging with high-purity nitrogen gas for 10 minutes with vigorous stirring in a 3 mm thick soft glass cell, irradiation with mixed ultraviolet rays having a wavelength of 250 to 400 nm for 60 minutes is performed with a 300 W short arc xenon lamp (manufactured by Asahi Spectroscopic Co., Ltd., MAX-301). did. The irradiation intensity was adjusted to 2.5 mW / cm ² by attaching UVD-C405 (detection wavelength range of 320 to 470 nm) to UIT-150 manufactured by USHIO. The polymerization solution is concentrated with an evaporator, the polymer is reprecipitated with n-hexane, purified by repeating reprecipitation three times with a chloroform / n-hexane system, and n-hexane is evaporated and dissolved in a small amount of benzene. The solution was filtered through a 0.2 μm filter and then freeze-dried to obtain a temperature-sensitive cationic homopolymer composed of the target 4-branched star polymer (pDMAEMAA). The number average molecular weight using polyethylene glycol of this thermosensitive cationic homopolymer as a standard substance was measured by GPC as 11,000 (Mw / Mn = 1.4). In addition, the molecular weight per branched chain was found to be 2570 by calculation.

The measurement result of ¹ H-NMR (in CD ₃ OD) is as follows: δ 0.8-1.2 ppm (br, 3H, —CH ₂ —CH ₃ —), δ 1.6-2.0 ppm (br, 2H, —CH _{2 -CH 3 -), δ2.2-2.4ppm (} br, 6H, N-CH 3), δ2.5-2.7ppm (br, 2H, CH 2 -N), δ4.0-4.2ppm (br, 2H, O-CH 2) was.

iii) Photopolymerization of a non-thermosensitive cationic homopolymer comprising a 4-branched star polymer 1,2,4,5-tetrakis using 3-N, N-dimethylaminopropylacrylamide as a cationic monomer Non-thermosensitive cationic homopolymer comprising [(N, N-diethyldithiocarbamyl) poly (3-N, N-dimethylaminopropylacrylamide) -methyl] benzene (hereinafter sometimes referred to as pDMAPAAm) Was synthesized.

Namely, 45.6 mg of 1,2,4,5-tetrakis (N, N-diethyldithiocarbamylmethyl) benzene synthesized by i) above was dissolved in 20 mL of toluene, and 3-N, N-dimethylaminopropylacrylamide was dissolved. 3.4 g of (3-N, N-DMAPAAm) was added and mixed, and the total amount was adjusted to 50 mL with toluene. After purging with high-purity nitrogen gas for 5 minutes with vigorous stirring in a 3 mm thick soft glass cell, irradiation with mixed ultraviolet rays having a wavelength of 250 to 400 nm was performed for 15 minutes with a 300 W short arc xenon lamp (manufactured by Asahi Spectroscopic Co., Ltd., MAX-301). . The irradiation intensity was adjusted to 2.5 mW / cm ² by attaching UVD-C405 (detection wavelength range of 320 nm to 470 nm) to UIT-150 manufactured by USHIO. The polymerization solution is concentrated with an evaporator, the polymer is reprecipitated with diethyl ether, purified by repeated reprecipitation three times with chloroform / diethyl ether system, ether is evaporated and dissolved in a small amount of water, 0.2 μm After filtering with a filter, it was freeze-dried to obtain a non-thermosensitive cationic homopolymer composed of the target 4-branched star polymer (pDMAPAAm). The molecular weight of this non-thermosensitive cationic homopolymer was measured by GPC as 13,000 (Mw / Mn = 1.3). Moreover, it turned out that the molecular weight per branch chain is 3070 by calculation.

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-).

iv) LCST measurement of thermosensitive cationic homopolymer and non-thermosensitive cationic homopolymer
A temperature-sensitive cationic homopolymer synthesized in ii) and a non-temperature-sensitive cationic homopolymer synthesized in iii) are prepared, and the temperature at which the aqueous solution becomes cloudy by increasing the temperature of the aqueous solution (LCST: The cloud point was measured.

  That is, 3% by weight aqueous solutions of each homopolymer were prepared, and the absorbance of light having a wavelength of 600 nm in the temperature range of 20 to 40 ° C. of these aqueous solutions was measured. From this measurement result, the temperature-sensitive cationic homopolymer (pDMAEMAA) synthesized in ii) has LCST (Lower Critical Solution Temperature) around 32 ° C and is hydrophobic in a cell culture environment (37 ° C). It was found to be a temperature-sensitive polymer that becomes an uncharged polymer and is suspended in water. On the other hand, the non-thermosensitive cationic homopolymer (pDMAPAAm) synthesized in iii) showed a transmittance of 100% in the range of 20 to 40 ° C. without turbidity. I knew it wasn't something to do.

v) Partial hydrolysis of the side chain of a thermosensitive cationic homopolymer comprising a 4-branched star polymer
50 mL of a 10% by weight aqueous solution of the thermosensitive cationic homopolymer synthesized in ii) was prepared. About 10 mL each of this preparation solution was quickly transferred to five dialysis cellulose tubes (“UC36-32” manufactured by Viscose) to prepare samples A to E, and dialyzed at 25 ° C. using demineralized water as a dialysis solution. Sample A was dialyzed for 1 hour from the dissolution of the polymer, and Samples B to E were the same as Sample A except that the dialysis times were 2 hours, 4 hours, 24 hours, and 120 hours, respectively. Dialysis was performed. Each sample was taken out from the dialysate after a predetermined dialysis time, and rapidly frozen at -70 ° C., then the upper end of the tube was opened and transferred to a vacuum, and freeze-dried.

  3 wt% aqueous solutions of powders after freeze-drying of samples A to E were prepared, and the absorbance of light at a wavelength of 600 nm in the temperature range of 20 to 60 ° C. of these aqueous solutions was measured. The LCST of each sample was 33.0 ° C for sample A, 34.1 ° C for sample B, 36.1 ° C for sample C, 38.9 ° C for sample D, and 41.6 ° C for sample E. From this result, the longer the dialysis time, the more the hydrolysis of the DMAEM part of the side chain proceeds, the amount of anionic functional groups increases, and the solvation of the branched chain occurs when the temperature of the solvent rises. The amine that had been desolvated and the branched chain changes to hydrophobic, but the anionic functional group generated by hydrolysis makes the entire gene transfer agent hydrophilic and the solubility of the gene transfer agent in water. It turns out that becomes high.

  The chemical formula for the expected hydrolysis of the branched chain of the thermosensitive cationic homopolymer synthesized in ii) is shown. This chemical formula shows hydrolysis of one structural unit derived from 2-N, N-dimethylaminoethyl methacrylate contained in the branched chain.

vi) Evaluation of gene transfer activity Gene transfer activity was evaluated according to the following procedures (1) to (3).
(1) Preparation of a polymer solution of a gene introduction agent and evaluation of gene introduction activity were performed.
COS-1 derived from African green monkey kidney cells was used for the cells, and pGL3 control vector was used for the DNA. The number of COS-1 cells was adjusted to 40,000 cells / mL, seeded in a 24 Well culture dish, and gene transfer was performed after 24 hours of culture. The non-thermosensitive cationic homopolymer synthesized in the above iii) is used as the gene introduction agent of Comparative Example 1, the thermosensitive cationic homopolymer synthesized in the above ii) is used as the gene introduction agent of Comparative Example 2, and the above Sample E hydrolyzed in v) (with LCST of 41.6 ° C.) was used as the gene introduction agent of Example 1, dissolved in physiological saline, and the concentration (polymer / saline) was 8 μg / 60 μL. A polymer solution was obtained. The DNA was dissolved in TE buffer, and the concentration (DNA / buffer) was adjusted to 3 μg / 90 μL.

(2) Evaluation of gene introduction activity in medium containing serum The gene introduction activity of the gene introduction agents of Example 1 and Comparative Examples 1 and 2 in a medium containing serum was evaluated.
Each polymer solution prepared in (1) above and the DNA solution were mixed at room temperature (25 ° C.), and then the solution was heated to 37 ° C. to form a nucleic acid complex. 25 μL of this nucleic acid complex was added to 1 mL of complete medium (DMEM + 10 wt% FCS + antibiotics) and incubated for 30 minutes. About 1 mL of the nucleic acid complex complete medium solution was brought into contact with the cultured cells washed with PBS at 37 ° C., and cultured for 48 hours. After 48 hours, 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.

(3) Evaluation of gene transfer activity in a medium not containing serum The gene transfer activity of the gene transfer agents of Example 1 and Comparative Examples 1 and 2 in a medium not containing serum was evaluated.
Each polymer solution prepared in (1) above and the DNA solution were mixed at room temperature (25 ° C.), and then the solution was heated to 37 ° C. to form a nucleic acid complex. 25 μL of this nucleic acid complex was added to 200 μL of serum-free medium (OPTI-MEM) and incubated for 30 minutes. About 200 μL of the OPTI-MEM solution of this nucleic acid complex was added to cultured cells washed with PBS at 37 ° C., and after 3 hours of transfection, 1 mL of complete medium was added, followed by further incubation for 45 hours. After 45 hours, 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 evaluation results of (2) and (3) are shown in FIG.

[Discussion]
The gene introduction agent of Comparative Example 1 showed high gene introduction activity in a medium not containing serum, but the gene introduction activity was reduced in a medium containing serum. This is because the gene introduction agent of Comparative Example 1 is composed of a high pKa cationic polymer, so that DNA can be condensed efficiently. However, in a medium containing serum, it is affected by anionic proteins in the serum. This is probably because of this. On the other hand, the gene introduction agent of Comparative Example 2 showed the same level of gene introduction activity in both the medium containing serum and the medium not containing serum, but the gene introduction activity was lower in the medium containing serum. It has become. This is because the gene introduction agent of Comparative Example 2 is composed of a temperature-sensitive cationic homopolymer, so that the gene introduction agent changes to hydrophobic when heated, and the influence of anionic proteins contained in the serum is affected. Although it has become difficult to receive, the influence of components that solubilize hydrophobic substances such as cholesterol and albumin contained in serum, as well as hydrophobic substances contained in serum and gene transfer agents, or gene transfer agents aggregate together It is thought to be for receiving.

  In contrast, the gene introduction agent of Example 1 showed lower gene introduction activity than the gene introduction agents of Comparative Examples 1 and 2 in the medium not containing serum, but was compared in the medium containing serum. The gene transfer activity was higher than that of the gene transfer agents of Examples 1 and 2. This is probably because the gene introduction agent of Example 1 exhibits hydrophilicity. That is, in the gene introduction agent of Example 1, like the gene introduction agent of Comparative Example 2, the amine solvated to the branched chain is desolvated by the increase in temperature, and the charge of the branched chain is reduced to zero. Although it approaches, it changes to hydrophobicity, but it is thought that it is because the anionic functional group produced | generated by hydrolysis shows hydrophilicity as a whole gene introduction agent, As a result, the gene introduction agent of Example 1 is contained in serum. It is presumed that high gene transfer activity was exhibited without being affected by the hydrophobic substances and components that solubilize the hydrophobic substances contained in serum.

Claims (12)

In a gene introduction agent comprising a polymer material having a branched chain,
The branched chain is a polymer obtained by polymerizing at least 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof, and the 2-N, N-dimethylaminoethyl methacrylate of the polymer and / or its A gene transfer agent, wherein a part of the structural unit derived from a derivative is a hydrolyzed polymer chain.   The gene introduction agent according to claim 1, wherein the gene introduction agent has an LCST (Lower Critical Solution Temperature) of 40 to 60 ° C.   3. The gene introduction agent according to claim 1, wherein the gene introduction agent is a compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule as an iniferter, and at least the 2-N, N-dimethylaminoethyl A gene transfer agent, which is obtained by hydrolyzing a branched polymer obtained by light-irradiating living polymerization of methacrylate and / or a derivative thereof.   The gene introduction agent according to claim 3, wherein the hydrolysis is performed by dialysis of the branched polymer.   5. The compound according to claim 3 or 4, wherein the compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule has a benzene ring as a nucleus and three or more N, N as branched chains in the nucleus. -A gene introduction agent characterized by having a dialkyldithiocarbamylmethyl group bonded thereto.   The polymer obtained by polymerizing at least 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof according to any one of claims 1 to 5, wherein 2-N, N-dimethylaminoethyl methacrylate and / or Alternatively, a gene introduction agent characterized by being a polymer composed only of a derivative thereof.   The gene introduction agent according to any one of claims 1 to 6, wherein the molecular weight per one of the branched chains is 300 to 60,000.   The molecular weight of the structural unit derived from 2-N, N-dimethylaminoethyl methacrylate and / or its derivative contained in the gene introduction agent according to any one of claims 1 to 7 is 1,200 to 290,000. A gene introduction agent characterized by being.   The gene introduction agent according to any one of claims 1 to 8, wherein the gene introduction agent has a molecular weight of 2,000 to 300,000. A method for producing the gene introduction agent according to any one of claims 1 to 9,
A branched polymer production process for producing a branched polymer by introducing a plurality of branched chains extending radially from the aromatic ring into the aromatic ring;
By hydrolyzing the obtained branched polymer, a part of the structural unit derived from 2-N, N-dimethylaminoethyl methacrylate and / or its derivative contained in the branched chain of the branched polymer is hydrolyzed. A method for producing a gene introduction agent, comprising a hydrolysis step.   The method for producing a gene introduction agent according to claim 10, wherein the hydrolysis step is a step of dialysis of the branched polymer.   A nucleic acid complex obtained by combining the gene introduction agent according to any one of claims 1 to 9 and a nucleic acid.

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