JP2003265171A - Gene dry powder and method for preparing the same and apparatus for preparing gene dry powder - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide gene dry powder which has high stability and high gene expression efficiency, to provide a method for preparing the same, and to provide an apparatus for preparing the gene dry powder. <P>SOLUTION: This method for preparing the gene dry powder comprises simultaneously charging carbon dioxide and an auxiliary solvent such as ethanol into a carbon dioxide/auxiliary solvent mixing column to obtain the supercritical carbon dioxide/ auxiliary solvent mixture liquid in the column, supplying the supercritical carbon dioxide/auxiliary solvent mixture liquid from the carbon dioxide/auxiliary solvent mixing column to a fine particle-producing column in a supercritical state, simultaneously supplying water from a water-supplying source to the fine particle-producing column, stabilizing the preparation apparatus, injecting and pouring a gene solution containing the complexes of plasmid genes with a polycation such as chitosan and an excipient such as mannitol from a gene solution injection port into the carbon dioxide/auxiliary solvent mixture liquid, thereby extracting water contained in the gene solution with the carbon dioxide/auxiliary solvent mixture liquid, and accumulating the left plasmid gene-polycation complex and the excipient in the fine particle-producing column as the dry powder. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a gene dry powder used for gene direct administration into the lung in gene therapy for local pulmonary diseases, a method for preparing the same, and an apparatus for preparing the gene dry powder.

[0002]

2. Description of the Related Art The gene therapy that has been performed so far includes an indirect method in which a patient's lymphocytes and bone marrow cells are once taken out of the body, a gene contained therein is cloned, and the gene is returned to the patient's body. There is a direct method of administering the clone directly to the diseased tissue. Of these, the direct method is becoming mainstream. In most cases, gene administration by the direct method is performed by intravenous administration of a solution containing the gene regardless of whether it is a clinical test or a basic test. In the case of gene therapy for local pulmonary diseases, genes administered intravenously are delivered to the lungs via other organs such as the liver, so that the efficiency of delivery to the lungs decreases accordingly. In addition, genes are decomposed by enzymes in the blood,
Therefore, the delivery efficiency to the lung becomes lower. The direct administration of the gene into the lung is performed by directly administering the solution containing the gene into the lung or administering it into the lung using a nebulizer. Direct administration of a solution containing the gene into the lungs imposes a burden on the patient. When the solution containing the gene is made into fine particles with a nebulizer, the gene is decomposed by ultrasonic energy.

In recent years, various studies have been conducted in order to efficiently deliver a gene to a target cell in a living body. Since the respiratory system from the nasal cavity to the alveoli can deliver the gene directly into the lung, direct administration of the gene into the lung causes lung cancer, pulmonary fibrosis, antigen-induced airway hyperreactivity, etc.
It is said to be effective for treating local lung diseases.

The key to successful gene therapy depends on how genes are delivered into the cells of interest in the body. So far, it is said that it is a viral vector that has a high gene expression efficiency, but in the case of a viral vector, the safety is not sufficiently ensured. Therefore, using a non-viral vector that has no risk of viral infection,
Studies are underway to improve gene expression efficiency. Among non-viral vectors, cationic polymers have the ability to form a complex with genes, and have begun to attract attention as useful non-viral vectors. Chitosan, polylysine, and polyethyleneimine are used as cationic liposomes and cationic microspheres. Is being studied as a gene delivery system.

[0005] Further, there is a system for administering dry powder into the lung, which is considered as a promising system for gene administration into the lung. By applying the supercritical extraction technology, it became possible to prepare dry powder with a particle size suitable for inhalation and needle-free injection. So far, preparations of lactose, steroids, protein powders, biodegradable microspheres, liposomes and the like using supercritical carbon dioxide have been performed.

[0006]

An object of the present invention is to provide a gene dry powder having high stability and high gene expression efficiency, a method for preparing the same, and an apparatus for preparing the gene dry powder.

[0007]

The invention according to claim 1 solves the problem relating to a gene dry powder, which comprises a complex of a plasmid gene and a cationic vector, and an excipient as a carrier of the complex. The dry powder included as

In the gene dry powder according to the present invention, pCMV-Luc in which CMV (gene expression promoter of cytomegalovirus) is incorporated as a promoter of a plasmid gene can be used.
When a plasmid gene in which the promoter CMV is incorporated is used, transduction occurs and a phenotypic effect is exhibited. In the present invention, any plasmid is applicable without being limited to pCMV-Luc. In addition, p
Since CMV-Luc itself has no medicinal effect, it is necessary to use a gene for the purpose of treating local lung disease in the case of practical application.

Further, in the gene dry powder according to the present invention, chitosan can be used as a cationic vector. Chitosan suppresses the degradation of the plasmid gene in the dry powder preparation process and enhances the transduction efficiency. Further, the recovery rate of the complex of the plasmid gene and the cationic vector at the time of preparing the dry powder is also improved.

The gene dry powder according to the present invention not only has high stability and high gene transfer efficiency,
It has a high gene expression efficiency and exhibits an excellent gene delivery action in which the action lasts for a long time.

In the dry gene powder according to the present invention, CMV is used as a promoter as a plasmid gene.
When pCMV-Luc in which is incorporated and chitosan is used as the cationic vector, and the molar ratio of nitrogen in chitosan to phosphorus in the plasmid gene is 5, the luciferase activity in the lung becomes maximum and the most gene The introduction efficiency is high.

In the gene dry powder according to the present invention, typical mannitol can be applied as an excipient, but in addition, sugars such as lactose commonly used in pharmaceuticals, starch, and cellulosic polymers (hydroxycellulose, hydroxy). Excipients generally used as pharmaceutical additives such as propyl cellulose) can be applied.

The invention according to claim 6 solves the problem relating to the method for preparing a gene dry powder of the present invention, wherein a supercritical carbon dioxide / auxiliary solvent mixture is prepared by adding an auxiliary solvent such as ethanol to supercritical carbon dioxide. A fluid is produced, and the mixed fluid is caused to flow into the fine particle generation column and water is caused to flow into the fine particle generation column to continue injection of the supercritical carbon dioxide / co-solvent mixed fluid and water into the fine particle generation column. After a predetermined time has passed, an aqueous solution of an excipient such as mannitol containing a complex of a plasmid gene and a cationic vector while continuing to supply supercritical carbon dioxide, water and a co-solvent into the particle generation column for a predetermined time. Excipient containing a complex of a plasmid gene and a cationic vector, which was injected into the particle generation column and simultaneously injected into the particle generation column The water contained in the aqueous solution was extracted with supercritical carbon dioxide, it is summarized as method for preparing a gene dry powder, characterized in that to obtain a complex of plasmid genes and cationic vectors carried in the vehicle.

In the method for preparing a gene dry powder according to the present invention, pCMV-Luc having CMV incorporated as a promoter as a plasmid gene can be used. When a plasmid gene in which the promoter CMV is incorporated is used, transduction occurs and a phenotypic effect is exhibited. Since pCMV-Luc itself has no medicinal effect, it is necessary to use a gene for the purpose of treating local lung disease in the case of practical application.

In the dry powder preparation method according to the present invention, chitosan can be used as a cationic vector. Chitosan suppresses the degradation of the plasmid gene in the dry powder preparation process and enhances the transduction efficiency. Further, the recovery rate of the complex of the plasmid gene and the cationic vector at the time of preparing the dry powder is also improved.

In the method for preparing dry powder according to the present invention, pC in which CMV is incorporated as a promoter is used.
MV-Luc was used, chitosan was used as the cationic vector, and mannitol and the like containing a complex of the plasmid and the gene-cationic vector were adjusted so that the molar ratio of nitrogen in the chitosan to phosphorus in the plasmid gene was 5. An aqueous solution of the excipient is used. Thus, when the molar ratio of nitrogen in chitosan to phosphorus in the plasmid gene is 5,
The luciferase activity in the lung is maximized and the gene transfer efficiency is highest.

In the method for preparing a gene dry powder according to the present invention, when a fine particle production column having a V-shaped nozzle is used as the fine particle production column, a carbon dioxide / ethanol mixed fluid and water are collided at the tip of the V-shaped nozzle. To promote mixing and dispersion.

An eleventh aspect of the present invention solves the problem relating to the gene dry powder preparation device of the present invention, and comprises a carbon dioxide cylinder, an auxiliary solvent supply source, a water supply source, and a fine particle generation column. And the auxiliary solvent source are respectively connected to a carbon dioxide / auxiliary solvent mixing column via a pump, the carbon dioxide / auxiliary solvent mixing column is connected to a particle generation column, and the water source is via a gene solution inlet and a pump. Is connected to the outside through a pressure control valve, and the carbon dioxide / auxiliary solvent mixing column and the particle generation column are provided in a thermostatic chamber. The temperature and pressure in the solvent mixing column and the fine particle generation column are adjusted by the thermostatic chamber and the fully automatic pressure control valve, and the carbon dioxide exceeds It is summarized as apparatus for preparing a gene dry powder, characterized in that is configured to be maintained to the field conditions.

In the apparatus for preparing gene dry powder of the present invention, it is desirable that the fine particle production column has a V-shaped nozzle. In that case, the carbon dioxide / ethanol mixed fluid and water can be collided at the tip of the V-shaped nozzle to promote mixing and dispersion.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION The dry powder of a gene according to the present invention is a dry powder containing a complex of a plasmid gene and a cationic vector, and an excipient as a carrier of the complex.

In the present invention, pCMV-Luc (a plasmid gene into which a gene expression promoter possessed by cytomegalovirus is incorporated) can be applied as a typical plasmid gene, but theoretically any other plasmid gene can be applied. Applicable. This is because the gene carrier uses a cationic polymer rather than a virus. When a virus is used as a gene carrier, the size of the plasmid gene limits its size, but since the cationic polymer electrostatically forms a complex with the gene, the size of the plasmid gene is not limited. Further, pCMV-Luc itself has no medicinal effect. pCMV-Luc is a reporter gene for confirming whether the gene is taken up by lung tissue and expresses a protein (luciferase in this case). In the case of practical application, it is necessary to use a gene for the purpose of treating local lung disease.

In the present invention, chitosan can be typically used as the cationic polymer, but polylysine, polyethyleneimine, etc., which are generally studied, can also be applied.

In the present invention, mannitol can be typically applied as an excipient, but in addition, saccharides such as lactose generally used in pharmaceuticals, starch, and cellulosic polymers (hydroxycellulose, hydroxypropylcellulose, etc.) are generally used. Excipients conventionally used as pharmaceutical additives can be applied.

Next, a method for preparing the gene dry powder of the present invention will be described.

A supercritical carbon dioxide / auxiliary solvent mixed fluid is prepared by adding an auxiliary solvent such as ethanol to supercritical carbon dioxide, and the mixed fluid is caused to flow into the fine particle generation column and water is caused to flow into the fine particle generation column. Then, after the supercritical carbon dioxide / co-solvent mixed fluid and water are continuously injected into the fine particle generation column and a predetermined time has passed, the supercritical carbon dioxide, water and co-solvent in the fine particle generation column are maintained for a predetermined time. While continuing the supply, an aqueous solution of an excipient containing the complex of the plasmid gene and the cationic vector was injected into the particle generation column, and at the same time, the excipient containing the complex of the plasmid gene and the cationic vector injected into the particle generation column was injected. The water contained in the aqueous solution of the excipient is extracted with supercritical carbon dioxide, and the plasmid gene and the cationic vector carried on the excipient are extracted. Get coalescence.

In the method for preparing a gene dry powder according to the present invention, pCMV-Luc having CMV incorporated as a promoter can be typically used as a plasmid gene. However, in the present invention, pCM
Any other plasmid gene is applicable without being limited to V-Luc. Promoter CMV
When a plasmid gene into which is incorporated is used, transduction occurs and a trait expression effect is exhibited.

Since pCMV-Luc itself has no medicinal effect, it is necessary to use a gene for the purpose of treating local pulmonary disease in the case of practical use.

Further, in the method for preparing dry powder according to the present invention, chitosan can be used as a cationic vector. Chitosan suppresses the degradation of the plasmid gene in the dry powder preparation process and enhances the transduction efficiency. Further, the recovery rate of the complex of the plasmid gene and the cationic vector at the time of preparing the dry powder is also improved. In the present invention, the cationic vector is not limited to chitosan, and polylysine, polyethyleneimine and the like which are generally studied are also applicable.

In the method for preparing a gene dry powder according to the present invention, pCMV-Luc in which CMV is incorporated as a promoter is used as a plasmid gene, and chitosan is used as a cationic vector. It is preferable to use an aqueous solution of an excipient containing the complex of the plasmid gene and the cationic vector so that the molar ratio of phosphorus is 5. When the molar ratio of nitrogen in chitosan to phosphorus in the plasmid gene is 5, the luciferase activity in the lung becomes maximum and the gene transfer efficiency becomes highest.

Next, the apparatus for preparing gene dry powder of the present invention will be described with reference to FIG. This preparation device
A carbon dioxide cylinder (1), an auxiliary solvent supply source (2), a water supply source (3) and a fine particle generation column (4) are provided. The carbon dioxide cylinder (1) and the auxiliary solvent supply source (2) are connected to the carbon dioxide / auxiliary solvent mixing column (6) via first and second pumps (5a) and (5b), respectively.
Further, the carbon dioxide / auxiliary solvent mixing column (6) communicates with the nozzle (8) of the particle generation column (4). On the other hand, the water source (3) is the gene solution inlet (7) and the third
It is connected to the nozzle (8) of the fine particle production column (4) via the pump (5c). Further, the fine particle production column (4) is connected to the outside air via a pressure regulating valve (9). The carbon dioxide / auxiliary solvent mixing column (6) and the fine particle production column (4) are arranged in a thermostat (10). The temperature and pressure in the carbon dioxide / auxiliary solvent mixing column (4) and the fine particle generation column (4) are adjusted by the thermostatic chamber (10) and the fully automatic pressure control valve (9) to maintain carbon dioxide in a supercritical state. Is configured to.

Carbon dioxide is sent from the carbon dioxide cylinder (1) to the carbon dioxide / auxiliary solvent mixing column (6), and at the same time, the auxiliary solvent such as ethanol is fed from the auxiliary solvent supply source (2) to the carbon dioxide / auxiliary solvent mixing column ( 6), and a supercritical carbon dioxide / cosolvent mixed fluid is obtained in the carbon dioxide / cosolvent mixed column (6). Here, the auxiliary solvent such as ethanol is an auxiliary solvent for dispersing the water supplied from the water supply source (3) in carbon dioxide in the fine particle production column (4). The temperature and pressure in the particle generation column (4) are regulated by the thermostatic chamber (10) and the fully automatic pressure control valve (9), and the carbon dioxide / auxiliary solvent mixture sent from the carbon dioxide / auxiliary solvent mixing column (6) is mixed. In the supercritical state, the fluid passes through the nozzle (8) and the particle generation column (4)
Supplied within. At the same time, water is supplied to the nozzle (8) from the water supply source (3). After the preparation device is stabilized, a solution (gene solution) containing a complex of a plasmid gene and a polycation such as chitosan and an excipient such as mannitol is injected from the gene solution inlet (7). The gene solution is poured into the carbon dioxide / cosolvent mixed fluid from the tip of the nozzle (8). The water in the gene solution is extracted into the carbon dioxide / cosolvent mixed fluid, and the remaining plasmid gene-polycation complex and excipients become dry powder (11) and accumulate in the fine particle generation column (4).
After sending the whole amount of the gene solution into the particle generation column (4), stop the supply of the auxiliary solvent and water and let only the supercritical carbon dioxide flow, so that the water and the auxiliary solvent in the particle generation column (4) are completely removed. Will be removed. The supply of supercritical carbon dioxide is stopped, the fully automatic pressure adjusting valve (9) is opened, and carbon dioxide in the particle generation column (4) is removed, and then the gene dry powder generated is taken out.

In the gene dry powder preparation apparatus of the present invention, it is desirable that the fine particle production column (4) is provided with a V-shaped nozzle (8). In that case, the carbon dioxide / ethanol mixed fluid and water can be collided at the tip of the V-shaped nozzle to promote mixing and dispersion.

(Example) pCMV-Luc used as a sample plasmid gene was replicated in large quantities by Escherichia coli strain DH5, and Qiegen Plasmid Giga
Mass replication was performed using the Kit (Qiegen GmbH, Hilden, Germany). The concentration of pCMV-Luc was measured by the absorbance at a wavelength of 260 nm. Also, the purity of pDNA is A26.
0 / A280 ratio (absorbance at wavelength 260nm vs. wavelength 2
The absorbance ratio at 80 nm) was used for evaluation.

Water-soluble chitosan (indicated molecular weight 3000-3
0000, manufactured by Wako Pure Chemical Industries, Ltd., mannitol (manufactured by Wako Pure Chemical Industries, Ltd.), sodium acetate (manufactured by Kishida Pharmaceutical Co., Ltd.)
Is a non-viral vector, dry powder carrier,
Used as an additive.

Restriction enzyme HndIII and DNA size marker λH
An indIII digest (manufactured by Toyobo Co., Ltd.) was used. As a luciferase assay (luciferase quantification kit), Picagene (manufactured by Toyo Ink Co., Ltd., Piccagene is a registered trademark by Toyo Ink Co., Ltd.) was used. Special grade products were used for other reagents and solvents.

Preparation of Chitosan-pDNA Complex Dry Powder A chitosan-pDNA complex dry powder was prepared by supercritical carbon dioxide crystallization method using the apparatus for preparing chitosan-pDNA complex dry powder shown in FIG. 1 as follows. did.

The preparation device is a carbon dioxide cylinder (1) to 2
Carbon dioxide pump (SCF-Get, maximum liquid transfer rate 10 ml /
min), auxiliary solvent pump (PU-1580), aqueous solution pump (PU-15
80), oven (GC353B, GL Science) to maintain the temperature of the constant temperature bath at the required temperature (35 ° C), and pressure control device (SCF- to maintain the carbon dioxide at supercritical pressure (15Mpa). Bpg, having a pressure control range of 1-50 Mpa) was used. Further, a column having a V-shaped nozzle attached was used as the particle generation column. From the carbon dioxide cylinder through two carbon dioxide pumps connected in parallel to the carbon dioxide cylinder, liquid was sent to the carbon dioxide / cosolvent mixture column at a rate of 14 ml / min, and at the same time, ethanol was used as a cosolvent from the cosolvent source. At a rate of 0.665 ml / min, the solution was sent to a carbon dioxide / auxiliary solvent mixing column and ethanol was mixed with supercritical carbon dioxide (35 ° C, 15 MPa) to produce carbon dioxide /
An ethanol mixed fluid was obtained. This mixed fluid is supplied from one side of the V-shaped nozzle to a fine particle generation column (inner diameter 2.0 cm, height 1
4 cm) while water was dispersed at a rate of 0.035 ml / min from the other end of the V-shaped nozzle into the fine particle production column. Chitosan-pDNA complex aqueous solution (0.4 ml)
Was injected into a water pipe connecting a V-shaped nozzle with a water supply source through a manual injector which is a gene injection port. Thirty minutes after the injection, the feeding of water and ethanol was stopped, and supercritical carbon dioxide was kept flowing for 90 minutes to remove the water and ethanol in the particle generation column. The dry powder was recovered from the inside of the particle generation column after depressurizing the inside of the particle generation column. The dry powder formulation prepared this time is as shown in Table 1.

[0038]

[Table 1]

An aqueous solution of chitosan-pDNA complex was prepared by dissolving pDNA (120 μg) and chitosan (1) in 0.4 ml of purified water.
42 μg, 283 μg, 586 μg) and N
/ P ratio (nitrogen in chitosan / molar ratio of phosphorus in pDNA)
Was 2.5, 5 and 10.

Examination of particle size distribution and particle shape The particle size distribution of the dry powder obtained as described above is determined by laser micron sizer (LMS-30, Seishin Enterprise Co., Ltd.) using laser diffraction scattering method (laser diffraction method). The device for measuring the particle diameter of fine particles is used. For particle size distribution measurement, an administration device for administering dry powder as shown in FIG. 2 into the lungs of mice was used. This device is a 1 ml syringe (a), 3
It is provided with a direction cock (b), a dry powder filling disposable tip (c), a compressed air cylinder (d) and a dosing handle. The dry powder spray-dispersed from this device was directly measured by laser light. The particle shape is scanning electron microscope (JSM-120, JOEL).
Was observed using.

Observation of Stability of pDNA by Electrophoresis Dry powder (1 mg) was dissolved in purified water with 8 μl of purified water, HindIII restriction enzyme was added, and after incubation at a temperature of 37 ° C. for 30 minutes, as described above. PDNA obtained in
A solution containing 0.06 μg was added to an agarose gel containing 0.6% ethidium bromide and electrophoresed. Electrophoresis is 24.2 g of TAE buffer (Tris (buffer component))
Glacial acetic acid (5.71 ml) and EDTA (10 ml) were added to the mixture to make 100 ml with purified water and at the same time diluted 50 times with purified water).
In 100 V for 2 hours.

In Vivo Lung Tissue Transduction Experiment Using Mice About 20 g of ICR female mice were anesthetized by intraperitoneally administering bentobarbital (50 mg / Kg, ip) to the mice, and fixed on the back. Expose the trachea and make a notch between the 5th and 6th tracheal cartilage from the thyroid cartilage to the tail and insert a 3cm PE-60 polyethylene tube to 1cm
Inserted only the length of.

Pulmonary administration of pDNA solution is carried out by
μL was administered using a microsyringe.

The dry powder was administered into the lungs of mice using the administration device shown in FIG. Dry powder
(1.5 mg) was filled in a disposable chip, and the disposable chip was connected to a polyethylene tube inserted into the trachea. Compressed air in the compressed air cylinder (0.2
(5 mL) was instantly opened, and dry powder was dispersed and administered into the lungs of the mouse.

After 3 hours, 6 hours and 9 hours from the administration, the aorta of the mouse was cut and the lung was extracted. The lungs were washed twice with ice-cold PBS and homogenized with cell lysate. The cell lysate was formulated in a 0.1 M Tris / Hcl buffer containing 0.05% Triton X-100 and 2 mM EDTA.
(pH 7.8). Cell lysate is 4m against lung weight
It was added at a rate of l / g. After freeze-thawing three times, the homogenate was centrifuged at 4 ° C., 15,000 rpm, 5 minutes. Luciferase assay was performed using 20 μL of homogenate supernatant.

Statistical Calculation The statistically significant difference in luciferase activity of each test group was analyzed by one-way analysis of variance (ANOVA). The least significant difference test was used for the significant difference between each average value. The p value is 0.
When it was less than 05, it was considered that a significant difference was seen.

Characteristic Evaluation of Chitosan-pDNA Complex Dry Powder A chitosan-pDNA complex dry powder was prepared using a chitosan-pDNA complex dry powder preparation device equipped with a V-shaped nozzle. As shown in Fig. 3, dry powder is 10 μm as observed by scanning electron microscope.
It was found to have a rectangle with the following short sides and long sides of 10 μm or more. In addition, FIG. 3 shows a scanning electron micrograph of a DNA dry powder having mannitol as an excipient, (a) is a micrograph of only mannitol,
(B) is a micrograph of pDNA dry powder, (c)
Is chitosan-pDNA complex dry powder (N / P =
2.5) micrograph, (d) chitosan-pDNA complex dry powder (N / P = 5) micrograph,
(E) is a dry powder of chitosan-pDNA complex (N
/ P = 10) micrograph, (f) sodium acetate-
It is a microscope picture of pDNA complex dry powder. Also, as shown in Table 1, the average particle shape of the dry powder measured using a laser micron sizer is 12-13 μm.
It was m. The recovery rate of pDNA / mannitol dry powder and pDNA + sodium acetate / mannitol dry powder was 70% or less. On the other hand, the recovery rate of the chitosan-pDNA complex dry powder was about 80% (Table 1).

FIG. 4 shows the effect of chitosan on the integrity of pDNA-Luc in dry powder prepared with supercritical carbon dioxide, lane 1 shows the molecular weight standard, lane 2 shows the aqueous solution of pDNA-Luc. If lane 3 is pDNA dry powder,
Lane 4 is chitosan-pDNA complex dry powder (N / P = 2.5), lane 5 is chitosan-pD.
In the case of NA complex dry powder (N / P = 5), lane 6 is chitosan-pDNA complex dry powder (N / P = 5).
The case of P = 10) is shown. In the dry powder containing no chitosan, a band showing the degradation of pDNA during the preparation of the dry powder was confirmed (Fig. 4, lane 3). On the other hand, when chitosan is added at a ratio of N / P = 2.5, pDN
It was confirmed by electrophoresis that the decomposition of A was reduced. Further, when the N / P ratio was increased to 5 and 10, no band indicating DNA degradation could be confirmed (FIG. 4, lane 5, lane 6). Also, when sodium acetate was added, no degradation of pDNA that occurred during the preparation of dry powder was found.

Examination of pCMV-Luc Dose in Transduction Experiment In carrying out the transduction experiment, pDNA in the mouse lung was examined.
The relationship between the dose and the expressed luciferase activity in lung tissue was examined. 1 μg, 3 μg, 10 μg or 30 μg pDN
A was dissolved in purified water to 50 μL, and luciferase activity was measured 6 hours after intrapulmonary administration. As a result,
P on the luciferase activity in the lungs of mice
A dose of 3 μg to show the effect of CMV-Luc administration,
There was no significant difference in luciferase activity by intrapulmonary administration of 10 μg or 30 μg of pDNA, but the dose of 1 μg showed significantly lower luciferase activity than that of other doses. Therefore, when pDNA is administered intrapulmonaryly, pD
It was considered that 3 μg of NA was sufficient, and it was decided to administer 3 μg of pDNA per mouse except for the intravenous administration experiment.

As for the luciferase activity, the daily fluctuation of the activity value of the standard solution was observed. In order to correct this variation, mice 4-5 were used as a control group in each experiment.
50 μL of an aqueous solution containing 3 μg of pDNA was intraperitoneally administered to a rat. The luciferase activity value in the lung tissue of the control group 6 hours after the administration was set to 100%, and the luciferase activity value of the other groups was shown.

FIG. 6 shows the time course of luciferase activity after intravenous administration of pCMV-Luc and after intrapulmonary administration.
In the figure, □ indicates pCMV-Luc (30 μg) dissolved in 200 μL of 5% mannitol intravenously, and hatched □ indicates p dissolved in 50 μL of water.
Intravenous administration of CMV-Luc (3 μg), and ■ is 50 μm
Chitosan-pDNA dissolved in L water (N / P = 2.5,
(corresponding to 3 μg of pCMV-Luc). As shown in FIG. 6, pCMV-L was added to 200 μL of 5% mannitol.
When 30 μg of uc was dissolved and intravenously administered in a volume of 0.2 mL, the luciferase activity 3 hours after the administration was only 8% of the control, and the luciferase activity decreased with time. On the other hand, pDNA aqueous solution or chitosan-pDNA (N / P = 2.5, pCMV-L
uc) (corresponding to 3 μg of uc) reached a maximum 6 hours after administration, and a significant difference from the activity 3 hours and 9 hours after administration was observed.

Relationship between N / P ratio of chitosan-pDNA complex solution and lung luciferase activity. The effectiveness of chitosan as a vector was demonstrated by chitosan-pDN.
A (N / P = 2.5, corresponding to 3 μg of pCMV-Luc)
It was examined by intrapulmonary administration of an aqueous solution of. FIG. 7 shows chitosan-pDNA (N / P = 2.5, p dissolved in 50 μL of water).
N / E on luciferase activity expressed in the lungs of mice 6 hours after intrapulmonary administration of CMV-Luc (corresponding to 3 μg)
The effect of P ratio is shown. Addition of N / P = 1.25 chitosan had no effect on luciferase activity. However, as the N / P ratio increased, the luciferase activity in the lung also increased. When the N / P ratio of the chitosan-pDNA complex was 2.5, the luciferase activity showed the maximum, which was about 360% of the control.

Relationship between luciferase activity and time by intrapulmonary administration of pDNA dry powder Chitosan-pDNA (N / P = 5, pCMV, which could avoid degradation of pDNA during preparation of dry powder)
In order to examine the usefulness of dry powder (corresponding to 3 μg of -Luc) and sodium acetate-pDNA complex dry powder, the powder was administered into the lungs of mice using the administration device shown in FIG. FIG. 8 shows the time course of luciferase activity after intrapulmonary administration of chitosan-pDNA. As shown in FIG. 8, chitosan-pDNA (N / P = 5, pCMV-
The lung luciferase activity of the mice after administration of dry powder (corresponding to 3 μg of Luc) increased with time and showed about 2700% of the control 9 hours after administration.
By adding sodium acetate, the degradation of pDNA during the preparation of dry powder could be suppressed, and in this case as well, the luciferase activity increased with time. However, the luciferase activity 9 hours after the administration was only about 300% of the control. Sodium acetate-chitosan compared to pDNA dry powder-
The luciferase activity of dry powder of pDNA (N / P = 5) was significantly higher over the whole time.

Chitosan-pDNA dry powder N /
Relationship between P ratio and pulmonary luciferase activity Chitosan pDNA dry powder with various N / P ratios was administered into the lungs of mice, and luciferase activity was measured 9 hours after administration. As shown in FIG. 9, the chitosan-pDNA dry powder with N / P = 5 showed higher luciferase activity than the chitosan-pDNA dry powder with other N / P ratios. In addition, even dry powder containing no chitosan showed high luciferase activity. This is because dry powder dissolves in a small amount of body fluid on the lung surface,
It seems that the local gene concentration in the lung becomes much higher than that of the administration of the solution, which may suppress the gene degradation in the lung or in the transduction process.

As is clear from the above description, the gene dry powder of the present invention can suppress degradation of pDNA during preparation of dry powder by (a) complexing pDNA with chitosan.

(B) By complexing pDNA with chitosan, the recovery rate of dry powder can be increased.

(C) Chitosan-pDNA dry powder is pDNA dry powder or pDN which does not contain chitosan.
Compared with the A solution or the chitosan-pDNA complex solution, it has advantages such as increased luciferase activity in mouse lung.

[0058]

INDUSTRIAL APPLICABILITY As described in detail above, the dry powder according to the present invention contains a complex of a plasmid gene and a cationic vector, and contains an excipient as a carrier of the complex, and therefore pDNA is cationic. PDNA when preparing dry powder by combining with vector
Can be obtained with a high recovery rate of dry powder while suppressing decomposition of Also, cationic vector-pDN
The A complex dry powder has an advantage that it can increase the luciferase activity in the mouse lung as compared with pDNA dry powder containing no cationic dry powder, pDNA solution, or chitosan-pDNA complex solution.

Further, in the method for preparing a gene dry powder according to the present invention, an auxiliary solvent such as ethanol is added to supercritical carbon dioxide to prepare a supercritical carbon dioxide / auxiliary solvent mixed fluid, and the mixed fluid is placed in a fine particle production column. Water into the fine particle generation column, the supercritical carbon dioxide / co-solvent mixed fluid and water are continuously injected into the fine particle generation column, and after a predetermined time elapses, the fine particle generation column While continuously supplying supercritical carbon dioxide, water, and a co-solvent, the aqueous solution of the excipient containing the complex of the plasmid gene and the cationic vector is injected into the particle generation column, and at the same time injected into the particle generation column. The water contained in the aqueous solution of the excipient containing the complex of the prepared plasmid gene and the cationic vector is extracted with supercritical carbon dioxide, Since characterized in that to obtain a complex of the supported plasmid genes and cationic vectors, pDN in dry powder prepared by combining pDNA and cationic vectors such as chitosan
The decomposition of A can be suppressed, and the recovery rate of dry powder can be increased.

The gene dry powder preparation apparatus according to the present invention is equipped with a carbon dioxide cylinder, an auxiliary solvent supply source, a water supply source, and a fine particle production column, and the carbon dioxide cylinder and the auxiliary solvent supply source are each supplied with carbon dioxide through a pump. /
Connected to a co-solvent mixing column, the carbon dioxide / co-solvent mixing column connected to a fine particle generation column, the water supply source connected to the fine particle generation column through a gene solution inlet and a pump, and the fine particle generation column pressure. The carbon dioxide / auxiliary solvent mixing column and the fine particle production column are connected to the outside through a regulating valve, and the carbon dioxide / auxiliary solvent mixing column and the fine particle production column are kept at a constant temperature and pressure. It is characterized in that it is regulated by a tank and a fully automatic pressure control valve, and that carbon dioxide is maintained in a supercritical state, which enables the preparation of the gene dry powder of the present invention. Yes, p
By complexing DNA with a cationic vector such as chitosan, it is possible to suppress the decomposition of pDNA during the preparation of dry powder and to prepare the dry powder with a high recovery rate.

[Brief description of drawings]

FIG. 1 is a schematic diagram of an apparatus for preparing gene dry powder.

FIG. 2 is a schematic diagram of a dry powder dosing device.

Figure 3: DN with mannitol as scanning excipient
It is a scanning electron micrograph of A dry powder.

FIG. 4 shows the effect of chitosan on pDNA-Luc integrity in a dry powder prepared with supercritical carbon dioxide.

FIG. 5 shows the effect of pCMV-Luc administration on luciferase activity in the lungs of mice.

FIG. 6 is a diagram showing the time course of luciferase activity after intravenous administration of pCMV-Luc and after intrapulmonary administration.

FIG. 7: Chitosan-pDNA (p
N / E on luciferase activity expressed in the lungs of mice 6 hours after intrapulmonary administration of CMV-Luc (corresponding to 3 μg)
It is a figure which shows the effect of P ratio.

FIG. 8 is a graph showing the time course of luciferase activity after intrapulmonary administration of chitosan-pDNA.

FIG. 9 is a graph showing the effect of N / P ratio on luciferase activity expressed in mouse lungs 9 hours after intrapulmonary administration of pDNA-Luc dry powder.

[Explanation of symbols]

1 carbon dioxide cylinder 2 Auxiliary solvent 3 Water supply source 4 Fine particle purification column 5a, 5b, 5c pumps 6 Auxiliary solvent mixing column 7 Gene solution injection port 8 nozzles 9 Pressure control valve 10 constant temperature bath 11 dry powder

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 48/00 A61P 11/00 4C087 A61P 11/00 35/00 35/00 C12M 1/00 A C12M 1 / 00 C12N 15/00 A (72) Inventor Seigo Sato 256-17 F-term (reference) Koizumi, Ageo-shi, Saitama 4B024 AA01 CA01 DA02 EA04 FA02 GA11 HA17 4B029 AA23 BB20 4C076 AA29 BB25 BB27 CC27 CC29 DD38 DD41 EE13 4084 FF04 FF04 AFF MA43 MA56 NA10 NA11 ZA59 ZB26 4C086 AA01 AA02 EA16 MA02 MA03 MA05 MA43 MA56 NA10 NA11 ZA59 ZB26 4C087 AA01 AA02 BC83 MA05 MA43 MA56 NA10 NA11 ZA59 ZB26

Claims (12)

[Claims] 1. A gene dry powder comprising a complex of a plasmid gene and a cationic vector, and an excipient as a carrier for the complex. 2. The gene dry powder according to claim 1, wherein the plasmid gene is pCMV-Luc in which CMV is incorporated as a promoter. 3. The gene dry powder according to claim 1, wherein the cationic vector is chitosan. 4. The plasmid gene is pCMV-Luc in which CMV is incorporated as a promoter, the cationic vector is chitosan, and the molar ratio of nitrogen in chitosan to phosphorus in the plasmid gene is 5. The gene dry powder according to claim 2, which is characterized in that. 5. The gene dry powder according to claim 2, further comprising a gene for the purpose of treating local lung diseases and the like. 6. A supercritical carbon dioxide / auxiliary solvent mixed fluid is prepared by adding an auxiliary solvent such as ethanol to supercritical carbon dioxide, and the mixed fluid is allowed to flow into the fine particle generation column and water is flowed into the fine particle generation column. Of the supercritical carbon dioxide / auxiliary solvent and water are continuously injected into the particle generation column, and after a predetermined time has passed, the supercritical carbon dioxide, water and auxiliary gas are supplied into the particle generation column for a predetermined time. While continuously supplying the solvent, an aqueous solution of an excipient such as mannitol containing a complex of the plasmid gene and the cationic vector was injected into the particle generation column, and at the same time, the plasmid gene and the cationic vector were injected into the particle generation column. The water contained in the aqueous solution of the excipient containing the complex was extracted with supercritical carbon dioxide, and the plasmid gene and A method for preparing a gene dry powder, which comprises obtaining a complex of an on vector. 7. The method for preparing a gene dry powder according to claim 6, wherein pCMV-Luc in which CMV is incorporated as a promoter is used as a plasmid gene. 8. The method for preparing a gene dry powder according to claim 6, wherein chitosan is used as the cationic vector. 9. A pCMV-Luc having CMV incorporated as a promoter is used as a plasmid gene, and chitosan is used as a cationic vector so that the molar ratio of nitrogen base in chitosan to phosphorus in the plasmid gene is 5. The method for preparing a gene dry powder according to claim 6, wherein an aqueous solution of an excipient containing a complex of a plasmid gene and a cationic vector is used. 10. A fine particle generation column having a V-shaped nozzle is used as the fine particle generation column, and the carbon dioxide / ethanol mixed fluid and water are bumped and mixed at the tip of the V-shaped nozzle to promote mixing and dispersion. The method for preparing a gene dry powder according to claim 6, which is characterized in that. 11. A carbon dioxide cylinder, an auxiliary solvent supply source,
A water supply source and a fine particle generation column are provided, the carbon dioxide cylinder and the auxiliary solvent supply source are respectively connected to the carbon dioxide / auxiliary solvent mixing column via a pump, and the carbon dioxide / auxiliary solvent mixing column is connected to the fine particle generation column. ,
The water supply source is connected to the fine particle generation column via the gene solution inlet and the pump, the fine particle generation column is connected to the outside air via the pressure control valve, and the carbon dioxide / auxiliary solvent mixing column and the fine particle generation column are at a constant temperature. It is installed in the tank, and the temperature and pressure in the carbon dioxide / auxiliary solvent mixing column and the fine particle production column are adjusted by the thermostatic bath and the fully automatic pressure control valve so that carbon dioxide is maintained in the supercritical state. An apparatus for preparing gene dry powder, which is characterized in that 12. The gene dry powder preparation device according to claim 11, wherein the particle generation column is provided with a V-shaped nozzle.