JP5944622B2 - Gene transfer agent composition - Google Patents

Description

  The present invention relates to a novel gene transfer agent composition using a cationic polymer and adenovirus in combination as a gene transfer agent.

  In recent years, gene therapy research has become more and more important as the molecular genetic factors of human diseases become clear. Gene therapy is aimed at the expression of DNA at the target site, how to make the DNA reach the target site, how to efficiently introduce the DNA into the target site and make it functionally expressed at the site It becomes important.

There are roughly two gene transfer techniques, viral vectors and non-viral vectors.
Gene transfer using a viral vector is a modification of an adenovirus, adeno-associated virus, Sendai virus, retrovirus, etc. (made by detoxifying a wild-type virus and inserting the desired nucleic acid), and then acting on the cell to create a foreign gene. It is a technique for introduction into cells and is widely practiced among those skilled in the art.
Many non-viral vectors make use of phagocytosis that allows a polymer polymer or a complex formed of a cationic lipid and a nucleic acid to be incorporated into cells, and others mechanically break through cell membranes such as electroporation and gold colloid guns. There are also techniques for inserting nucleic acids.

  With respect to non-viral vectors, the present inventors have developed a gene transfer technique using a star polymer in which a polymer chain extends radially from a benzene ring, and have made many proposals (for example, Patent Documents 1 and 2). The star polymer vector has improved gene transfer activity depending on the number of linear polymer chains bonded to the benzene ring, and a star polymer having a maximum of 6 polymer chains has the highest gene transfer activity. Become.

  In addition, even in the case of a polymer of the same monomer, the star-type polymer can be introduced with a gene without causing cytotoxicity even at a high CA ratio (ratio of the positive charge number of the vector to the negative charge number of the DNA). Yes, even if the particle size of the nucleic acid complex obtained by conjugating nucleic acid to the star polymer is large (that is, the smaller the particle size, the more advantageous it is for the permeation of the cell membrane) 200 μm to 300 μm, and the linear polymer is about 100 μm, and the star type polymer is considered disadvantageous in terms of particle diameter), but has high cell membrane permeability; Have.

However, compared with viral vectors, non-viral vectors such as polymer vectors and cationic lipid vectors have the drawback of low gene transfer activity.
In particular, they could hardly be introduced into stem cells such as hematopoietic stem cells, myoblasts, osteoblasts, ES cells, iPS cells, and cells with low phagocytosis such as nerve cells and cardiomyocytes.
For this reason, in the past, studies using virus vectors have been the mainstream of examples of use in living organisms (animals), and in vivo use with polymer vectors has hardly been reported. This is because high-molecular vectors can also be applied to cell lines (cells that can be easily cultured for experiment in a test tube and have been treated so that there is no change in cell properties due to passage). Primarily cells (cells immediately after being collected from an animal organ or the like) can hardly be introduced with a gene.

In contrast, a viral vector has the advantage of being able to introduce a nucleic acid into various cells using its strong infectivity, but has the following drawbacks.
(1) Risk of creating a new virus (for example, (1) In general, a recombinant gene is created before creating a viral vector, but there is a mutation in the target gene during the creation of this recombinant gene. The risk of entering and the digestion of the E1 region and the ability to self-renew, and (2) homology with the DNA of the packaging cell during viral gene replication during viral vector passage There is a risk that the E1 region will be obtained by recombination and a virus that has acquired the self-propagating ability may be produced) and the researchers themselves will be infected.
(2) Since the titer (titer) decreases with time even when stored at a temperature of -70 ° C or lower, in order to obtain a high titer, a titer confirmation test, re-preparation and purification A process is required, and preparation of a virus vector with strong infectivity requires a great deal of time and labor cost.
(3) Since the virus itself has a size, there is a limit to the size of the nucleic acid that can be physically inserted inside, and large DNA cannot be inserted.
(4) Since a viral vector enters via a receptor on the cell membrane surface (for example, CAR (Cossackie adenovirus receptor)), it cannot be used for cells that do not have a receptor.
(5) In many cases, an application for approval is required as a recombinant gene experiment, and a great deal of time and dedicated facilities are required from the planning to the experiment.

WO2004 / 092388 JP 2007-70579 A

  It is an object of the present invention to provide a gene introduction agent composition that solves the disadvantages of the non-viral vector and the viral vector as described above, and has both advantages of the gene introduction activity and is remarkably excellent in gene introduction activity.

  As a result of intensive studies to solve the above problems, the present inventors have used a cationic polymer vector that is a non-viral vector and an adenovirus as a viral vector to eliminate or reduce the disadvantages of viral vectors. In addition, the gene transfer activity has been dramatically improved and exhibits high gene transfer activity that cannot be achieved by the cationic polymer vector and the adenovirus vector alone, and the gene transfer is impossible or difficult only with the cationic polymer vector. The present inventors have found that an excellent gene transfer agent composition capable of transferring genes to stem cells and primary cells (cells immediately after being separated from a living tissue) can be realized.

  The present invention has been achieved on the basis of such findings, and the gist thereof is as follows.

[1] Host cells containing a star-type cationic polymer, nucleic acid, and adenovirus having four or more polymer chains (hereinafter referred to as “cationic polymer chains”) mainly composed of cationic vinyl monomers in a medium. A gene introduction agent composition comprising an unpurified adenovirus which is a fraction obtained by culturing with and sonicating and culturing the culture .

[ 2 ] The gene transfer agent composition according to [ 1 ], wherein the cationic polymer has a benzene ring as a nucleus, and the cationic polymer chain as a branched chain is bonded to the nucleus.

[ 3 ] The gene introduction agent composition according to [1] or [2] , wherein the unpurified adenovirus includes an inactivated individual.

[ 4 ] In any one of [1] to [ 3 ], a gene introduction agent composition comprising adenovirus 10 × 10 ¹² TCID ₅₀ or less in terms of per 1 g of the cationic polymer. .

[ 5 ] In any one of [1] to [ 4 ], the nucleic acid complex obtained by complexing the cationic polymer and the nucleic acid is further mixed with at least a part of unpurified adenovirus. A gene transfer agent composition.

[6] A combination of a star-type cationic polymer having four or more polymer chains mainly composed of cationic vinyl monomers (hereinafter referred to as “cationic polymer chains”) and at least a part of unpurified adenovirus. An in vitro gene transfer method comprising the steps of:

  According to the present invention, by using adenovirus together with a cationic polymer, the above-mentioned drawbacks of adenovirus, which is a viral vector, are eliminated or reduced, and the gene transfer activity is dramatically increased. In addition to high gene transfer activity that cannot be achieved with each adenovirus vector alone, it can be used for stem cells and primary cells (cells immediately after separation from living tissue) where gene transfer is impossible or difficult only with cationic polymer vectors. Can also introduce genes.

  Although the details of the mechanism of action of the above-mentioned effect produced by using the cationic polymer of the present invention in combination with adenovirus are not clear, the following (1) to (5) are considered, and these act synergistically. Therefore, it is estimated that high gene transfer activity can be obtained.

(1) Adenovirus enters the cell via a receptor (receptor) present on the surface of the cell membrane (some Sayida viruses, for example, enter membrane fusion). Unlike the phagocytosis of cells called endocytosis (called pinocytosis), this invasion route is likely to be taken up as a useful component for the cell, and quickly enters the cell from the endoplasmic reticulum. It may have escaped and diffused into the cytoplasm. On the other hand, many non-viral vector nucleic acid complexes incorporated by phagocytosis (endocytosis) as foreign substances are decomposed in the endoplasmic reticulum and are considered to have poor transport efficiency.
In the present invention, adenovirus (even if debris) helps the endosomal escape of the complex of cationic polymer and nucleic acid.
(2) Adenovirus (even debris) is adsorbed on the surface of the cationic polymer vector nucleic acid complex, and the nucleic acid complex can enter the cell via the receptor, and / or When a single adenovirus enters the cell via a receptor, the nucleic acid complex of the cationic polymer vector is piggybacked and taken into the cell.
(3) Adenovirus functions to escape from endosomes.
(4) The adenovirus-derived protein contains a component that promotes the transcription of the nucleic acid carried by the cationic polymer vector.
(5) Cell proliferation is promoted by infection with adenovirus, and the number of cells in the binuclear cell phase, which is the only timing at which a cationic polymer vector can enter the nucleus, is increasing.

In the present invention, it is not necessary to use an adenovirus with an activity as in gene transfer with a normal viral vector, and a titer of 10 recommended in an adenovirus gene transfer experiment protocol well known to those skilled in the art. ⁹
TCID ₅₀ / mL (TCID ₅₀ statistically 50% cytopathic end point) or more must ^not, to obtain the effect of sufficiently adenovirus combined with very low-activity of about 10 _{6 TCID} 50 / mL Can do. In other words, the effect of improving gene transfer activity can be obtained by using at a low concentration so that the adenovirus does not substantially function, and it is also effective with sterilized or inactivated adenovirus (remnant) or adenovirus degradation fragments. In this way, it is effective even with a very small amount of adenovirus, sterilized or inactivated adenovirus, and adenovirus degradation fragments, so re-preparation that is a drawback of viral vectors The problem of requiring labor such as titer confirmation and purification and the risk of infection are eliminated or alleviated. In addition, since the gene to be introduced is mounted on the nucleic acid complex of the cationic polymer vector, there is no restriction on the size of the nucleic acid to be introduced.

  Therefore, according to the present invention, the cationic polymer vector, which is a non-viral vector, and the adenovirus, which is a viral vector, are used together to eliminate or reduce the disadvantages of the viral vector, and then the cationic polymer. Provided is an excellent gene transfer agent composition that exhibits high gene transfer activity that cannot be achieved by each of a vector and an adenovirus vector, and that can transfer a gene to various cells with high gene transfer efficiency.

  In the present invention, the cationic polymer has a polymer chain mainly composed of a cationic vinyl monomer, and in particular, such a cationic polymer has a star-type cationic property having four or more of the cationic polymer chains. Polymers, particularly those having a benzene ring as a nucleus and a cationic polymer chain as a branched chain bound to this nucleus are preferred.

It is a graph which shows the gene transfer activity to FSHfb of an adenovirus / 6 branch star type cationic polymer nucleic acid complex. It is a graph which shows the gene transfer activity to the Hela cell of an adenovirus / 6 branch star type cationic polymer nucleic acid complex. It is a graph which shows the gene introduction | transduction activity to FSHfb of an adenovirus single-piece | unit, an adenovirus / linear type cationic polymer nucleic acid complex, and an adenovirus / 6 branch star type cationic polymer nucleic acid complex. It is a graph which shows the gene introduction | transduction activity to FSHfb of the adenovirus / 6 branch star type cationic polymer nucleic acid complex using refined adenovirus or unpurified adenovirus.

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

[Gene transfer agent composition]
The gene introduction agent composition of the present invention is characterized in that it contains at least a part of a cationic polymer, a nucleic acid, and an adenovirus, and usually adenovirus is adsorbed to a complex of a nucleic acid and a cationic polymer. Provided as an aqueous dispersion.

<Cationic polymer>
The cationic polymer used in the present invention has a polymer chain mainly composed of a cationic vinyl monomer (hereinafter referred to as “cationic polymer chain”). “To” means that 50 mol% or more, particularly 80 to 100 mol% of the structural unit derived from the vinyl monomer constituting the cationic polymer chain is a structural unit derived from the cationic vinyl monomer.

  The cationic vinyl monomer is not particularly limited, but 3-N, N-dimethylaminopropylacrylamide, 2-N, N-dimethylaminoethylacrylamide, 2 from the viewpoint of easy synthesis of the cationic polymer. -N, N-dimethylaminoethyl methacrylate, 4-N, N-dimethylaminostyrene, 4-aminostyrene or derivatives thereof are preferably used. These cationic vinyl monomers may be used alone or in combination of two or more.

The cationic polymer chain according to the present invention may contain a constitutional unit derived from a vinyl monomer other than the cationic vinyl monomer. Examples of the vinyl monomer other than the cationic vinyl monomer include acrylic acid derivatives, Vinyl monomers such as styrene derivatives are suitable, and specific examples include vinyl monomers such as N, N-dimethylacrylamide, methoxyethyl (meth) acrylate, and N-isopropylacrylamide. These other vinyl monomers may be used alone or as a mixture of two or more. These other vinyl monomers are vinyl monomers constituting a cationic polymer chain. It is necessary to use so that 50 mol% or more, particularly 80 to 100 mol%, of the structural unit derived from the structural unit is derived from the cationic vinyl monomer.
When these other vinyl monomers are used, the cationic polymer chain may be a block copolymer chain or a random copolymer chain.

  The cationic polymer used in the present invention may be a linear cationic polymer having only one linear cationic polymer chain, but since it has excellent gene transfer activity, a plurality of cationic polymer chains are used. In particular, a polymer having 4 or more is preferable, and in particular, a star-type cationic polymer having a benzene ring as a nucleus and the cationic polymer chain as a branched chain bound to this nucleus is preferable.

  The method for producing such a star-type cationic polymer is not particularly limited, but at least an iniferter composed of a compound having three or more N, N-disubstituted dithiocarbamylmethyl groups in the same molecule, and a cationic vinyl A method of producing a solution containing a monomer based on light irradiation is preferred.

  Here, the iniferter is a polymerization initiator that generates radicals by light irradiation, a function as a chain transfer agent, a function that stops growth by binding to the growth terminal, and a polymerization start that stops polymerization when light irradiation stops.・ Molecules that function as polymerization terminators.

  As the aromatic compound having three or more N, N-disubstituted dithiocarbamylmethyl groups in the same molecule as the iniferter, the N, N-disubstituted dithiocarbamylmethyl group, preferably N, Those in which three or more N-dialkyldithiocarbamylmethyl groups are bonded as a branched chain are preferred, 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.

  To react the iniferter with the cationic vinyl monomer, prepare a raw material solution containing the iniferter and the cationic vinyl monomer, and irradiate it with light so that the cationic vinyl monomer is in contact with the iniferter. A reaction product in which polymer chains are bonded is generated.

  The concentration of the cationic vinyl monomer in the raw material solution is preferably 0.5M or more, for example, 0.5 to 2.5M. The concentration of the iniferter is preferably about 0.1 to 100 mM.

The light irradiation conditions are preferably a wavelength of 250 to 400 nm, an irradiation time of 1 to 150 minutes, and an irradiation intensity of about 100 to 10,000 μW / cm ² .

  This light irradiation produces a branched cationic polymer in which a branched chain composed of a polymer chain of a cationic vinyl monomer is bonded to an iniferter in the reaction solution, and is used after purification as necessary.

When other vinyl monomers are used, the cationic polymer chain of the random copolymer can be formed by allowing other vinyl monomers to exist together with the cationic vinyl monomer in the raw material solution. Moreover, the cationic polymer chain of a block copolymer can be formed by performing two-stage polymerization of photopolymerization of a cationic vinyl monomer and photopolymerization of another vinyl monomer.
In order to increase the molecular weight of the resulting cationic polymer, a high molecular weight cationic polymer is produced by performing photopolymerization in two or more stages using only a cationic vinyl monomer as in Example 1 described later. You can also

  The molecular weight per cationic polymer chain of the thus obtained star-type cationic polymer having a benzene ring as a nucleus and a cationic polymer chain as a branched chain bound to this nucleus is 1,500 to About 150,000, especially about 3,000 to 100,000 are preferable. The molecular weight of the star-type cationic polymer is preferably about 10,000 to 10,000,000, and more preferably about 100,000 to 600,000, although it depends on the number of cationic polymer chains. This molecular weight can be adjusted by controlling the light irradiation time. That is, by increasing the irradiation time, the polymerization reaction can be advanced to obtain a star-type cationic polymer having a large molecular weight. As described above, by performing this light irradiation in two or more stages, the total irradiation time can be extended to obtain a star-type cationic polymer having a large molecular weight.

  The higher the molecular weight of the star-type cationic polymer, the higher the gene transfer activity, which is preferable. However, an excessively high molecular weight may cause cytotoxicity, so it may be appropriately selected according to the purpose of gene transfer. . That is, in the gene introduction agent using only the cationic polymer that does not use adenovirus, if the molecular weight is excessively large, the gene introduction agent may be recognized as a foreign substance in a living body. Although the above-described problem does not occur by inclusion, the above-described molecular weight range is considered preferable from the viewpoint of metabolic degradation and excretion.

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

  In order to select and use a high molecular weight cationic polymer obtained by photopolymerization, molecular weight fractionation can also be performed using a size exclusion column or the like.

In the gene introduction agent composition of the present invention, the content (concentration) of the cationic polymer is preferably about 0.01 μg / μL to 0.5 μg / μL. If the concentration of the cationic polymer is too low,
(1) The ionic bond between the cationic polymer and the nucleic acid is not sufficient to form a nucleic acid complex, and / or the aggregation of the nucleic acid in the nucleic acid complex is insufficient to protect from the enzymatic reaction.
(2) The ζ potential (zeter potential) on the surface of the nucleic acid complex is low, and the adsorptivity to the cell membrane becomes low, or adenovirus adsorption to the nucleic acid complex does not occur.
It is not preferable because of problems such as. On the other hand, if the concentration of the cationic polymer is too high, it may cause cytotoxicity, which is not preferable.

<Adenovirus>
The present invention is characterized in that the gene introduction agent composition contains an adenovirus together with a cationic polymer.

  The adenovirus may be in an inactivated state, may be a sterilized individual, an inactivated individual (remnant), or a part thereof. In the case of an inactivated adenovirus, it may be a nucleic acid complexed (introduced with a gene) or a nucleic acid uncomplexed. However, it is preferable that the viral recombinant gene genome does not contain the E1 region and is not self-replicating.

  In the case of a gene-transferred adenovirus complexed with a nucleic acid, the nucleic acid may be the same as or different from the nucleic acid targeted by the gene transfer agent composition of the present invention. That is, an object of the present invention is to introduce a gene in a nucleic acid complex by a cationic polymer, and there is no particular limitation on the gene in adenovirus. In addition, since the titer of adenovirus used in the gene transfer agent composition of the present invention is lower than the titer used in gene transfer using a normal adenovirus vector, the gene introduced into the adenovirus is introduced into the cell. It is thought that it is hardly introduced.

  In the gene introduction agent composition of the present invention, since a cationic polymer and adenovirus are used in combination as a gene introduction agent, the amount of adenovirus used is very small compared to the case where adenovirus is used as a virus vector. Since even a part of the adenovirus which has been deactivated and remains is effective, the risk derived from the conventional virus vector can be eliminated or greatly reduced.

If the content of adenovirus in the gene introduction agent composition of the present invention is small, the effect of the present invention due to the use of adenovirus cannot be obtained sufficiently, but if it is too much, cytotoxicity derived from adenovirus is expressed. In other words, the gene transfer activity is reduced.
Therefore, the content of adenovirus in the gene introduction agent composition of the present invention is 10 × 10 ¹² TCID ₅₀ or less, particularly 10 × 10 ¹⁰ TCID ₅₀ or less, especially 1 × it is preferable to _{^{10 7 TCID 50 ~1 × 10 10}} TCID 50.

In other words, the titer of adenovirus (potency) is (1) plaque forming method, (2) TCID ₅₀ method, measured in (3) DNA methods, such as methods well known to those skilled in the art, _"TCID 50 / mL (Kerber It is expressed in units of “statistical 50% cell degeneration end point” or “PFU (Plaqua Formin Unit)”. In the present invention, a gene introduction agent composition is prepared by adding and mixing the necessary amount of titer adenovirus-containing liquid expressed in such units to a liquid containing a complex of a cationic polymer and a nucleic acid. Therefore, the amount of adenovirus used per 1 g of the cationic polymer is calculated by the following formula.
Adenovirus consumption (TCID ₅₀ ) = A × M / K
A: The titer of the adenovirus-containing solution used (TCID ₅₀ / mL)
K: the amount of the cationic polymer in the liquid containing the cationic polymer / nucleic acid complex (g)
M: Volume of adenovirus-containing liquid (mL) mixed with liquid containing cationic polymer / nucleic acid complex

  In this invention, it is preferable to use so that the usage-amount of adenovirus calculated by the said formula may be below the said upper limit.

In addition, when using the deactivated adenovirus (remnant), since the titer is not measured, the lower limit of the amount of adenovirus used represented by the above-mentioned TCID ₅₀ units is almost zero.

<Nucleic acid>
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.

  The nucleic acid is preferably a polynucleotide such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), particularly DNA, but may be a ribonucleoprotein.

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

  In addition to repressing by antisense, it can also be used for mRNA destruction by RNA interference using double-stranded RNA of 21 to 23 bases.

<Method for preparing gene transfer agent composition>
In order to produce the gene introduction agent composition of the present invention, it is preferable to first combine a cationic polymer and a nucleic acid to form a nucleic acid complex, and add adenovirus to this and mix.

  In order to combine the cationic polymer and the nucleic acid, the nucleic acid may be added to and mixed with a solution of the cationic polymer having a concentration of about 0.1 to 1000 μg / mL. It is preferable to add an excessive amount of the cationic polymer to the nucleic acid, to saturate the cationic polymer with respect to the nucleic acid, and to complex the cationic polymer and the nucleic acid.

  The particle diameter of the complex of the cationic polymer and the nucleic acid is preferably about 50 to 400 nm. This particle diameter is measured by, for example, a dynamic light scattering method using a laser. If the particle size is smaller than this, there is a risk that 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.

  After the nucleic acid complex is formed in this way, the adenovirus-containing solution is added and mixed to prepare the gene introduction agent composition of the present invention. The added adenovirus is considered to adsorb to the nucleic acid complex. The nucleic acid complex adsorbed with adenovirus maintains a stable dispersion state in a liquid such as water.

[Gene transfer method]
In the gene transfer method of the present invention using a cationic polymer and adenovirus as a gene transfer agent, the gene is introduced into the cell by bringing the gene transfer agent composition prepared as described above into contact with the cell in the presence of serum. To do. By contacting the nucleic acid complex and the cell in the presence of serum, the nucleic acid can be transferred to the cell without imposing a burden on the cell.

  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 gene introduction agent composition 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 agent comprising this gene transfer agent composition as an active ingredient further comprises a pharmaceutically acceptable carrier (osmotic pressure regulator, stabilizer, preservative, solubilizer, pH adjuster, thickener, etc. if necessary) ). Any known carrier can be used.

  Moreover, the medicine which uses this gene introduction agent composition as an active ingredient includes what contains the gene introduction agent composition containing 2 or more types of nucleic acids from which the kind of nucleic acid contained differs. 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 gene introduction agent composition is preferably applied to gene therapy. Applicable diseases vary depending on the type of nucleic acid contained in the gene transfer composition, but may be diseases in the cardiovascular region that cause lesions such as peripheral artery disease, coronary artery disease, and restenosis after arterial dilation. In addition, cancer (malignant melanoma, brain tumor, metastatic malignant tumor, breast cancer, etc.), infection (HIV, etc.), single genetic disease (cystic fibrosis, chronic granulomas, α1-antitrypsin deficiency, Gaucher disease, etc.) ) And the like.

  Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples.

  In the following, the devices described below were used for the production and evaluation of the cationic polymer.

<Light source>
300W xenon lamp MAX-301 manufactured by Asahi Spectroscopic Co., Ltd. (corresponding to obtaining a filter function by using an alkali glass cell and irradiating mixed light with a wavelength of 330 to 400 nm)
<Irradiation intensity meter>
USING USHIO INC. Integrating Photometer UIT-150 with Light Sensor UVD-405 <GPC>
Mobile phase: DMF + 30 mM LiBr (manufactured by Kanto Chemical Co., Inc.)
Apparatus: LC-10Avp manufactured by Shimadzu Corporation
Column: Connect Asahipack GF-710HQ manufactured by Shodex and Asahipack GF-510HQ Calibration curve: PEG standard TSK series manufactured by Shodex <NMR>
Varian's Gemini-300
<Reaction vessel>
3mm thick rectangular glass cell made of alkali glass <Freeze drying equipment>
EYELA FUD-2200 and ULVAC vacuum pump GCD-136X
<Molecular weight fraction>
Apparatus: LC-10Avp manufactured by Shimadzu Corporation
Column: GPCK-2005 + GPC K-2004 manufactured by Shodex
Mobile phase: chloroform + 30 mM triethylamine

As the reagent for synthesis, a special grade reagent manufactured by Aldrich or Kanto Chemical Co. was used after purification by recrystallization or vacuum distillation.
As the adenovirus, a commercially available recombinant adenovirus production kit was used. A full-length DNA (coding LacZ) is inserted to prepare a plasmid, and the plasmid is introduced into 293 cells (hereinafter referred to as “Ad virus” or “Adv”).
Moreover, the plasmid DNA which codes commercially available firefly luciferase was used for the nucleic acid for introduction | transduction.

[Example 1]
{Synthesis of 6-branched star-type cationic polymer}
(1) Synthesis of 6-branch iniferter 1,2,3,4,5,6-hexakis (N, N-diethyldithiocarbamylmethyl) benzene as an iniferter was synthesized as follows.

  5.0 g of 1,2,3,4,5,6-hexakis (bromomethyl) benzene and 30.0 g of sodium N, N-diethyldithiocarbamate were added to 300 mL of ethanol and stirred at room temperature for 4 days under light shielding. The precipitate was collected by filtration, poured into 3 L of methanol, stirred for 10 minutes and filtered. This operation was repeated 4 times in total. The precipitate was dissolved in 200 mL of chloroform, 100 mL of methanol was added and heated to 50 ° C., stored in a refrigerator for 12 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 1,2,3,4,5,6-hexakis (N, N-diethyldithiocarbamylmethyl) benzene needle-like crystals (yield 90%).

¹ H-NMR (inchloroform-d ₁ ): δ 1.26-1.31 ppm (m, 36 H, —CH ₂ —CH ₃ ), δ 3.71 to 3.73 ppm (q, 12 H, —N—CH ₂ — ), Δ 3.99-4.01 ppm (q, 12H, —N—CH ₂ —), δ 4.57 ppm (s, 12H, Ar—CH ₂ ).

(2) Synthesis of 6-branched star type cationic polymer The above 1,2,3,4,5,6-hexakis (N, N-diethyldithiocarbamyl) using 2-N, N-dimethylaminoethyl methacrylate as a monomer Synthesis of a 6-branched star type cationic polymer comprising methyl) benzene was carried out by the following two-stage polymerization.

Dissolve 1,2,3,4,5,6-hexakis (N, N-diethyldithiocarbamylmethyl) benzene in 20 mL of chloroform in a dark room and mix with 2-N, N-dimethylaminoethyl methacrylate To 50 mL with chloroform. Final concentration is 1,2,3,4,5,6-hexakis (N, N-diethyldithiocarbamylmethyl) benzene 5 mM (as photocleavable functional group), 2-N, N-dimethylaminoethyl methacrylate 1.0M. This was transferred to a reaction vessel and purged with high purity nitrogen gas for 10 minutes (flow rate: 2 L / min). The reaction vessel was sealed and irradiated with light while stirring vigorously. The irradiation intensity was adjusted to 2.5 mW / cm ² . After irradiation for 60 minutes, the solution was concentrated with an evaporator and then reprecipitated with n-hexane / ether = 50/50 (V / V) (first stage polymerization).
To this, 7.9 g of 2-N, N-dimethylaminoethyl methacrylate was added and dissolved, and the total amount was adjusted to 50 mL with chloroform, and the same operation as above was performed (second stage polymerization).
The reprecipitation was repeated 6 times, vacuum-dried at room temperature for 1 hour, dissolved in benzene, and lyophilized for 48 hours for purification.

GPC: Mn = 25,000 (Mw / Mn = 1.40)
¹ H-NMR (inchloroform-d ₁ ): δ 0.8-1.2 ppm (br, 3H, —CH ₃ , m / r = 1/2), 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).

(3) Molecular weight fractionation treatment of 6-branch star type cationic polymer The 6-branch star type cationic polymer with Mn = 25,000 synthesized in the above (2) was fractionated in a size exclusion column.
After fractionation, the product was purified by the same method as described above and freeze-dried to obtain an ultrahigh molecular weight monodisperse fraction with Mn = 150,000 (Mw / Mn = 1.1).

{Gene transfer experiment}
As cells, Hela cells derived from human cervical cancer, which are cell lines, and primary cells of cardiac fibroblasts derived from goat fetus (hereinafter referred to as “FSHfb”) were used.
The number of 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 number of positive charges per unit weight in the 6-branch star-type cationic polymer (Mn = 150,000) obtained in the above (3) is the monomer unit (2-N, N-dimethylaminoethyl methacrylate) was calculated from the molecular weight of 156. On the other hand, the number of negative charges per unit weight in DNA was calculated from the number of base pairs by the sequence MAP and the average molecular weight 660 of nucleobases.
This 6-branched star-type cationic polymer was dissolved in physiological saline to adjust the concentration to 0.26 μg / μL. The DNA was dissolved in a TE buffer solution to adjust the concentration to 0.33 μg / μL. Six-branched star type cationic polymer solution 60 μL and DNA solution 90 μL were mixed and incubated for 30 minutes. The mixing ratio is adjusted so that the number of charges is 10 times the number of positive charges (CA ratio = 10).

An Ad virus solution (6 × 10 ⁶ ) diluted to a 1/20 concentration in this 150 μL solution (the content of the 6-branched star-type cationic polymer is about 16 μg).
TCID ₅₀ / mL) was mixed with 0 μL (not added), 10 μL, 20 μL, 40 μL, 60 μL or 80 μL, added to 100 μL of OPTI-MEM culture solution, mixed, incubated for 30 minutes, and then added to each cultured cell. (The addition amount was adjusted so that 0.5 μg of DNA was administered to any well).
The added amount of Ad virus converted per 1 g of the cationic polymer in each added amount of Ad virus solution is as follows.
Addition amount 0 μL = 0 TCID ₅₀
Addition amount 10 μL = 3.8 × 10 ⁹ TCID ₅₀
Addition amount 20 μL = 7.5 × 10 ⁹ TCID ₅₀
Addition amount 40 μL = 1.5 × 10 ¹⁰ TCID ₅₀
Addition amount 60 μL = 2.3 × 10 ¹⁰ TCID ₅₀
Addition amount 80 μL = 3.0 × 10 ¹⁰ TCID ₅₀

After 3 hours of culture, OPTI-MEM was removed, and after washing with PBS, complete medium was added and further cultured for 48 hours. Forty-eight hours later, gene transfer activity was evaluated using a luciferase assay kit. Correction (normalization) was performed with the total protein concentration, and total protein quantification was performed with the Bradford reagent of BioRad.
The result of introduction into FSHfb is shown in FIG. The results of introduction into Hela cells are shown in FIG.

[ Reference example ]
{Synthesis of linear cationic polymer}
In Example 1, chloromethylbenzene was used instead of 1,2,3,4,5,6-hexakis (bromomethyl) benzene, and a linear cationic polymer having only one cationic polymer chain was synthesized.

  That is, in 50 mL of an ethanol solution containing sodium N, N-diethyldithiocarbamate (10.3 g, 46 mmol), 10 mL of an ethanol solution containing chloromethylbenzene (4.8 g, 38 mmol) was added dropwise at 0 ° C. in a nitrogen atmosphere to react. After the solution was stirred at room temperature for 23 hours, 150 mL of water was added and extracted with diethyl ether (200 mL × 3 times). The organic layer was washed with water (100 mL × 3 times) and dried over sodium sulfate. The solvent was distilled off under reduced pressure using an evaporator to obtain N, N-diethyldithiocarbamylmethylbenzene (colorless liquid, yield 17.6 g (yield 93%)).

¹ H-NMR: δ 7.407 to 7.271 ppm (m, 5H, Ar—H) δ 4.540 ppm (s, 2H, Ar—CH ₂ S), δ 4.082 to 4.012 ppm (q, 2H, —N) —CH ₂ —), δ 3.763 ppm to 3.692 ppm (q, 2H, —N—CH ₂ —), δ 1.311 to 1.252 ppm (m, 6H, —CH ₂ —CH ₃ ).

Next, a mixture of 2-N, N-dimethylaminoethyl methacrylate (3.9 g, 24.96 mmol) and N, N-dimethyldithiocarbamylmethylbenzene (23.94 mg, 0.1 mmol) was diluted with methanol, A total volume of 20 mL solution was prepared. The solution was stirred while blowing nitrogen gas, and irradiated with ultraviolet light (light amount: 1 mW / cm ² ). After 30 minutes of ultraviolet light irradiation, the polymerization solution was concentrated with an evaporator and dropped into a large amount of diethyl ether to precipitate a cationic polymer. The supernatant was removed by decantation, and the cationic polymer was dissolved in water and lyophilized. When the molecular weight of the cationic polymer obtained by GPC was measured after lyophilization, Mn was about 25,000.

¹ H-NMR: δ 7.8 to 7.4 ppm (br, 1H, —NH), δ 3.43 to 3.0 ppm (br, 2H, —NH—CH ₂ —CH ₂ —), δ 2.4 to ₂ . 2 ppm (br, 2H, —CH ₂ —CH ₂ —NR ₂ ), δ 2.2 to 2.1 ppm (br, 6H, —N—CH ₃ ), δ 1.8 to 1.5 ppm (br, 2H, —CH ₂ -CH ₂ -CH ₂ -)

{Gene transfer experiment}
The cells used were primary cells (FSHfb) of myocardial fibroblasts derived from goat fetuses.
The number of 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 number of positive charges per unit weight in the linear cationic polymer (Mn = 25,000) obtained above is the monomer unit (2-N, N-dimethylaminoethyl) constituting the linear cationic polymer. (Methacrylate) was calculated from the molecular weight of 156. On the other hand, the number of negative charges per unit weight in DNA was calculated from the number of base pairs by the sequence MAP and the average molecular weight 660 of nucleobases.
This linear cationic polymer was mixed with DNA in 150 μL of TE buffer solution and incubated for 30 minutes. The mixing ratio was adjusted so that the number of charges was 10 times the number of positive charges (CA ratio = 10) (15.8 μg of linear cationic polymer and 3.0 μg of DNA).
To this 150 μL solution, an Ad virus solution diluted to 1/20 concentration (6 × 10 ⁶
TCID ₅₀ / mL) is mixed with 60 μL (Ad virus mixed amount converted to 1 g of linear cationic polymer is 2.3 × 10 ¹⁰ TCID ₅₀ ), and then added to 100 μL of OPTI-MEM culture solution and mixed, 30 After incubating for a minute, it was added to the cultured cells (the addition amount was adjusted so that 0.5 μg of DNA was administered to any well). After 3 hours of culture, OPTI-MEM was removed, and after washing with PBS, complete medium was added and further cultured for 48 hours. Forty-eight hours later, gene transfer activity was evaluated using a luciferase assay kit. Correction was performed with the total protein concentration, and total protein quantification was performed with Bradford reagent from Bio Rad.
The result of introduction into FSHfb is shown in FIG.

[Example 3]
A gene transfer experiment was conducted in the same manner as in the Reference Example except that the 6-branch star type cationic polymer (Mn = 25,000) obtained in (2) of Example 1 was used instead of the linear cationic polymer. The results are shown in FIG.

[Comparative Example 1]
Without using the nucleic acid complex, use only Ad virus, add 60 μL of Ad virus solution (6 × 10 ⁶ TCID ₅₀ / mL) to 100 μL of OPTI-MEM culture solution, mix for 30 minutes, and then incubate to cultured cells Except for the addition, a gene introduction experiment was conducted in the same manner as in the Reference Example , and the results are shown in FIG.

[Examination of Ad virus titer]
The Ad virus produced with a commercially available kit was used after diluting the concentration to 1/20 in an unpurified or purified state. Purification was performed as follows.
293 cells were cultured in a 225 mm2 flask, 300 μL of 6 × 10 ⁶ TCID ₅₀ / mL Ad virus solution and 5 mL of 5% FCS-containing DMEM medium were added, and cultured at 37 ° C. for 4 days. The culture solution was centrifuged at 3000 rpm and 4 ° C. for 15 minutes, the precipitate was pulverized by sonication, and centrifuged at 10,000 rpm and 4 ° C. for 15 minutes. The virus stock solution was separated by density gradient centrifugation of 4.0 M cesium chloride solution and 2.2 M cesium chloride solution, and the virus band placed between the two cesium solutions was collected, dialyzed against PBS solution and chlorinated. Cesium was removed to obtain a purified virus solution. The titer was 3 × 10 ⁸ TCID ₅₀ / mL. Furthermore, an experiment was conducted to compare and evaluate gene transfer activity by lowering the titer by diluting the unpurified product to 1/80, 1/160, 1/320, 1/640, or 1/1280 concentration.

The cells used were primary cells (FSHfb) of myocardial fibroblasts derived from goat fetuses.
The number of 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 number of positive charges per unit weight in the 6-branched star type cationic polymer (Mn = 25,000) obtained in the above (2) of Example 1 is the monomer unit constituting the 6-branched star type cationic polymer ( 2-N, N-dimethylaminoethyl methacrylate) was calculated from the molecular weight of 156. On the other hand, the number of negative charges per unit weight in DNA was calculated from the number of base pairs by the sequence MAP and the average molecular weight 660 of nucleobases.
This 6-branched star-type cationic polymer was mixed with DNA in 150 μL of TE buffer solution and incubated for 30 minutes. The mixing ratio was adjusted so that the number of charges was 15 times the number of positive charges (CA ratio = 15).
The 150 μL solution was mixed with 60 μL of the diluted Ad virus solution, added to 100 μL of OPTI-MEM culture solution, mixed, incubated for 30 minutes, and then added to the cultured cells (0.5 μg of DNA in each well). Was administered). After 3 hours of culture, OPTI-MEM was removed, and after washing with PBS, complete medium was added and further cultured for 48 hours. Forty-eight hours later, gene transfer activity was evaluated using a luciferase assay kit. Correction (normalization) was performed with the total protein concentration, and total protein quantification was performed with the Bradford reagent of BioRad.
The result of introduction into FSHfb is shown in FIG.

In addition, the mixing amount converted per 1g of 6 branch star type cationic polymers of each Ad virus solution is as follows.
Refined product 1/20 times dilution: 6.3 × 10 ¹² TCID ₅₀
Undiluted product 1/20 times dilution: 7.6 × 10 ⁸ TCID ₅₀
Undiluted 1/80 dilution: 1.9 x 10 ⁸ TCID ₅₀
Undiluted product 1/160 times dilution: 9.5 × 10 ⁷ TCID ₅₀
Undiluted product 1/320 dilution: 4.8 × 10 ⁷ TCID ₅₀
Undiluted product 1/640 times dilution: 2.4 × 10 ⁷ TCID ₅₀
Undiluted product 1/1280 times dilution: 1.2 × 10 ⁷ TCID ₅₀

[Discussion]
1-4 shows the following.

  From FIG. 1, it can be seen that gene introduction into FSHfb by a cationic polymer vector became possible only by mixing Ad virus into the nucleic acid complex. However, the effect of ad virus mixing does not tend to improve in a concentration-dependent manner. Rather, a remarkable effect is obtained with a smaller mixing amount, and the DNA inserted into the ad virus is a gene encoding LacZ. Since it was unrelated to the activity of this firefly luciferase, it was confirmed that it was not a synergistic effect with the introduction of DNA inserted into the Ad virus.

  From FIG. 2, it can be seen that gene transfer is possible even with a 6-branch star polymer alone without mixing Ad virus in the established Hela cells. This is a known fact that the present inventors have reported. On the other hand, mixing Ad virus improves the gene transfer activity even in Hela cells, and it can be seen that the present invention is effective even when introduced into cell lines.

From FIG. 3, in the structure of the cationic polymer vector in which the introduction activity cannot be obtained with FSHfb without mixing Ad virus, there is a difference in gene introduction activity between the linear type and the 6-branched star type, and the same molecular weight. This suggests that a branched structure is advantageous.
Moreover, from the results of FIG. 1 and FIG. 3, it can be seen that, even with the same 6-branch star structure, the higher the molecular weight, the higher the possibility of obtaining high gene transfer activity.
In addition, in the activity evaluation of the Ad virus alone, the activity of firefly luciferase was not recognized, and LacZ (β-galactosidase) inserted into the Ad virus used in this example contributed to the firefly luciferase activity evaluation reaction nonspecifically. It was also shown that they did not.

  FIG. 4 shows that even if the Ad virus is not purified, the effect can be obtained by mixing it with the nucleic acid complex. From the fact that purification is usually performed and used as a high titer for gene transfer into cells, it is epoch-making that high activity is expressed in unpurified products. Since the activity was expressed by diluting the unpurified product to a concentration of 1/160, it was found that the Ad virus expressed the effect in a state of being mixed in a small amount into the nucleic acid complex, It is presumed that there is a possibility that some action is applied to the cell to promote the gene transfer activity of the nucleic acid complex itself.

Claims (6)

  A star-type cationic polymer, nucleic acid, and adenovirus having four or more polymer chains mainly composed of cationic vinyl monomers (hereinafter referred to as “cationic polymer chains”) are cultured together with host cells in a medium. A gene introduction agent composition comprising an unpurified adenovirus which is a fraction obtained by sonication and centrifugation of a culture.   2. The gene introduction agent composition according to claim 1, wherein the cationic polymer has a benzene ring as a nucleus, and the cationic polymer chain as a branched chain is bound to the nucleus.   3. The gene introduction agent composition according to claim 1 or 2, wherein the unpurified adenovirus comprises an inactivated individual. The gene transfer agent composition according to any one of claims 1 to 3, wherein adenovirus is 10 × 10 ¹² TCID ₅₀ or less in terms of conversion per gram of the cationic polymer.   The gene according to any one of claims 1 to 4, wherein the nucleic acid complex obtained by complexing the cationic polymer and the nucleic acid is further mixed with at least a part of an unpurified adenovirus. Introducing agent composition.   A star-type cationic polymer having four or more polymer chains mainly composed of cationic vinyl monomers (hereinafter referred to as “cationic polymer chains”) and adenovirus are cultured together with host cells in a medium, and the culture An in vitro gene transfer method comprising using in combination with unpurified adenovirus, which is a fraction obtained by sonication and centrifugation.

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