JP4765061B2 - Method for producing molecular complex for gene transfer - Google Patents

Description

  The present invention belongs to the technical field of molecular complexes (non-viral vectors) for introducing genes such as DNA into cultured cells or cells that make up living organisms.

  The technology for introducing genes into cells is extremely important in clinical applications such as gene therapy in which structural genes, antisense nucleotides, decoy-type nucleotides, ribozymes, etc. are introduced into cells. Therefore, it is excellent in safety and stability, and Development of a method for introducing a highly efficient gene into cells is strongly desired.

  Conventionally, when a gene is introduced into a cell, many viral vectors such as adenovirus and retrovirus have been used. In particular, in vivo, viral vectors have become useful materials due to their high introduction efficiency and reproducibility by diverting the natural phenomenon of infection. However, when it is used for gene therapy, it cannot be applied to the introduction of large genes, there are problems with stability of the virus, restriction of frequent administration by the immune mechanism, etc. Also, the safety of the virus itself should be improved The point is left. Therefore, development of non-viral vectors using nanoparticles composed of a complex of a polycation such as a dendrimer and a polycation such as a liposome, which is an aggregate of lipids, and a gene is underway.

  As an example of the above-mentioned non-viral vector, (Patent Document 1) includes phospholipid and quaternary ammonium salt as main membrane constituents, N-acylated asialofetin is contained in the membrane, liver disease A liposome vector for treatment of liver disease comprising a therapeutic peptide expression gene is described.

  (Patent Document 2) discloses a technique for forming a complex by mixing a non-viral vector composed of magnetic particles carrying a cationic polymer and a nucleic acid. This nucleic acid-containing complex is administered into a living body, and then is collected at a specific site of a blood vessel by applying a magnetic force from a device inserted outside or inside the body to treat an intravascular disease or the like. Furthermore, it is known that a complex of polylysine and a gene is also effective for gene introduction into a cell.

  The method using a non-viral vector such as a liposome as described above generally has a problem that gene introduction efficiency is low. In order to improve the efficiency of the vector, there is a conflicting function of forming a stable complex until an external gene is introduced into the cell, and reducing the stability and releasing the gene into the nucleus. Although required, a non-viral vector that sufficiently satisfies this requirement has not been proposed yet.

JP 7-69933 A JP 2004-129602 A

  In view of the above-described conventional circumstances, an object of the present invention is to provide a non-viral vector (molecular complex) that is excellent in stability and safety and has high gene transfer efficiency.

To solve the above problems, the present invention provides, as claimed in claim 1, a method for producing a molecular complex for transferring genes to cells constituting the cultured cell or organism, initially, the hydrogen-bonding functional It has a group or a bond, and a polymer capable of forming a gene and hydrogen bonds, by mixing the crystals of hydroxyapatite (calcium phosphate hydroxide) to bind the two, followed by the addition of genes, mixtures thereof 10000 by treatment with high pressure ～15000 atm, there is provided a method for producing a molecular complex you and forming a molecular complex of a nano or micrometer size.

  According to the above configuration, a stable molecular complex containing a gene is formed by high-pressure treatment. This molecular complex is in the form of a gel or fine particles, and when it is taken into cells by endocytosis or the like, the gene is easily separated by the action of an inorganic salt.

  According to the above configuration, the hydrogen bonding property is emphasized by the treatment under ultra high pressure, and the hydration state around the functional group is changed to form a strong structure.

  According to the above configuration, hydroxyapatite has a polar group on the crystal surface and forms a stable molecular complex by interaction with the polymer. Moreover, in the cell, it becomes calcium ion and phosphate ion, and the osmotic pressure changes to destabilize the molecular complex.

  According to the above configuration, in order to form a stable molecular complex of nano or micrometer size that can be used for gene introduction into cells, the range of pressure for treating the mixture is optimized.

According to the present invention, a molecular complex that has high gene transfer efficiency and can be used as a non-viral vector can be obtained. Since this molecular complex has nucleic acid enzyme resistance and exhibits gene expression over a long period of time, it can function as a “persistent” vector.
In addition, the gene can be introduced into cells even in the presence of serum, and therefore can be applied to gene therapy.

Embodiments of the present invention are described in detail below.
The molecular complex of the present invention is formed by treating a mixture of a polymer, an inorganic salt that can be ionized in a cell, and a gene with high pressure. By this high-pressure treatment, the hydration structure around the molecule is destroyed, the entire entropy is reduced, and a stable molecular complex in the form of gel or fine particles is obtained.

  As the polymer, a polymer having a hydrogen bonding functional group or bond is preferably used. These form a stable structure in which hydrogen bonds between genes are emphasized by high-pressure treatment and are stable even when the pressure is returned to normal pressure, or the half-life of decay is very long.

  Synthetic and natural polymers can be used as the above-described polymer having a hydrogen bonding functional group or bond, and both water-soluble and water-insoluble are applicable. Here, the hydrogen bondable functional group is a hydroxyl group, an amide group, a carboxyl group, an amino group, a sulfone group, an acyl group, an aldehyde group, an acetyl group, a nitrile group, etc., and the hydrogen bondable bond is an amide A bond, an ether bond, an ester bond or the like. In addition to polymers having these, polymers containing alkaloids (nitrogen), polymers having electrophilic and nucleophilic sites, polymers having conjugated sites, polymers having heteroatoms in the main chain may be used. it can.

  Specifically, as a synthetic polymer, polyvinyl alcohol, polyethylene glycol, polylactic acid-polyethylene glycol copolymer, polyhydroxyethyl methacrylate, polyvinyl acetamide, polyacrylamide, polyacrylic acid, polyvinyl amine, polyvinyl sulfate, polyvinyl sulfonic acid, Polymethylmethacrylate, Nylon, Polyamide, Polyimide, Polyphosphazene, Polylactic acid, Polyglycols, Polyεcaprolactam, Poly-N-isopropylacrylamide, Poly-γ-benzyl-L-glutamate, Polyorthoester, Poly Acid anhydride, polyacrylonitrile, polysulfone, polyallylamine, polymethacryloylphosphatidylcholine, polyglucosyloxyethyl methacrylate, poly-N-paravinylbenzyl- Examples include 4-o-β-galactopyranosyl-D-gluconamide, polyethylene terephthalate, polycarbonate, silicone resin, and fluororesin. In particular, polyvinyl alcohol is preferably used because the molecular complex to be formed is stable and the gene expression efficiency after incorporation into cells is excellent. Polyethylene glycol has been approved for administration to the human body, and is suitably used as a substance with excellent safety when applied to gene therapy.

Furthermore, examples of natural polymers include protein, sugar, microorganism-derived mucilage, plant-derived mucilage, starch, and the like. Specifically, antibodies such as dextran and IgG, collagen, gelatin, agarose, arginic acid, Starch, pectin, fibroin, cellulose, γ-polyglutamic acid, fibrin, lignin, elastin, chitin, chitosan and the like can be mentioned. Examples of the semi-synthetic polymer include cellulose and starch, and specific examples include regenerated cellulose, oxidized cellulose, cellulose acetate, and sucrose ester.
In the present invention, it is preferable to use a hydrogen-bonding polymer as described above, but besides that, it is excellent in molecular chain flexibility, and interacts with a gene or an inorganic salt by high-pressure treatment to cause entropy. Any polymer that tends to decrease can be used as appropriate. Specific examples include polyolefins such as polyethylene.

  The molecular weight of the polymer is not particularly limited and varies depending on the pressure applied to the mixture and the type of polymer, but it affects the size of the molecular complex to be formed. Is set as appropriate. In general, when the molecular weight is large, the size of the complex increases. Conversely, when the molecular weight decreases, the size of the complex decreases. For example, when polyvinyl alcohol is treated at about 10,000 atmospheres, the final molecular complex is formed into a size (200 nm or less) that can enter a capillary vessel, depending on the degree of saponification and the concentration dissolved in the liquid medium. Preferably has a molecular weight of about 100,000 to 300,000 in order to form a molecular weight of 1 to 50,000 and a molecular complex with a size of about 1 μm or less.

  Next, the present invention is characterized in that an inorganic salt is added. This inorganic salt stabilizes the molecular complex by interacting with the molecular chain of the polymer. For example, when hydroxyapatite (calcium hydroxide phosphate) is added, a more stable composite is obtained by the interaction between the surface of phosphoric acid in a hydroxyapatite crystal and a polymer (particularly a hydrogen-bonding polymer such as PVA). Form the body. The present inventor has also found that gene expression efficiency increases when an inorganic salt is added. This is because when the molecular complex is taken into the cell by an action such as endocytosis and then transported by the endosome, the endosome is acidic (about pH 5), so hydroxyapatite has calcium ions and phosphate ions. This is thought to be caused by the fact that water is flown into the molecular complex, whereby water flows into the molecular complex, the osmotic pressure changes and the molecular complex collapses, and as a result, the gene is easily released. As a specific example of such an inorganic salt, any inorganic salt that can be ionized with intracellular pH change (environmental change from pH 7 to pH 5) can be applied. Specifically, hydroxyapatite Other examples include sodium chloride, ammonium sulfate, and lithium bromide.

  Further, the gene in the present invention refers to DNA, RNA, or synthetic nucleotides that form a hybrid with DNA or RNA, and their modified ones. Also, a mixture of two or more of these may be used. Furthermore, genes include genes such as antisense genes, decoy-type genes, and ribozymes in addition to structural genes.

  When a mixture of the above substances is subjected to high pressure treatment, it is usually performed by adding a predetermined polymer, inorganic salt, and gene into a liquid medium and applying pressure to the solution. There is no restriction | limiting in particular as a liquid medium, Water or an organic solvent can be used. As the organic solvent, either a polar or nonpolar solvent can be applied. Specific examples of the organic solvent include benzene, toluene, xylene, chloroform, methylene chloride, carbon tetrachloride, DMF, acetonitrile, DMSO, MEK, acetone, lower alcohol, THF and the like.

  The concentration of the polymer and the inorganic salt in the liquid medium affects the size of the molecular complex, and is appropriately set in consideration of the application. In general, when the concentration is increased, the size of the molecular complex tends to increase. The specific concentration differs depending on the type of polymer or liquid medium and is not particularly limited. For example, polyvinyl alcohol having a molecular weight of about 170,000 is used as the polymer, hydroxyapatite is used as the inorganic salt, and water is used as the liquid medium. In this case, when a molecular complex having a size of 200 nm or less is to be formed by applying 10,000 atmospheres, the polyvinyl alcohol is about 0.005 to 0.015 wt% and the inorganic salt is about 0.0005 to 0.0015 wt%. Is appropriate.

  The pressure applied to the mixture is suitably 1000 to 15000 atm, preferably 3000 to 15000 atm, and more preferably 4000 to 15000 atm in a state dissolved in a liquid medium, and is performed using an ultra-high pressure treatment apparatus or the like. When adding a mixture to a liquid medium, a polymer, an inorganic salt, and a gene may be added at the same time. First, the polymer and the inorganic salt crystal are mixed, and ultrasonic vibration is applied to both. It is preferable to bind them, and then add the gene of interest followed by high-pressure treatment because the stability of the complex increases. The form of the molecular complex formed by the high-pressure treatment is gel or fine particles, and fine particles are generated when the concentration of the mixture is low. The temperature during the high-pressure treatment is preferably from room temperature to about 50 ° C., but may be treated at a higher or lower temperature as necessary.

  The target molecular complex is obtained by the above procedure. Genes can be introduced by allowing this molecular complex to act on cells. The cell into which the gene is to be introduced is appropriately selected according to the gene to be introduced. Specific examples include, but are not limited to, vascular endothelial cells, bone marrow cells, blood stem cells, blood cells, bone cells, cardiomyocytes, smooth muscle cells, fibroblasts, skeletal muscle cells, and the like. .

  In the method for transforming cells using the molecular complex of the present invention, when introduced into cultured cells, the molecular complex is added to the medium of cultured cells cultured in an appropriate culture solution, and the cells are appropriately cultured at an appropriate temperature for an appropriate time. By treatment (incubation) under various conditions. At this time, in the present invention, it is known that even when FCS (fetal calf serum) or the like is used as a medium, high gene expression is exhibited. Since gene delivery into cells is possible even in the presence of serum, the antigenicity is low, it can withstand frequent administration, suggesting that it is effective for gene therapy.

  The gene itself is generally unstable in vivo and is degraded by nucleic acid enzymes. However, the molecular complex of the present invention is protected by a polymer and an inorganic salt, and maintains sufficient gene expression. However, it also has high nucleic acid enzyme resistance. Therefore, it can be suitably used as a “persistent” non-viral vector.

  In addition, the molecular complex of the present invention can be directly inoculated not only into the above cultured cells but also directly into a living body and introduce a gene into cells constituting the living body. Although any method can be adopted for administration to a living body, parenteral administration is preferred for sustained and local gene expression. That is, if necessary, a pharmaceutically acceptable carrier such as a stabilizer, a preservative, a solubilizer, a pH adjuster, or a thickener is added to the molecular complex of the present invention, and this is injected into a living body. Can be performed. Intravenous or intraarterial injection is preferred, but intramuscular, adipose tissue, intralymph node, subcutaneous, body cavity (pericardial cavity, thoracic cavity, abdominal cavity, cerebrospinal cavity), intramuscular administration, lesions It can also be administered directly into the tissue. The dose is appropriately set according to the type of gene, the site to be treated, and the like.

  When administered parenterally, not only when a liquid drug is prepared and administered, it may be a solid or semi-solid preparation. In this case, the method of directly embedding the preparation at the site for gene transfer, the method of injecting the paste preparation with a syringe, the method of injecting the particulate preparation by suspension injection, the catheter is inserted percutaneously transluminally, Examples thereof include a method of indwelling a molecular complex attached to a stent in a blood vessel, a method of injecting fine particles of the molecular complex with a catheter, and a method of localizing the site where gene expression is expected.

  The molecular complex of the present invention can be suitably applied as a non-viral vector to gene therapy in addition to transformation of cultured cells. Applicable diseases include, but are not limited to, cancer (brain tumor, metastatic malignant tumor, breast cancer, etc.), infectious diseases such as HIV, genetic diseases, diseases in the cardiovascular region, etc. .

Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
Polyvinyl alcohol (PVA117, manufactured by Kuraray Co., Ltd., saponification degree 99.3%, molecular weight 74800) and crystals of hydroxyapatite (HAp) of about 50 nm were added to water and subjected to ultrasonic vibration for 3 minutes. The concentrations of PVA and HAp were 1% by weight and 0.1% by weight, respectively. Thereafter, a 1 kb ladder DNA marker was added, and treatment was performed for 10 minutes at 40 ° C. and 10,000 atmospheres using an ultrahigh pressure treatment apparatus (Dr. CHEF; Kobe Steel, Ltd.). The obtained ultrahigh pressure treatment solution was observed visually and under a microscope.
As a result of the experiment, particles of about 200 nm were observed by SEM observation of the solution after high pressure treatment, suggesting the formation of a molecular complex of PVA-HAp-DNA.
In addition, in order to confirm that the interaction of the obtained molecular complex is hydrogen bonding, an interaction inhibition experiment was conducted using urea. When urea was added and the ultra-high pressure treatment was performed, complexation could not be confirmed. This is the result of the inhibition of hydrogen bonding by urea, suggesting that the interaction of the molecular complex is hydrogen bonding.

(Example 2)
The efficiency of gene expression was evaluated using a fluorescent agent (rhodamine) labeled pEGFP as a gene. A molecular complex to be incorporated into cells was formed by the same method as in Example 1 except that the Rh-labeled pEGFP was used instead of the DNA ladder. In addition, monkey-derived kidney cells (COS7) were used as cells for gene transfer.
As comparative examples, (1) DNA (Rh-pEGFP) alone was treated with high pressure, (2) PVA and DNA alone were treated with high pressure, (3) Conventional liposome vector (Lipofectamine 2000) and DNA were mixed (No pressure applied), (4) a mixture of HAp and DNA only (no pressure applied), and (5) a mixture of PVA, HAp and DNA (no pressure applied), which is incorporated into cells and gene expression Evaluated. The results of fluorescence microscope observation after gene expression are shown in FIG. 1 for Example 2 and FIG. 2 for the comparative example (3).
As shown in the figure, it was revealed that the molecular complex of the present invention exhibits a gene expression efficiency equal to or higher than that of a conventional liposome vector. In addition, the other comparative examples did not show gene expression ability or were very low in efficiency.

It is a figure which shows the result of the fluorescence microscope observation in Example 2. FIG. It is a figure which shows the result of the fluorescence microscope observation in a comparative example.

Claims (1)

A method for producing a molecular complex for introducing a gene into cultured cells or cells constituting a living body,
First, a polymer that has a hydrogen bonding functional group or bond and can form a hydrogen bond with a gene and a hydroxyapatite (calcium hydroxide phosphate) crystal are mixed together, and then the gene is bound. in addition, by treating these mixtures with a high pressure of 10,000 to 15,000 atmospheres, method for producing a molecular complex you and forming a molecular complex of a nano or micrometer size.