JP2001335489A - Nucleic acid-introducing pharmaceutical preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a nucleic acid-introducing pharmaceutical preparation for promoting the introduction efficiency of the nucleic acid by a direct introduction method for a naked DNA into a skeletal muscle cell or a myocardial cell. SOLUTION: This nucleic acid-introducing agent comprises a mixture of the desired nucleic acid with a polyethylene glycol and is capable of introducing the nucleic acid into the skeletal muscle cell or myocardial cell with a high efficiency.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the introduction of nucleic acid into skeletal muscle cells or cardiomyocytes by direct introduction of naked DNA, and particularly to gene therapy. More specifically, the invention is naked
The present invention relates to a nucleic acid transfection preparation for efficiently and safely transducing nucleic acid into skeletal muscle cells or cardiomyocytes by a direct DNA transfection method.

[0002]

2. Description of the Related Art Gene therapy is a method of treating diseases using genes as drugs. In recent years, research on gene therapy has been actively conducted, and 3,000
Gene therapy has been attempted in more than one patient. Genetic diseases such as ADA deficiency, familial cholesterol, hemophilia, and Gaucher's disease, and diseases such as malignant brain tumor, leukemia, neuroblastoma, and renal cancer, enhance immune or drug resistance genes, Gene therapy by introduction of a sense gene or the like is being studied. In clinical application of such gene therapy, development of a method for efficiently and safely introducing a target gene into cells has become one of the important technical issues.

[0003] Gene transfer methods can be broadly classified into virus vector methods and non-viral vector methods. The viral vector method is more efficient than the non-viral vector method, and the viral vector method is widely used in current gene therapy. The virus vector method includes a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, and the like. Among them, the retrovirus vector, which has been most developed, has many clinical cases. Retroviral vectors generally have a broad host range and can treat many types of cells. However, conversely, the gene is introduced into cells other than the target cells, and is not suitable for in vivo administration from the viewpoint of side effects. Therefore, since the target cell is once taken out of the body, the gene is transferred, and then the body is returned to the body again,
Facilities that can perform such treatment are limited. In addition, since the gene introduced by the retroviral vector is integrated into the chromosome of the host cell, the transgene is stably maintained for a long time without being lost by cell division. However, since the integration site into the chromosome is random, there is a concern that the proto-oncogene or the like may be activated. Adenovirus vectors have the advantages of extremely high gene transfer efficiency and the ability to transfer genes into non-dividing cells, but they are derived from pathogenic viruses and elicit an immune response against the transfected cells. There are drawbacks such as Adeno-associated virus vectors are derived from non-pathogenic viruses and have high safety, but studies on vector preparation methods and the like have been delayed. In addition, producing a viral vector required for treatment requires culturing a large amount of cells, which is very costly.

[0004] Instead of the above-described virus vector method, there is a demand for the development of a gene transfer method that is safer, lower in cost, and has a higher gene transfer efficiency.
With respect to safety, non-viral vectors are superior, and development of non-viral vectors with higher gene transfer efficiency is expected. Non-viral vector methods include calcium phosphate coprecipitation method, DEAE-dextran method, electroporation method, lipofection method, gene transfer method using polymer micelle vector, gene transfer method using particle gun, direct gene transfer method ( naked DNA). The most promising of such non-viral vector methods is the lipofection method using liposomes.However, the gene transfer method using liposome vectors can transfer genes extremely efficiently in cultured cells, When used for gene transfer in tissues, the expected effect is not obtained, and the gene transfer efficiency is often extremely low. Similarly to the liposome vector, a polymer micelle vector is a vector for delivering a nucleic acid into a cell by utilizing the endocytosis of the cell. The polymer micelle vector is a vector formed by binding a nucleic acid to a molecule called a block copolymer in which a hydrophilic polymer compound and a hydrophobic polymer compound are bound.

[0005] One of the methods classified as a gene transfer method using a non-viral vector is a direct transfer method of naked DNA. This gene transfer method uses molecules such as liposomes and polymer micelles that form a complex with nucleic acids, unlike the above-described method using cell endocytosis such as a liposome vector as a gene transfer mechanism into cells. Not. The mechanism of gene transfer into cells is a method utilizing properties specific to skeletal muscle cells and cardiomyocytes, and is a method for gene transfer using naked nucleic acids as they are. The actual operation at the time of gene transfer according to the present method is a very simple method by simply injecting a solution in which the nucleic acid to be introduced is dissolved into skeletal muscle tissue or myocardial tissue using a syringe or the like. this,
Therapeutic treatment of arteriosclerosis obliterans (S. Takesita, et al., Circulatio
n, 90, II-228, 1994; JMIsner, etal., Hum.Gene.Ther.,
7,989,1996; I.Baumgartner, Circulation, 31,1114,199
8), but this clinical trial of gene therapy is considered to be the most successful gene therapy trial. J. Wolff et al. First reported the results of a study using mouse skeletal muscle on the direct transfer method of naked DNA, a gene transfer method used in gene therapy (JAWolf
f, et al., Science, 247, 1465, 1990). Na to skeletal muscle cells
The mechanism of introduction of ked DNA has been studied by JA Wolff et al. (JAWolff, et al., J. Cell. Sci., 1
03,1249,1992), it has been reported that naked DNA binds to T-tubule which is specifically present in skeletal muscle cells and cardiomyocytes, and is taken up into cells via T-tubule. In addition, when performing gene transfer into skeletal muscle, the transgene must first reach the cell membrane of skeletal muscle cells through the periosteum and intimal membrane, which are present around the skeletal muscle cells and serve as barriers, It has also been reported that naked DNA can pass through these barriers relatively easily and reach the skeletal muscle cell membrane surface. These are the results of naked DN in skeletal muscle cells.
A It is thought that the gene transfer by the direct transfer method has been enabled.

[0006] Gene therapy by the method of directly introducing naked DNA into skeletal muscle has a great advantage in safety since no viral vector is used. That is, the possibility that the introduced gene is integrated into the chromosome of the host cell is extremely rare, and there is almost no risk of mutation in the gene of the host cell. further,
Since it is a direct transfer method, the possibility of gene transfer to an unexpected organ or site is extremely low. In addition, since the nucleic acid itself has no toxicity or immunogenicity, there is no danger of causing an inflammatory reaction, and an immune reaction is unlikely to occur, so that repeated administration is possible. In addition, there is an advantage that there is no limitation on the length of a nucleic acid such as a plasmid to be introduced. In short, naked DNA
In the direct transfer method, as long as nucleic acids such as plasmids for transfer can be produced, no special facilities or techniques are required, large-scale adjustment is easy, and gene therapy can be performed very easily at low cost and safely. A possible way. Current,
Studies on renal disease, erythropoietin replacement and immunotherapy have also been conducted (Y.Isaka, et al., Nature Med.,
2,4,418,1996; SK.Tripathy, et al., Proc. Natl Acad Sc
i USA, 93, 10876, 1996; M. Chattergoon, et al., FASEB
J., 11, 753, 1997), and can be expected to be applied to gene therapy in a wide range of fields in the future.

However, the low gene transfer efficiency is a major problem that restricts the use of the present gene transfer method.
Studies on the treatment of muscular dystrophy indicate that gene expression is low for clinical application (G. Acsa
di, et al., Nature, 29, 352, 815, 1991). To solve this problem, various conditions have been studied for a method of directly introducing naked DNA into skeletal muscle (S. Jiao, et al., Hum. Gen.
e.Ther., 3,21,1992; M.Manthorope, et al., Gene.Ther.,
4,419,1993; V. Budker, et al., Hum. Gene. Ther., 3,593,1
996). JAWolff et al. Found that the use of multiple needles, which increase the damage to muscles during gene transfer, reduces the efficiency of gene transfer, and that the amount of injection solution, the rate of injection into the muscle, and the size of the needle used, There is no change in the transfection efficiency, the solution that dissolves DNA is a solution that does not damage the living body such as physiological saline, or the gene to be transfected is pelletized and embedded in muscle to improve the transfection efficiency. (Wolff, Bi
otechniques, 11,474,1991). However, a method for dramatically improving gene transfer efficiency has not been developed.

Although the efficiency of gene transfer into skeletal muscle cells can be safely improved by using liposome vectors and the like, it has been found that liposome vectors have good gene transfer in gene transfer into cultured skeletal muscle cells. On the other hand, it has been reported that the efficiency of gene transfer into skeletal muscle cells in vivo is low. The reason for this is that, in the case of gene transfer into skeletal muscle cells, the liposome vector must reach the sarcolemma, the cell membrane of skeletal muscle cells, through the periosteum and intimal membrane around the skeletal muscle cells. It is believed that the positive charge of cationic lipids used in many liposome vectors is disadvantageous for passing through membranes existing around skeletal muscle cells (JAWolff, et al., JC
ell. Sci., 103, 1249, 1992). In other words, in order to efficiently and safely transfer genes into skeletal muscle cells and cardiomyocytes, use T-tubules that exist specifically in skeletal muscle cells and cardiomyocytes and can transport nucleic acids into cells The gene transfer method described above is advantageous, and it is expected that the efficiency of gene transfer by the direct transfer method of naked DNA will be improved.

[0009]

The introduction of nucleic acid into skeletal muscle cells or cardiomyocytes by direct introduction of naked DNA involves
It is a safe and low cost method. However, the low gene transfer efficiency of this method limits its use.
An object of the present invention is to enhance the gene transfer efficiency of the naked DNA direct transfer method and to enable safe, low-cost and high gene transfer efficiency gene transfer.

[0010]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, have desired to improve the efficiency of gene transfer into skeletal muscle cells or cardiomyocytes by the method of directly introducing naked DNA. It has been found that a desired preparation can be efficiently introduced into skeletal muscle cells or cardiomyocytes by preparing a preparation in which the above nucleic acid and polyethylene glycol are mixed, and the present invention has been completed. That is, the gist of the present invention is as follows. (1) A preparation for introducing a nucleic acid into cells having a T-tubule of a living body or tissue, comprising a mixture containing a desired nucleic acid and polyethylene glycol. (2) A preparation for introducing a nucleic acid into a skeletal muscle cell or a cardiomyocyte of a living body or tissue, comprising a mixture containing a desired nucleic acid and polyethylene glycol. (3) The preparation according to the above (1) or (2), wherein the nucleic acid is a gene expression vector containing a gene sequence to be expressed in cells into which the nucleic acid has been introduced. (4) The nucleic acid-introduced preparation according to any one of (1) to (3), wherein the polyethylene glycol concentration is 0.5 (w / v)% to 20 (w / v)%.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The molecular weight of polyethylene glycol used in the present nucleic acid-introduced preparation is not particularly limited, but is preferably from 200 to 20,000, which has been used as a pharmaceutical additive. For example, polyethylene glycol 4000 having an average molecular weight of 4000 can be used. The concentration of polyethylene glycol used in the nucleic acid-introduced preparation is not particularly limited, but as shown in the examples, only the raw food used for the control at a concentration of 1% (W / V) to 10% (W / V) is used. Β-galactosidase activity is about 8 to 18 times as high as that when used, and it is preferable to use 0.5% (W / V) or more and 20% (W / V) or less for efficiency of gene transfer.

The solution for dissolving the nucleic acid and polyethylene glycol of the present nucleic acid-introduced preparation may be any solution as long as it can be administered to a living body. For example, distilled water, physiological saline, phosphate buffer and the like may be used. Can be.

The nucleic acid that can be introduced into cells by the nucleic acid-introduced preparation of the present invention is not particularly limited by the size of the nucleic acid, the amount of the nucleic acid, the type of the nucleic acid and the like. As the type of nucleic acid, for example, linear double-stranded DNA, circular double-stranded DNA, oligonucleotide,
RNA, antisense, ribozyme, triplex and the like. For example, a structural gene encoding a useful protein can be introduced using the present nucleic acid-introduced preparation, and the gene can be expressed. The form of the gene to be used may be any form as long as the gene can be expressed in a living body. For example, a plasmid in which a structural gene is incorporated into a mammalian cell expression vector is preferable. The expression vector is
For example, pSVL expression vector (Pharmacia), pSG expression vector (Japanese Patent Application No. 6-117506, Japanese Patent Application No. 6-169238), pKCR expression vector (K.O'Hare, et al., Proc. Natl. Acad. Sci. USA) , 7
8,1527,1981), pCAGGS expression vector (H. Niwa, et al., G
ene, 108, 193, 1991), but is not limited by the type of expression vector. When an expression vector incorporating such a structural gene is introduced by the nucleic acid-introduced preparation of the present invention, extremely high gene expression is exhibited as shown in the Examples below.

The structural gene to be used is not particularly limited. For example, vascular endothelial cell growth factor which has an angiogenic activity and is considered to be effective for the treatment of atherosclerosis obliterans
(VEGF, vascular endothelial growth factor), hepatocyte growth factor (HGF, hepatocyte growth factor) and fibroblast growth factor (FGF) gene. Obstructive arteriosclerosis causes obstruction and stenosis of peripheral blood vessels, and severely requires amputation of the lower leg. Such a patient is intramuscularly injected with a plasmid vector for mammalian cell expression in which the gene having the angiogenic activity described above has been incorporated as a structural gene, and the proteins having these angiogenic activities are produced in the muscle cells. The formation and treatment of collateral circulation can be promoted. Similarly, myocardial infarction can be treated by injecting a mammalian cell expression vector having a gene for an angiogenic factor as a structural gene into myocardium to promote the formation of collateral circulation.

Other structural genes include, for example, the dystrophin gene deficient in muscular dystrophy, the erythropoietin gene which is a blood-growing factor acting on erythroid cells and is useful for renal anemia, and the treatment of hemophilia patients. Coagulation factor VIII / IX gene, insulin gene effective for the treatment of insulin-dependent diabetic patients, and transforming growth factor (TGF-β), which is thought to promote fibrosis in various fibrosis. , transf
For example, a decorin gene that inhibits the activity of orming growth factor-β) can be used. In addition, reporter genes such as β-galactosidase, luciferase, chloramphenicol acetyltransferase and the like can be used. further,
If a gene of a constituent protein of an infectious pathogenic microorganism such as a virus or a bacterium is used as a structural gene, it can be used as a DNA vaccine. Antisense can also be used, for example, Duchenne muscular dystrophy due to exon deletion can be treated by using antisense of a splicing promoting sequence.

The method for administering the gene-introduced preparation is not limited, but can be preferably carried out by injection. In the tissue to be transfected, the cells that make up the tissue
There is no particular limitation as long as it has a T-tubule, but skeletal muscle, cardiac muscle and the like are preferred. The dosage of the nucleic acid transfer preparation of the present invention is not limited, but the nucleic acid transfer preparation containing the nucleic acid in an amount showing a satisfactory effect for treating the target disease is injected once at about 0.03 ml to about 20 ml. It is possible. The required amount of nucleic acid and the number of administrations vary depending on the disease to be treated, but when performing angiogenesis therapy for obstructive arteriosclerosis of the lower leg, for example, polyethylene glycol 4000 content in which about 2 mg of VEGF plasmid vector is dissolved A therapeutic effect can be expected by injecting 3 ml of a 1 (W / V)% nucleic acid-introduced preparation twice into the muscle at intervals of about 3 weeks. When angiogenesis therapy is performed for patients with myocardial infarction or angina, for example, about 500 μg of VE
A therapeutic effect can be expected by injecting about 3 ml of a nucleic acid transfection preparation having a polyethylene glycol 4000 content of 1 (W / V)% in which the GF plasmid vector is dissolved into myocardium.

[0017]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. 1. Construction of plasmid pCAGGS lac-Z The lac-Z gene (gene encoding β-galactosidase) was inserted downstream of the CMV-IE enhancer and the chicken β-actin promoter sequence, and the plasmid pCAG shown in FIG.
AGGS lac-Z was constructed.

2. Preparation of Plasmid pCAGGS lac-Z Nucleic Acid-Introduced Preparation Polyethylene glycol 4000 was dissolved in physiological saline so as to have various concentrations, and then sterilized by filtration through a filter (Millipore) having a pore size of 0.22 μm to prepare a polyethylene glycol solution. The above-mentioned 1. PCAGGS lac-Z constructed as shown in
Final polyethylene glycol concentration from 1% (W / V) to 10
% (W / V) and the concentration of the plasmid was 0.167 μg / μl to prepare a nucleic acid transfection preparation. As a control, the plasmid was dissolved in physiological saline to reduce the plasmid concentration.
A solution having a concentration of 0.167 μg / μl was prepared.

3. Gene transfer into mouse skeletal muscle Mice were given pentobarbital sodium (50 mg / kg)
Was administered intraperitoneally and anesthetized. After anesthesia, 2. 30 μl of the nucleic acid transfection preparation prepared in the above and the solution prepared as a control were injected into the tibialis anterior muscle using a 29G syringe (Mijector, Terumo Corporation).
After l muscle injection, they were bred.

[4] Measurement of β-galactosidase activity On day 5 after the intramuscular injection, the mouse injected with the nucleic acid-introduced preparation was exsanguinated and killed under ether anesthesia, and the tibialis anterior muscle was collected. The activity of β-galactosidase contained in the collected tibialis anterior muscle was measured. The results are shown in FIG.
Groups transfected with the nucleic acid-introduced preparation of the present invention, especially 1% polyethylene glycol 4000 / saline, 4%
A group using polyethylene glycol 4000 / saline and 10% polyethylene glycol 4000 / saline, and these groups showed significantly higher gene expression than the group in which gene transfer was performed using a solution in which a plasmid was dissolved in physiological saline used as a control. Β-galactosidase activity, which is an indicator of, was high. The above results indicate that the nucleic acid transfer preparation of the present invention
This indicates that the product is highly useful as a drug that improves gene transfer efficiency in the direct transfer method of A.

5. Safety test pCAGGS lac-Z 10 μg / μl, 10% polyethylene glycol 4000 / Nucleic acid transfection preparation prepared with a raw diet 70
μl was injected intramuscularly into the tibialis anterior muscle of mice. Ten days after the intramuscular injection, the animals were bled to death under ether anesthesia and dissected, and the tibialis anterior muscle was collected. Tissue sections of the collected tibialis anterior muscle were prepared and examined microscopically, and the tibialis anterior muscle tissue intramuscularly injected with the nucleic acid-introduced preparation showed a histological image similar to that of a normal mouse, and no abnormalities were observed. . The LD ₅₀ when polyethylene glycol 4000 was injected intravenously into mice was 16%.
g / kg. Further, LD ₅₀ when the pCAGGS lac-Z were intravenously injected into mice was 10 g / kg.

[0022]

The nucleic acid-introduced preparation of the present invention comprises naked DNA
And the efficiency of gene transfer into skeletal muscle cells and cardiomyocytes by the direct transfer method. Specifically, the nucleic acid introduction preparation of the present invention is a preparation which has high gene transfer efficiency, high safety, and enables gene transfer at low cost. Particularly, the present invention is a nucleic acid transfection preparation that enables high-efficiency gene transfer into skeletal muscle and cardiac muscle, which are mammalian tissues, and enables safe, low-cost gene transfer.

[Brief description of the drawings]

FIG. 1 shows a schematic diagram of the plasmid pCAGGS lac-Z.

FIG. 2 shows the β-galactosidase activity in the mouse tibialis anterior muscle when the plasmid pCAGGS lac-Z was transfected into the mouse anterior tibialis muscle for the nucleic acid transfection preparation to which polyethylene glycol 4000 of the present invention was added and the control.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C12N 15/09 C12N 15/00 A F term (Reference) 4B024 AA01 AA20 CA01 DA02 EA04 FA02 FA06 GA11 HA17 4C076 AA12 BB15 EE23 FF68 4C084 AA13 CA53 DB56 DB57 MA66 NA05 ZA361 ZA451 4C086 AA01 AA02 EA16 MA66 NA05 ZA36 ZA45

Claims (4)

[Claims] 1. A nucleic acid preparation comprising a mixture containing a desired nucleic acid and polyethylene glycol, which is used for introducing a nucleic acid into cells having a T-tubule of a living body or tissue. 2. A preparation for introducing a nucleic acid into a skeletal muscle cell or a cardiomyocyte of a living body or tissue, comprising a mixture containing a desired nucleic acid and polyethylene glycol. 3. The preparation according to claim 1, wherein the nucleic acid is a gene expression vector containing a gene sequence to be expressed in cells into which the nucleic acid has been introduced. 4. The polyethylene glycol concentration of 0.5 (w /
The nucleic acid-introduced preparation according to any one of claims 1 to 4, wherein the amount is from v)% to 20 (w / v)%.