JP2011207813A - Method for producing gene transfer agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a gene transfer agent, whereby the gene transfer agent exerting an excellent gene transfer activity in a medium containing blood serum can be efficiently produced by hydrolyzing a side chain in a branched chain within a short time.SOLUTION: The method for producing the gene transfer agent comprising a branched polymer having the branched chain comprises: a branched polymer preparation step of preparing the branched polymer by introducing into an aromatic ring a plurality of branched chains that extend radially from the aromatic ring and are obtained by polymerizing at least 2-N, N-dialkyl aminoalkyl methacrylate and/or a derivative thereof; and a hydrolysis step of hydrolyzing the obtained branched polymer. The hydrolysis step comprises a step of passing a solution containing the branched polymer through a size-exclusion column.

Description

  The present invention relates to a method for producing a gene introduction agent comprising a branched polymer having a branched chain, and more specifically, efficiently produces a gene introduction agent exhibiting excellent gene introduction activity in a medium containing serum. Regarding the method.

  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. Synthetic polymer vectors and cationic lipid vectors have been researched and developed as vectors for introducing foreign DNA.

  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 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. For this reason, it is possible to more strongly aggregate complexes with nucleic acids such as DNA and RNA, and to form polyplex particles that can more strongly protect nucleic acids from the action of enzymes and the like, improving gene transfer activity did. However, the gene introduction agent containing this cationic polymer does not show high gene introduction activity in a medium containing serum, which is an optimum environment for culturing cells. This is because the gene introduction agent containing the above-mentioned cationic polymer has a high zeta potential even under neutral conditions at a pH of about 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 agent capable of efficiently introducing a gene even in a medium containing serum, the present applicants show a cationic property at a temperature below a predetermined temperature and a hydrophilic property as a whole of the gene introduction agent at a temperature above a predetermined temperature. A gene introduction agent comprising a warm cationic polymer was found and a patent application was filed first (Patent Document 3).

  The gene introduction agent described in Patent Document 3 introduces a polymer obtained by polymerizing at least 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof as a branched chain of the branched polymer. Since a part of is hydrolyzed, the gene can be efficiently introduced even when the gene is introduced into the cell in a medium containing serum. That is, in the gene introduction agent of Patent Document 3, since the whole gene introduction agent becomes hydrophilic due to the effect of the anionic functional group generated by hydrolysis, a medium containing serum is thereby obtained. Also exhibits high gene transfer activity without adsorbing anionic proteins or lipids in serum. In the Example of this patent document 3, the above-mentioned hydrolysis is performed by dialyzing at 25 degreeC for 120 hours by using RO water as a dialysis solution.

WO2004 / 092388 JP 2007-70579 A Japanese Patent Application No. 2009-227405

  The gene introduction agent described in Patent Document 3 exhibits excellent gene introduction activity and is highly useful as a gene introduction agent. However, since the time required for production is long, the production time is reduced. It is desirable to shorten it. That is, in general hydrolysis, the reaction can be promoted by warming the reaction solution. However, when the branched polymer according to Patent Document 3 is heated, the branched polymer is desolvated in water. It is combined to form a globule colloidal particle and its affinity with water is reduced, making it impossible to hydrolyze the entire molecule uniformly, and in particular, hydrolysis of the inner part of the colloidal particle is difficult to proceed. Become. Therefore, in Patent Document 3, hydrolysis is performed by dialysis at a temperature at which the branched polymer is uniformly dissolved in water. However, at this temperature, hydrolysis does not proceed easily, and production takes time. Further improvement is desired.

  The present invention has been made in view of the above-described conventional situation, and shortens the time required for hydrolysis of a branched polymer as in Patent Document 3, and exhibits gene transfer activity excellent in a medium containing serum. It aims at providing the manufacturing method of the gene introduction agent which can manufacture an agent efficiently.

  The method for producing a gene introduction agent according to the present invention (Claim 1) is a method for producing a gene introduction agent comprising a branched polymer having a branched chain, 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 obtained by polymerizing N, N-dialkylaminoalkyl methacrylate and / or a derivative thereof, and the obtained branched polymer A method for producing a gene introduction agent comprising a hydrolysis step for hydrolysis, wherein the hydrolysis step includes a step of passing a solution containing the branched polymer through a size exclusion column. Is.

The method for producing a gene introduction agent according to claim 2 is characterized in that, in claim 1, the pressure in the size exclusion column is 30 to 80 kg / cm ² .

  According to a third aspect of the present invention, there is provided the method for producing a gene introduction agent according to the first or second aspect, wherein the size exclusion column is filled with porous polymer particles.

  The method for producing a gene introduction agent according to claim 4 is characterized in that, in claim 3, the porous polymer particles have a particle diameter of 10 μm or less.

  The method for producing a gene introduction agent according to claim 5 is the method according to claim 3 or 4, wherein the porous polymer particles are selected from the group consisting of styrene / divinylbenzene copolymer, polyvinyl alcohol, and polyacrylamide. It is characterized by being.

The method for producing a gene introduction agent according to claim 6 is the method according to claim 5, wherein the porous polymer particles are a styrene / divinylbenzene copolymer, and the pressure in the size exclusion column is 40 to 80 kg / cm ^2. It is a feature.

The method for producing a gene introduction agent according to claim 7 is characterized in that, in claim 5, the porous polymer particles are polyvinyl alcohol, and the pressure in the size exclusion column is 30 to 40 kg / cm ^2. is there.

  The method for producing a gene introduction agent according to claim 8 is characterized in that, in any one of claims 1 to 7, the number of theoretical plates of the size exclusion column is 1,000 / 10 cm or more.

  The method for producing a gene introduction agent according to claim 9 is characterized in that, in any one of claims 1 to 8, the gene introduction agent has an LCST (Lower Critical Solution Temperature) of 37 to 60 ° C. It is.

  The method for producing a gene introduction agent according to claim 10 is the method according to any one of claims 1 to 9, wherein the branched polymer has three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule. The compound is an iniferter and is obtained by light-irradiating living polymerized with at least the 2-N, N-dialkylaminoalkyl methacrylate and / or its derivative.

  The method for producing a gene introduction agent according to claim 11 is the method according to claim 10, wherein the compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule has a benzene ring as a nucleus and branches to this nucleus. It is characterized in that three or more N, N-dialkyldithiocarbamylmethyl groups are bonded as a chain.

  The method for producing a gene introduction agent according to claim 12 is characterized in that, in any one of claims 1 to 11, the molecular weight of the gene introduction agent is 2,000 to 600,000.

  In the method for producing a gene introduction agent of the present invention, at least 2-N, N-dialkylaminoalkyl methacrylate and / or a derivative thereof (hereinafter, 2-N, N-dialkylaminoalkyl methacrylate and / or a derivative thereof is referred to as “DAAAM”. The hydrolysis of the branched chain DAAAM-derived structural unit (hereinafter, the DAAAM-derived structural unit may be referred to as the “DAAAM unit”) obtained by polymerizing a size exclusion column This can be done in a short time. In addition, since the hydrolysis is performed using a size exclusion column, the gene introduction agent can be purified simultaneously with the hydrolysis.

The pressure is preferably 30 to 80 kg / cm ² (Claim 2). If the pressure is within the above range, the hydrolysis time can be further shortened.

  The size exclusion column is preferably packed with porous polymer particles (Claim 3), and the porous polymer particles preferably have a particle size of 10 μm or less (Claim 4). .

  The porous polymer particles are preferably at least one selected from the group consisting of a styrene / divinylbenzene copolymer, polyvinyl alcohol, and polyacrylamide (Claim 5).

When styrene / divinylbenzene copolymer particles are used as the porous polymer particles, the pressure in the size exclusion column is preferably 40 to 80 kg / cm ² (Claim 6). When polyvinyl alcohol particles are used, the pressure in the size exclusion column is preferably 30 to 40 kg / cm ² (Claim 7).

  The theoretical plate number of the size exclusion column is preferably 1,000 / 10 cm or more. If the number of theoretical plates is within the above range, the hydrolysis can be performed in a short time, and the alcohol generated by the hydrolysis and the target gene introduction agent can be separated.

  The gene introduction agent preferably has a LCST (Lower Critical Solution Temperature) of 37 to 60 ° C. (Claim 9). When the LCST of the gene introduction agent is 36 ° C. or higher, particularly 37 to 60 ° C., the gene introduction agent is partially hydrophobic under conditions of 35 to 37 ° C., which is the optimum temperature for culturing cells. As shown, it exhibits hydrophilicity as a whole 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.

  The branched polymer is preferably 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 DAAAM to light irradiation living polymerization. (Claim 10) The compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule has a benzene ring as a nucleus, and the nucleus has three or more N, N- It is preferable that a dialkyldithiocarbamylmethyl group is bonded (claim 11).

  The gene introduction agent preferably has a molecular weight of 2,000 to 600,000 (claim 12). 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.

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

[1] Method for producing gene introduction agent The method for producing a gene introduction agent of the present invention comprises a branched polymer production step for introducing a plurality of branched chains obtained by polymerizing DAAAM, which is radially extended into an aromatic ring, A hydrolysis step of hydrolyzing the branched polymer obtained in the step, wherein the hydrolysis step includes a step of passing a solution containing the branched polymer through a size exclusion column. It is characterized by.

  According to the method for producing a gene introduction agent of the present invention, a target gene introduction agent can be obtained in a short time by a simple operation of passing a solution containing a branched polymer through a size exclusion column. Purification can also be performed.

  In explaining the method for producing the gene introduction agent of the present invention, first, the hydrolysis step will be explained, and then the branched polymer production step will be explained.

[Hydrolysis step]
In the hydrolysis step according to the method for producing a gene introduction agent of the present invention, specifically, a solution containing a branched polymer is passed through a size exclusion column packed with porous polymer particles, whereby a branched type is obtained. Hydrolysis of the polymer can be performed.

The flow rate of the solution containing the branched polymer in the present invention is preferably 0.1 mL / min or more, for example, about 0.1 to 10 mL / min, particularly preferably about 0.5 to 5.0 mL / min. the pressure in the size exclusion column is generally 30kg / cm ² or more, it is preferable that the particular 50~70kg / cm ² approximately. Further, when particles made of a styrene / divinylbenzene copolymer are used as the porous polymer particles, it is preferable to adjust the pressure in the size exclusion column to 40 to 80 kg / cm ^2. When particles made of polyvinyl alcohol are used, it is preferable to adjust the pressure in the size exclusion column to 30 to 40 kg / cm ² . If the liquid flow rate and pressure are within the above ranges, hydrolysis can be performed in a shorter time. The concentration of the branched polymer in the solution is preferably about 1 to 300 g / L and about 10 to 100 g / L. If the concentration of the branched polymer is high, the pressure may become too high or the size exclusion column may be blocked. If the concentration is low, the amount of the branched polymer that can be hydrolyzed in one hydrolysis step It is not efficient because it decreases.

  The diameter of the size exclusion column used in the present invention is preferably about 5 to 40 mm, and the length of the size exclusion column is preferably about 150 to 600 mm. If the diameter and length of the column are larger than the above range, a large equipment is required and the amount of the solvent to be used increases, so that the cost increases. The production efficiency is low because of the limited amount.

  The particle size of the porous polymer particles is preferably 10 μm or less, particularly about 3 to 8 μm. When the particle size of the porous polymer particles is large, the pressure in the size exclusion column is difficult to increase. Even if the particle size exceeds the above range, the pressure can be increased by adjusting the flow rate and the length of the size exclusion column, but the amount of solvent used, the amount of porous polymer particles This is not preferable because it increases the number of devices and requires large equipment. In this hydrolysis step, it is considered that the side chain is broken by shearing stress in addition to the side chain decomposition by hydrolysis. This shear stress is considered to be generated more efficiently when porous polymer particles having a particle size within the above range are used.

  The number of theoretical plates of the size exclusion column is preferably about 1,000 to 6,000 / 10 cm. When the number of theoretical plates is large, a gene introduction agent with higher purity can be obtained. However, the time required for the hydrolysis process becomes long, and when the number of theoretical plates is small, the purity of the resulting gene introduction agent may be lowered. Such a size exclusion column may be used alone or in combination with two or more different types of size exclusion columns.

  The treatment conditions in the hydrolysis using the size exclusion column of the present invention differ depending on the type of porous polymer particles packed in the size exclusion column, and even when the same porous polymer particles are used, Depending on the solvent used, etc., it is necessary to set conditions so that efficient hydrolysis can be performed each time, and it is not possible to define the conditions in general. For example, the following treatments (1) and (2) Conditions can be used. The following (1) and (2) are examples of the treatment conditions in the present invention, and the hydrolysis step according to the present invention uses the size exclusion column to hydrolyze the aforementioned branched polymer. It is not limited to the following in the range which performs.

(1) When the porous polymer particles are made of a styrene / divinylbenzene copolymer << mobile phase (solvent) >>
Solvents when using a size exclusion column packed with porous polymer particles made of styrene / divinylbenzene copolymer include DMF (N, N-dimethylformamide), ethyl acetate, DMSO (dimethyl sulfoxide), dioxane, dimethyl Acetamide, xylene and toluene are preferred. Since the porous polymer particles are harder than the other porous polymer particles, the pressure in the size exclusion column can be easily increased by using such a solvent.

<< pore diameter of porous polymer particles >>
The pore diameter of the porous polymer made of a styrene / divinylbenzene copolymer is preferably about 5 to 1,000 nm, particularly about 30 to 600 nm.

≪Temperature≫
As a temperature of a size exclusion column, 30 degreeC or more, for example, about 40-80 degreeC, especially about 40-60 degreeC are preferable. By setting the temperature within the above range, the temperature control is easy because the difference from room temperature is appropriate, and stable separation can be performed without any trouble. In addition, although the viscosity of a mobile phase falls and column internal pressure falls by raising temperature, it can adjust suitably by those skilled in the art, such as raising a flow rate or using a mobile phase with a high viscosity.

(2) When the porous polymer is made of a hydrophilic polymer substrate such as polyvinyl alcohol or polyacrylamide << mobile phase (solvent) >>
As a solvent in the case of using a size exclusion column packed with a porous polymer made of polyvinyl alcohol or polyacrylamide, water, methanol, ethanol, acetonitrile, tetrahydrofuran, DMSO, and DMF are preferable. This porous polymer has a higher affinity with a polar solvent than other porous polymers, and by using such a solvent, it is easy to increase the pressure in the size exclusion column. Furthermore, mixing the inorganic salt such as lithium bromide in the range of 50 mM or less also increases the viscosity of the mobile phase, which easily increases the pressure in the size exclusion column. If inorganic salts are mixed excessively, purification of the recovered gene introduction agent is likely to be complicated, which is not preferable.

≪Porous polymer pore size≫
The pore diameter of the porous polymer made of polyvinyl alcohol or polyacrylamide is preferably about 50 to 5,000 nm, particularly preferably about 100 to 1,000 nm.

≪Temperature≫
The temperature of the size exclusion column is preferably set to the same temperature as that of the size exclusion column when the porous polymer composed of the styrene / divinylbenzene copolymer is used.

  In the present invention, a secondary amine such as diethylamine or a primary amine such as ethanolamine may be added to the mobile phase. In particular, it is preferable to add the amine when a nonpolar solvent such as toluene or chloroform is used as the mobile phase. Such an amine acts as a good proton donor in decomposing a part of the branched chain, and suppresses hydrogen abstraction reaction from the porous polymer and hydrogen abstraction reaction between the polymer chains. It is considered that the decomposition reaction takes place preferentially. In addition, the use of such an amine can protect the tertiary amine contained in DAAAM, and has a low molecular weight, a low boiling point, and does not chemically bond to the side chain of the cationic polymer. The collected gene introduction agent is preferable because it can be easily purified.

  In this invention, since it can manufacture without using water, the hydrolysis which arises after completion | finish of a hydrolysis process can be suppressed as much as possible. That is, when hydrolysis is performed by dialysis using water, an unintended hydrolysis reaction with water may proceed after completion of the hydrolysis step, and alcohol generated by this hydrolysis is mixed into the gene introduction agent. In some cases, however, the purity of the gene transfer agent is lowered, but according to the present invention, this problem can be avoided.

  As described above, the gene introduction agent produced by this method has a branched chain composed of a polymer chain containing a structural unit derived from DAAAM, and a part of the DAAAM unit in the branched chain is hydrolyzed. Therefore, the anionic functional group generated by the hydrolysis shows that the entire gene transfer agent is hydrophilic, hardly adsorbs anionic proteins and lipids in serum, and even in a medium containing serum. Can efficiently introduce genes.

[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 DAAAM 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.

  DAAAM used in this branched polymer production process, that is, 2-N, N-dialkylaminoalkyl methacrylate is a dialkylamino having two alkyl groups having about 1 to 3 carbon atoms and an alkylene group having 1 or 2 carbon atoms. An ester of an alkyl alcohol and methacrylic acid is preferred. As such a dialkylaminoalkyl methacrylate, the aforementioned 2-N, N-dimethylaminoethyl methacrylate and derivatives thereof are preferable.

  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, particularly preferably benzene, toluene, chloroform or methylene chloride, especially toluene.

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

  The concentration of DAAAM 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, so that the polymer chain composed of DAAAM units is introduced into the branched chain portion by purification as necessary, 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 100,000, particularly about 3,000 to 60,000. 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 consisting only of the above-mentioned DAAAM, but is a block copolymer or random copolymer into which one or more monomers different from DAAAM and DAAAM are introduced. Also good.

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 DAAAM and DAAAM, a raw material solution containing monomers other than the iniferter, DAAAM, and DAAAM is prepared in the same manner as in the case of reacting the iniferter with DAAAM. Is irradiated with light to obtain a random copolymer in which DAAAM and a monomer other than DAAAM are bonded to the iniferter.

  In addition, for the above iniferter, first, DAAAM is block-polymerized to form a homopolymer, and then 3-N, N-dimethylaminopropylacrylamide is block-polymerized to this homopolymer, and the base end side of the branched chain is A DAAAM block polymer may be a branched chain composed of a 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.

[2] Gene transfer agent The gene transfer agent (branched polymer after hydrolysis) produced by the method for producing a gene transfer agent of the present invention has an LCST (Lower Critical Solution Temperature) of 36 ° C. or higher. It is particularly preferable that the temperature is about 37 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 a nucleic acid and a complex in which 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. Therefore, if the LCST is within the above range, that is, 36 ° C. or higher, the gene can be efficiently introduced into the cell. In addition, the measuring method of LCST is as describing in the term of the below-mentioned Example.

  The molecular weight per branched chain of the gene introduction agent (branched polymer after the hydrolysis step) is preferably about 300 to 100,000, particularly about 1,000 to 60,000. The total molecular weight of the DAAAM units contained depends on the number of branched chains, but is preferably about 1,200 to 590,000, particularly preferably about 10,000 to 250,000. The total molecular weight of the DAAAM units contained in the gene introduction agent in this case is the total molecular weight of the unhydrolyzed DAAAM unit and the hydrolyzed DAAAM unit.

  Of the DAAAM units contained in the gene introduction agent (branched polymer after hydrolysis), the proportion of DAAAM that has been hydrolyzed is only required to satisfy the above-mentioned LCST, and is not particularly limited. Absent. 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.

  In addition, the molecular weight of the gene introduction agent is preferably about 2,000 to 600,000, particularly about 10,000 to 300,000.

[3] Nucleic acid complex The above gene introduction agent (vector) 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 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.

  “Cells” that are desirable as a target for introduction of nucleic acid are those that require functional expression of the nucleic acid, and such cells are variously selected depending on, for example, the nucleic acid to be used (that is, its function). Examples include cells, smooth muscle cells, fibroblasts, skeletal muscle cells, vascular endothelial cells, bone marrow cells, bone cells, blood cell stem cells, blood cells. 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.

  This nucleic acid complex can be administered to a living body by any method besides being used for a culture test.

  Intravenous or intraarterial injection is particularly preferred 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 with 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 δ0.8-1.2 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) Partial hydrolysis of side chain of thermosensitive cationic homopolymer comprising 4-branched star polymer <Hydrolysis by liquid chromatograph>
0.3 g of the thermosensitive cationic homopolymer synthesized in ii) above was dissolved in 3 mL of chloroform, and this solution was dissolved in Table 1 using a high performance liquid chromatograph (“LC-10Avp series” manufactured by Shimadzu Corporation). The partial hydrolysis of the side chain was carried out by treatment under the conditions of Protocol-A1 to A5 and B1 to B6 shown. In each Protocol, two columns manufactured by Shodex were connected and used. Table 2 shows the characteristics of each column.

  In any Protocol, the polymer components eluted at a time other than 10% from the start of elution and 10% before the end of elution were recovered from the total elution time of the polymer. Polymer component recovery was completed within 40 minutes.

<Purification of polymer>
In each Protocol, each recovered polymer solution is concentrated with an evaporator and reprecipitated with an n-hexane / diethyl ether system to obtain a polymer component. Each polymer is dissolved in a small amount of benzene, and then freeze-dried. Of powder was obtained. The molecular weight of the gene introduction agent obtained with each Protocol and the molecular weight per branched chain of the gene introduction agent were calculated from the molecular weight. The results are shown in Table 1, respectively. In addition, since the polymers of Protocol-A5 (Comparative Example 1-2) and B6 (Comparative Example 2-3) were not dissolved in the solvent, the measurement of molecular weight of these polymers and the cloud point described later were used. Measurement was not performed.

<Measurement of cloud point>
An aqueous solution of the polymer obtained by each Protocol was prepared, and the temperature at which the solution became cloudy (LCST: cloud point) was measured by increasing the temperature of the aqueous solution.

  That is, 3% by weight aqueous solutions of each homopolymer were prepared, and the absorbance of light having a wavelength of 600 nm in a temperature range of 20 to 60 ° C. of these aqueous solutions was measured. The results are shown in Table 1. The untreated polymer that had not been hydrolyzed had LCST around 32.0 ° C.

  As is clear from Table 1, the pressure in the column was greatly different in each Protocol. It is considered that the difference in particle diameter, type of mobile phase, viscosity, liquid flow rate and column temperature of the porous polymer packed in each column affected the pressure loss.

  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.

<Discussion>
In Protocol-A1, B1, and B2 (Comparative Examples 1-1, 2-1, and 2-2), the pressure in the column was low and the polymer side chain did not decompose, so the cloud point (LCST) did not change. It is thought. In contrast, Protocol-A3, A4, B3, B4, B5 (Examples 1-2, 1-3, 2-1, 2-2, 2-3) are high in pressure and decomposed in the column. It was considered that the cloud point (LCST) was raised due to the occurrence. That is, it is considered that the solubility in water was increased because the anionic functional group was exposed by partial hydrolysis of the side chain, and the hydrophilicity was maintained even when the amine was desolvated by a change in temperature.

  In Protocol-A5 and B6 (Comparative Examples 1-2 and 2-3), the recovered polymer component did not dissolve in water even at room temperature. This is because the hydrolysis of the side chains has progressed too much, and the polymers have been ionically crosslinked, aggregated, gelled, or a crosslinking reaction has occurred in the column simultaneously with the decomposition, resulting in a water-insoluble component. it is conceivable that. Polymers hydrolyzed by Protocol-A5 and B6 are considered to be difficult to use as gene introduction agents because of their low solubility in water.

  In the production method in which the hydrolysis step is performed by dialysis, it takes about 5 days from collecting the branched polymer to obtaining the target gene introduction agent. According to the method for producing a gene introduction agent of the present invention, 1 It takes about an hour. That is, according to the production method of the present invention, partial hydrolysis of the polymer side chain can be carried out easily and in a short time, and the intended gene introduction agent can be obtained.

Claims (12)

In a method for producing a gene introduction agent comprising a branched polymer having a branched chain,
Branch for producing a branched polymer by introducing a plurality of branched chains obtained by polymerizing at least 2-N, N-dialkylaminoalkyl methacrylate and / or a derivative thereof radially extending from the aromatic ring into the aromatic ring Mold polymer manufacturing process;
A hydrolysis step of hydrolyzing the obtained branched polymer;
A method for producing a gene transfer agent having
The method for producing a gene introduction agent, wherein the hydrolysis step includes a step of passing a solution containing the branched polymer through a size exclusion column. According to claim 1, method for producing a transgenic agent characterized in that the pressure of the size exclusion column is 30~80kg / cm ^2.   3. The method for producing a gene introduction agent according to claim 1, wherein the size exclusion column is filled with porous polymer particles.   4. The method for producing a gene introduction agent according to claim 3, wherein the porous polymer particles have a particle size of 10 μm or less.   5. The gene introduction agent according to claim 3 or 4, wherein the porous polymer particle is at least one selected from the group consisting of a styrene / divinylbenzene copolymer, polyvinyl alcohol, and polyacrylamide. Method. 6. The method for producing a gene introduction agent according to claim 5, wherein the porous polymer particles are styrene / divinylbenzene copolymer, and the pressure in the size exclusion column is 40 to 80 kg / cm ² . 6. The method for producing a gene introduction agent according to claim 5, wherein the porous polymer particles are polyvinyl alcohol, and the pressure in the size exclusion column is 30 to 40 kg / cm ² .   The method for producing a gene introduction agent according to any one of claims 1 to 7, wherein the theoretical plate number of the size exclusion column is 1,000 / 10 cm or more.   The method for producing a gene introduction agent according to any one of claims 1 to 8, wherein the gene introduction agent has an LCST (Lower Critical Solution Temperature) of 37 to 60 ° C.   The branched polymer according to any one of claims 1 to 9, wherein the branched polymer is an iniferter having a compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule. , N-dialkylaminoalkyl methacrylate and / or a derivative thereof, which is produced by light irradiation living polymerization, a method for producing a gene introduction agent.   The compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule according to claim 10 having a benzene ring as a nucleus and three or more N, N-dialkyls as branched chains in the nucleus. A method for producing a gene introduction agent, wherein a dithiocarbamylmethyl group is bound thereto.   The method for producing a gene introduction agent according to any one of claims 1 to 11, wherein the gene introduction agent has a molecular weight of 2,000 to 600,000.