JP2005065561A - Method for transferring gene to blood vessel cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently and quantitatively transferring a safe and inexpensive plasmid DNA to a blood vessel wall cell. <P>SOLUTION: In a gene transfer apparatus 1 constituted of electrode parts 2 composed of one or a plurality of cathodes and anodes for impressing a pulse voltage to a blood vessel, a pulse generator 3 for generating the pulse voltage, a power source 4 for supplying driving electric power and a computer 5 for controlling the impression conditions of pulse, a plasmid DNA to be transferred is injected to the blood vessel, the electrodes parts are arranged at both the sides of the blood vessel so as to nip the blood vessel, fixed voltage pulses are impressed to the arranged electrodes parts for given times and a hydrophilic hole for gene transfer is bored through the blood vessel cell. Consequently the plasmid DNA is preferentially transferred to the blood vessel cell at a position corresponding to the edge part of the cathode. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for introducing a gene into a vascular wall cell, particularly an electroporation method.

As gene therapy research advances, various methods for gene transfer into vascular wall cells have been developed. Currently, the adenovirus vector method is widely used because of its high efficiency, but side effects such as its antigenicity are problematic because the virus must act at a high concentration. On the other hand, plasmid DNA is advantageous in that it is inexpensive, has high quality and can be used safely, but its introduction efficiency is low. Recently, electroporation has been studied as one of the new plasmid DNA introduction methods (for example, Non-patent Reference 1). T.A. Matsumoto et al. Attempted to introduce a gene into a blood vessel wall by sandwiching a rabbit carotid artery with a T-shaped electrode and repeatedly applying a pulse voltage (10 to 40 V) 10 times. As a result, it was confirmed that the maximum introduction amount was obtained when the pulse application cycle was 20 ms On / 80 ms Off, the applied voltage was 20 V, and the maximum introduction amount was obtained when the DNA concentration was 200 μg / ml. In the confirmation using the LacZ gene, it was confirmed that it was introduced almost over the entire surface of the electrode.
T.A. Matsumoto et al. “Successful and optimized in vivo gene transfer to rabbit carrotid articulated by electronic pulse” Gene Therapy [2001] 8. 1174-1179

  T. mentioned above. In the method of Matsumoto et al., Introduction of genes by electroporation method has been confirmed, but efficient introduction of genes and quantitative introduction of genes have not been pursued, and they have been put to practical use. It can not be said. In view of the above problems, the present invention aims to provide an efficient and quantitative gene transfer method and to further promote its use.

  According to the present invention having the following configuration, efficient and quantitative gene introduction can be realized.

  (1) One or a plurality of cathodes and anodes for applying a pulsed voltage to the blood vessel, a pulse generator for generating the pulsed voltage, a power supply for supplying driving power, and pulse application In a gene introduction apparatus comprising a computer for controlling conditions, plasmid DNA to be introduced is injected into the blood vessel, and the electrode part is placed on both sides of the blood vessel so as to sandwich the blood vessel, and the placement electrode part The plasmid DNA was preferentially introduced into the vascular wall cell at the position corresponding to the edge of the cathode by applying a predetermined voltage pulse to the vascular wall cell a predetermined number of times to open a hydrophilic hole for gene introduction in the vascular wall cell. A gene introduction method characterized by the above.

  (2) The gene transfer method according to (1), wherein the voltage pulse is applied at 10 to 50 V, converted into the maximum electric field strength applied to the tissue, and applied at 17 to 82 kV / m for 20 milliseconds.

  (3) The gene introduction method according to (1), wherein the gene introduction device is further provided with a device for switching the polarity of a power supply so that gene introduction can be performed from both sides of the blood vessel.

  (4) The gene introduction according to (1), wherein the cathode is composed of two parallel electrodes, and the gene can be introduced linearly into blood vessel wall cells at a position corresponding to the edge portion of the cathode. Method.

  (5) The gene introduction method according to (1), wherein the cathode is composed of a plurality of small electrodes so that the gene can be introduced almost uniformly into the blood vessel wall.

  According to the present invention, an inexpensive and safe plasmid DNA can be efficiently and quantitatively introduced, so that an effect of further promoting gene therapy can be obtained.

  FIG. 1 shows a schematic configuration of a gene introduction apparatus using electroporation according to the present invention. The gene transfer device 1 includes an electrode unit 2 for applying a voltage to the blood vessel wall, a pulse generator 3 for generating an applied voltage to the electrode unit 2, a power source 4 for supplying power to the device, and a pulse generation cycle to be described later. The computer 5 is configured to control the applied voltage and its polarity.

  As shown in FIG. 2, the electrode part 2 is installed so as to sandwich the blood vessel, and a predetermined voltage is applied. In the electroporation method used in the present invention, a hydrophilic hole is formed in a cell membrane by the action of an electric field caused by an electric pulse from the electrode part 2, and a gene is introduced into the cell through the hole. .

  The gene was introduced by the following procedure. First, plasmid DNA is injected into a blood vessel using a small-diameter catheter and incubated for 20 minutes. As shown in FIG. 2, the electrode part 2 of the gene introduction apparatus is arranged so as to sandwich the blood vessel, and a pulse is applied 10 times every 20 msec at a predetermined voltage to open the hydrophilic hole in the cell membrane of the blood vessel and To introduce. In order to obtain a sufficient introduction effect, the effect of gene introduction was measured after standing for 2 days.

  In order to specify the gene introduction site, pGAGGS-LacZ plasmid DNA was used as the plasmid DNA and the above introduction operation was performed, followed by staining with 6-galactosidase to identify the expression site of the plasmid DNA. A photograph showing the stained expression site is shown in FIG. This photograph shows that the part corresponding to the edge part (peripheral part) of the electrode is stained and the gene is preferentially introduced therein.

  As shown in FIG. 4, the electric field simulation result in the state where the electrode is arranged around the blood vessel as shown in FIG. 2 has a high potential in the portion corresponding to the periphery of the electrode, and corresponds to the result of the above-mentioned stained photograph. doing. From this observation fact, it is possible to introduce a gene linearly by using two parallel electrodes shown in FIG. 5 (a), and using a large number of electrodes as shown in FIG. It becomes possible to introduce a gene at a substantially uniform concentration.

  Further, the stained part shown in Photo 2 of FIG. 3 was observed only on the cathode electrode side. Therefore, the gene can be introduced from both sides of the blood vessel without re-installing the electrode by switching the polarity of the power supply of the gene introduction device, and it is possible to easily introduce twice as much gene.

  Further, in order to examine the gene introduction amount, pGAGGS-Luc plasmid DNA was used as the plasmid DNA, and the introduction operation was performed, and then the plasmid DNA introduction amount was measured. The relationship between the measured introduction amount and voltage is shown in FIG. From this figure, it can be seen that the amount of introduction is maximized at an applied voltage of 30 V to the electrodes. When the voltage applied to the electrode is 50V, the blood vessels are observed to be occluded, and some of the cells are considered damaged. Therefore, the applied voltage is preferably 40 V or less in consideration of the safety of the introduction target, and 30 V is optimal from the viewpoint of introduction efficiency.

  As described above, in the electroporation method according to the present invention, the maximum introduction effect can be obtained at an electrode applied voltage of 30 V (corresponding to a maximum electric field strength of about 50 kV / m), and the introduction amount and the introduction site can be changed by changing the shape of the electrode. Can be controlled.

  According to the present invention, a safe and inexpensive plasmid gene can be efficiently introduced into a predetermined site, which can greatly contribute to the promotion of gene therapy in the medical field.

The figure which shows the schematic structure of the gene transfer apparatus by this invention. Diagram showing the placement of the electrodes of the gene transfer device Staining diagram showing the site of introduction of the gene into the blood vessel The figure which shows the simulation result of the electric potential distribution around the electrode of FIG. 1A and 1B are diagrams showing the form of an electrode according to the present invention, in which FIG. 1A shows two parallel electrodes, and FIG. Diagram showing the relationship between the voltage applied to the electrode and the amount of gene transfer

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Gene transfer apparatus 2 Electrode part 3 Pulse generator 4 Power supply 5 Computer

Claims (5)

  Controls one or more cathodes and anodes that apply a pulsed voltage to the blood vessel, a pulse generator that generates the pulsed voltage, a power source that supplies driving power, and pulse application conditions In a gene introduction apparatus comprising a computer, a plasmid DNA to be introduced is injected into the blood vessel, and the electrode portion is placed so as to sandwich the blood vessel on both sides of the blood vessel, and a predetermined voltage is applied to the placement electrode portion. A plasmid DNA is preferentially introduced into vascular wall cells at a position corresponding to the edge of the cathode by applying a pulse a predetermined number of times to open a hydrophilic hole for gene introduction into the vascular wall cell. Gene transfer method.   2. The gene introduction method according to claim 1, wherein the voltage pulse is applied at 10 to 50 V and converted into the maximum electric field strength applied to the tissue at 17 to 82 kV / m for 20 milliseconds.   2. The gene introduction method according to claim 1, wherein the gene introduction device further comprises a device for switching the polarity of a power source so that gene introduction can be performed from both sides of the blood vessel.   2. The gene introduction method according to claim 1, wherein the cathode is composed of two parallel electrodes, and the gene can be introduced linearly into blood vessel wall cells at a position corresponding to the edge portion of the cathode.   A gene introduction method characterized in that the cathode is composed of a plurality of small electrodes, and the gene can be introduced almost uniformly into the blood vessel wall.