WO2015133852A1

WO2015133852A1 - Gene packaging method using ultrasonic waves and apparatus for implementing same

Info

Publication number: WO2015133852A1
Application number: PCT/KR2015/002162
Authority: WO
Inventors: 서종범; 박진감; 김성배; 박동희; 손정우; 송길수; 차오름; 김진호; 김철우
Original assignee: 주식회사 퍼시픽시스템; 연세대학교 원주산학협력단; 서울대학교 산학협력단
Priority date: 2014-03-07
Filing date: 2015-03-06
Publication date: 2015-09-11
Also published as: KR20150105242A

Abstract

Disclosed are a gene packaging method using ultrasonic waves and an apparatus for implementing the same. The gene packaging method comprises the steps of: holding a solution containing a gene; determining ultrasonic irradiation conditions including the intensity and time of ultrasonic waves to be irradiated depending on the amount and length of the gene contained in the solution; and irradiating ultrasonic waves, corresponding to the ultrasonic wave irradiation conditions, to the solution such that the gene is wound or folded by radiation force to have a changed shape. Therefore, the cell transduction rate or delivery efficiency of the gene is improved, and thus the effect of the gene on disease treatment can be improved.

Description

Gene packaging method using ultrasound and apparatus for performing same

The present invention relates to genetic modification technology, and more particularly, to a method for packaging a gene using ultrasound to improve the effect of treating the disease, and to an apparatus for performing the same.

Recently, researches for treating various kinds of diseases from genetic diseases to cancer using genes such as DNA or RNA have been actively conducted. In order to use genes for the treatment of diseases, desired genes can be easily injected into specific cells. To this end, it is necessary to understand the morphological characteristics of genes.

1 is an exemplary view for explaining the form of a general gene.

Referring to FIG. 1, genes may be generally classified into a relaxed circle shape, a linear shape, and a supercoilded shape.

More specifically, as shown in (a) of FIG. 1, the relaxed circle shape refers to a state in which a double helix is twisted and closed. In addition, the linear form refers to a state in which the double helix of the gene is completely unfolded without twisting and a nick is present in one of the double helixes as shown in FIG. Finally, the supercoilded form refers to a state where the double helix is twisted several times as shown in (c) of FIG. 1.

At this time, as can be seen through the electrophoresis result shown in the upper part of Figure 1 it can be seen that the gene of the supercoilded type is relatively small in volume compared to other types of genes occupy less space.

Thus, in many mechanisms of gene transfer, supercoilded genes have higher permeability and delivery effects than other genes, based on the fact that a small volume of material can permeate the cell membrane relatively easily. In fact, the results showed that the supercoilded form of genes had a higher transfer effect than other genes under the same conditions.

However, in the related art, only a method for treating a genetic defect or a disease by using a method of replacing an abnormal gene with a normal gene or expressing an improved function by injecting a material such as a protein into the gene is described above. There is a limit in that the morphological characteristics of the genes are not reflected and thus the cell permeability or delivery efficiency of the genes may be reduced. In addition, there is a problem that this may reduce the therapeutic effect of the disease using the gene.

SUMMARY OF THE INVENTION An object of the present invention for solving the above problems is to provide a gene packaging method that can improve the cell permeability or delivery efficiency of a gene by packaging the gene small using ultrasound.

In addition, another object of the present invention is to provide a gene packaging device that can be easily applied to the treatment of all diseases at the same time to improve the disease treatment effect of the gene by packaging the gene small using ultrasound.

Genetic packaging method using ultrasonic waves according to an aspect of the present invention for achieving the above object is carried out in an ultrasonic stimulation system, the step of receiving a solution (gene) containing the gene, the amount and length of the gene contained in the solution Determining the ultrasonic irradiation conditions including the intensity and time of the ultrasonic waves to be irradiated according to the method, and the ultrasonic waves corresponding to the ultrasonic irradiation conditions in the solution so that the genes contained in the solution are wound or folded due to a radiation force. Investigating the step.

Here, the step of determining the ultrasonic irradiation conditions may adjust the ultrasonic irradiation conditions so that the gene contained in the solution is not destroyed.

Herein, the determining of the ultrasound irradiation condition may be performed by adjusting the intensity and time of the ultrasound including at least one of frequency, cycle, and ultrasound pressure according to the amount of the gene included in the solution. The frequency may be adjusted according to the position and length of the gene to be irradiated with ultrasound.

Here, when the solution is irradiated with ultrasonic waves corresponding to the ultrasonic irradiation conditions, the nonlinear radiation force is generated due to the difference in density and acoustic impedance between the gene contained in the solution and the fluid around the gene, and the gene is generated by the nonlinear radiation force. The method may further include packaging the gene by modifying the shape of the gene such that the volume is reduced by winding or folding the volume.

In addition, the gene packaging device according to another aspect of the present invention for achieving the above object is implemented in an ultrasonic stimulation system, the solution receiving portion for receiving a solution containing the gene, according to the amount and length of the gene contained in the solution Ultrasonic determination unit for determining the ultrasonic irradiation conditions including the intensity and time of the ultrasonic waves to be irradiated and ultrasonic waves irradiating the ultrasonic waves corresponding to the ultrasonic irradiation conditions on the solution so that the genes contained in the solution are wound or folded due to the radiation force It includes an output unit.

Here, when the solution is irradiated with ultrasonic waves corresponding to the ultrasonic irradiation conditions, the nonlinear radiation force is generated due to the difference in density and acoustic impedance between the gene contained in the solution and the fluid around the gene, and the gene is wound by the nonlinear radiation force. Or the gene packaging unit may be further configured to package the gene by modifying the shape of the gene so that the volume is reduced.

According to the gene packaging method using the ultrasonic wave and the apparatus for performing the same according to the embodiment of the present invention as described above, it is possible to improve the cell permeability or delivery efficiency of the gene by packaging the gene small using the ultrasonic wave (packaging).

In addition, by packaging the gene small using ultrasound, it is possible to improve the disease treatment effect of the gene and at the same time can be easily applied to all diseases treatment.

1 is an exemplary view for explaining the form of a general gene.

2 is a flowchart illustrating a gene packaging method using ultrasound according to an embodiment of the present invention.

3 is an exemplary diagram illustrating a change in the shape of a gene due to the intensity of ultrasound irradiation through electrophoresis results according to an embodiment of the present invention.

Figure 4 is an exemplary view for explaining the form of the gene before the irradiation of the ultrasound according to an embodiment of the present invention.

5 is an exemplary view illustrating a change in the shape of a gene due to the irradiation time of ultrasonic waves according to another embodiment of the present invention.

Figure 6 is an exemplary view for confirming the shape of the packaged gene according to an embodiment of the present invention.

7 is an exemplary view showing a result of analysis of a packaged gene according to an embodiment of the present invention.

8 is a block diagram illustrating a gene packaging apparatus using ultrasonic waves according to an exemplary embodiment of the present invention.

9 is an exemplary view illustrating the shape of a gene packaging apparatus using ultrasonic waves according to an embodiment of the present invention.

As the invention allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the drawings, similar reference numerals are used for similar elements.

Terms such as first, second, A, and B may be used to describe various components, but the components should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, the first component may be referred to as the second component, and similarly, the second component may also be referred to as the first component. The term and / or includes a combination of a plurality of related items or any item of a plurality of related items.

When a component is referred to as being "connected" or "connected" to another component, it may be directly connected to or connected to that other component, but it may be understood that other components may be present in between. Should be. On the other hand, when a component is said to be "directly connected" or "directly connected" to another component, it should be understood that there is no other component in between.

The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting of the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this application, the terms "comprise" or "have" are intended to indicate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, and one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art and shall not be construed in ideal or excessively formal meanings unless expressly defined in this application. Do not.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Referring to FIG. 2, a gene packaging method using ultrasound may be performed in an ultrasound stimulation system.

Here, the ultrasonic stimulation system refers to a system for stimulating specific organs or cells of the human body by converting an electrical signal into an ultrasonic signal to treat a disease.

Recently, researches for treating diseases using genes such as DNA or RNA have been actively conducted. However, conventional therapeutic techniques using genes do not consider the morphological characteristics of the genes. There is a limit that efficiency may be reduced. For this reason, there is a problem that the therapeutic effect of the disease using the gene may be reduced.

Accordingly, the present invention proposes a method of artificially packaging a gene so as to improve the cell permeability or delivery efficiency of the gene to improve the disease treatment effect of the gene.

Gene packaging method using the ultrasonic wave according to the present invention comprises the steps of receiving a solution containing the gene (S100), determining the ultrasonic irradiation conditions (S200) and irradiating the ultrasonic waves corresponding to the ultrasonic irradiation conditions (S300) ), And packaging the gene by modifying the shape so that the volume of the gene is reduced (S400).

In order to package a gene, first, a solution containing a gene may be accommodated (S100).

More specifically, the quantitative gene may be mixed in a solution and then housed in a container such as a well plate to irradiate ultrasonic waves.

If the solution containing the gene is accommodated, it is possible to determine the ultrasound irradiation conditions (S200).

Here, the ultrasonic irradiation conditions may include the frequency of the ultrasonic waves to be irradiated to the solution, the cycle (cycle), the intensity of the ultrasonic wave (ultrasound pressure) and the like, and the time when the ultrasonic waves are irradiated.

Ultrasonic irradiation conditions are freely adjustable so that the genes contained in the solution are not destroyed. More specifically, the intensity and time of the ultrasonic wave may be adjusted according to the amount of the gene included in the solution, and the frequency of the intensity of the ultrasonic wave may be adjusted within any particular region according to the position or length of the gene included in the solution.

In this case, the reason for adjusting the frequency according to the length of the gene is that the influence of the radiation force (radiation force) may appear differently depending on the length of the gene.

As the ultrasonic irradiation conditions are controlled according to the amount and length of the genes included in the solution, ultrasonic waves corresponding to the ultrasonic irradiation conditions may be irradiated to the solution (S300).

That is, when the electrical signal generator constituting the ultrasonic stimulation system generates an electrical signal corresponding to the ultrasonic irradiation conditions, the ultrasonic transducer converts the electrical signal into an ultrasonic signal and irradiates the solution.

When ultrasound is irradiated to the solution, the gene can be packaged by modifying the shape so that the volume of the gene contained in the solution is reduced (S400).

More specifically, when ultrasonic waves are applied to a solution, nonlinear radiation may be generated due to a difference in density and acoustic impedance between the gene and the fluid in the solution. As a force is applied to a gene by nonlinear radiation force, the gene moves. The force is not applied uniformly based on the center of gravity of the gene, which causes rotation.

Thus, the gene may be changed in shape, such as winding or folding, and thus, the volume of the gene may be reduced and packaged smaller.

Hereinafter, the experimental results to confirm that the gene is packaged according to the ultrasonic irradiation in Figures 3 to 7 will be described in more detail.

In addition, Figure 4 is an exemplary view for explaining the shape of the gene before the irradiation of the ultrasound according to an embodiment of the present invention, Figure 5 illustrates the change in the shape of the gene by the irradiation time of the ultrasound according to another embodiment of the present invention This is an example diagram.

In addition, Figure 6 is an exemplary view for confirming the shape of the packaged gene according to an embodiment of the present invention, Figure 7 is an exemplary view showing the analysis results of the packaged gene according to an embodiment of the present invention.

Referring to Figure 3, it can be confirmed through the electrophoresis experiment that the shape of the gene is modified according to the intensity of the ultrasound. To easily distinguish this, coiled DNA and uncoiled DNA were used in the experiment.

Here, the uncoiled DNA may refer to a heated pDNA that is naturally colled for 30 minutes by heating the pEGFP solution to 94 ℃ for 180 seconds, electrophoresis may be performed in a constant voltage mode (5V / cm) at 100V, 95mA have.

Thus, (1) shows the results of electrophoresis experiments using coiled DNA, and (2) to (4) can show the results of electrophoresis experiments using uncoiled DNA. Particularly, (2) shows that when the ultrasonic wave is not irradiated, (3) shows a sound pressure of 400 kPa and a duty of 1% for 30 seconds, and (4) shows a sound pressure of 400 kPa and a duty of 10%. Results are shown when lasting for 30 seconds.

Through this, it can be seen that the band signal region of (3) is similar to the band signal region of (1). This means that the uncoiled DNA is coiled and packaged during ultrasonic irradiation. However, in the case of (4), as the ultrasonic energy of too strong energy is irradiated to the gene, a large number of DNA is destroyed and the band signal is weakly displayed.

Therefore, it can be confirmed that the gene is more efficiently packaged when ultrasonic waves of appropriate intensity are irradiated.

4 and 5, the process of changing the shape of the gene according to the irradiation time of the ultrasound can be confirmed through an AFM (Atomic Force Microscope) image.

First, the gene form before irradiation of ultrasound can be confirmed through FIG. At this time, the gene was used plasmid DNA, it can be seen that it has an uncoiled form as can be confirmed through the gene form of A, B, C shown in FIG.

When irradiated with ultrasonic waves of the same intensity, it can be transformed into a coiled gene as shown in FIG. 5. In this case, (1) is the gene form after irradiating the uncoiled form of plasmid DNA for 30 seconds, (2) is the form of the gene after irradiating the uncoiled form of plasmid DNA for 120 seconds, and (3) is Uncoiled plasmid DNA shows the shape of the gene after 300 seconds of ultrasound irradiation.

That is, when ultrasonic waves are irradiated to an uncoiled type gene through FIGS. 4 and 5, it is packaged as a coiled type gene, and when the ultrasonic wave is irradiated to the gene for a longer time, it can be confirmed that the package is smaller.

6 and 7, the shape of the packaged gene and the analysis result of the packaged gene may be confirmed by controlling the time for which the ultrasound is irradiated differently.

In FIG. 6, A is not irradiated with ultrasound, B is irradiated with ultrasound for 30 seconds, C is irradiated with ultrasound for 60 seconds, D is irradiated with ultrasound for 120 seconds, and E is 300 seconds. The gene morphology in the case of ultrasound irradiation can be confirmed.

In order to analyze the packaged gene by controlling the irradiation time differently, the folding index was calculated by analyzing the coiled degree of the AFM (Atomic Force Microscope) images of pEGFPs based on the quantitative image processing method.

To obtain the folding index, first measure the degree of coiling according to the amount of local curvature and convert the color image into a sum gray scale image of the red, green, and blue channels. In addition, a single DNA is accessed and processed by the ROI, which is Gaussian lowpass filtered to remove noise. The processed image is then parametrized using splines, and the local index can be finally determined by obtaining the second-order differential equation of the spline function.

Thus, when comparing the folding index values of A to E, the folding index of the gene is increased as the time of ultrasonic irradiation increases, so that the coiled shape is stronger as the time of ultrasonic irradiation is longer.

FIG. 8 is a block diagram illustrating a gene packaging apparatus using ultrasonic waves according to an exemplary embodiment of the present invention, and FIG. 9 is a diagram illustrating a shape of a gene packaging apparatus using ultrasonic waves according to an exemplary embodiment of the present invention.

Referring to FIG. 8, the gene packaging apparatus 100 using ultrasonic waves may be implemented by an ultrasonic stimulation system, and includes a solution receiving unit 110, an ultrasonic determination unit 120, an ultrasonic output unit 130, and a gene packaging unit. 140 may be included.

The solution receiving unit 110 may receive a solution containing a gene.

The ultrasound determination unit 120 may determine the ultrasound irradiation condition including the intensity and time of the ultrasound to be irradiated according to the amount and length of the gene included in the solution.

Here, the ultrasonic irradiation condition may include the intensity of the ultrasonic wave including the frequency of the ultrasonic wave to be irradiated to the solution, a cycle, an ultrasonic pressure, and the like, and a time for irradiating the ultrasonic wave.

In particular, the ultrasound determination unit 120 may adjust the ultrasound irradiation conditions so that the genes contained in the solution are not destroyed. More specifically, the intensity and time of the ultrasonic wave can be freely adjusted according to the amount of the gene included in the solution, and the frequency of the intensity of the ultrasonic wave can be freely adjusted in any particular region according to the length of the gene included in the solution. In this case, the reason for adjusting the frequency according to the length of the gene is that the influence of the radiation force (radiation force) may appear differently depending on the length of the gene.

The ultrasonic output unit 130 may radiate ultrasonic waves corresponding to ultrasonic irradiation conditions to the solution.

That is, when an electric signal corresponding to the ultrasonic irradiation condition is generated, the ultrasonic output unit 130 converts the electrical signal into an ultrasonic signal and irradiates the solution. Thus, the ultrasonic output unit 130 may refer to an ultrasonic transducer for generating an ultrasonic probe.

The gene packaging unit 140 may package the gene by modifying the shape of the gene included in the solution.

More specifically, when ultrasonic waves are applied to a solution, nonlinear radiation may be generated due to a difference in density and acoustic impedance between the gene and the fluid in the solution. As the force is applied to the gene by the nonlinear radiation force, the gene moves. At this time, the force is not applied uniformly based on the center of gravity of the gene, and rotation may also be induced.

FIG. 9 illustrates the above-described gene packaging apparatus, and a method of artificially small pEGFP-C1 vector packaging using the gene packaging apparatus will be described as an example.

In order to package the pEGFP-C1 vector, first, a solution 113 containing a quantitative amount of the pEGFP-C1 vector 111 may be accommodated in the well receiving solution 110 in a well plate form about 1 mm. In this case, the pEGFP-C1 vector may refer to a gene grown in Escherichia coli, isolated and purified using an Endo-free plamid giga kit, and quantitatively analyzed through Nano Drop ND-1000.

As the solution 113 including the pEGFP-C1 vector 111 is accommodated in the solution receiving unit 110, the ultrasonic probe 130 may be irradiated with a specific type of ultrasonic probe. For example, the solution may be irradiated with an intensity of ultrasonic waves corresponding to a center frequency of 1.12 MHz, a period of 10 ms and a sound pressure of 400 KPa for a time of about 30 to 300 seconds.

In this case, although not separately illustrated in FIG. 9, the pEGFP-C1 vector 111 included in the solution 113 through the ultrasonic crystal determiner 120 so that the pEGFP-C1 vector 111 included in the solution 113 is not destroyed. The intensity and time of the ultrasound may be adjusted according to the amount and length of the ultrasonic wave.

As a result, the pEGFP-C1 vector 111 included in the solution 113 may be deformed in a shape such as being folded or folded to reduce its volume.

Although described above with reference to a preferred embodiment of the present invention, those skilled in the art will be variously modified and changed within the scope of the invention without departing from the spirit and scope of the invention described in the claims below I can understand that you can.

Claims

In the gene packaging method performed in the ultrasonic stimulation system,

Receiving a solution containing a gene;

Determining ultrasonic irradiation conditions including intensity and time of ultrasonic waves to be irradiated according to the amount and length of genes included in the solution; And

And irradiating ultrasonic waves corresponding to the ultrasonic irradiation conditions on the solution such that the genes contained in the solution are wound or folded due to a radiation force to deform the shape.
The method according to claim 1,

Determining the ultrasonic irradiation conditions,

Gene packaging method, characterized in that the ultrasonic irradiation conditions can be adjusted so that the gene contained in the solution is not destroyed.
The method according to claim 2,

Determining the ultrasonic irradiation conditions,

According to the amount of the gene contained in the solution, the intensity and time of the ultrasonic wave including at least one of frequency, cycle and ultrasound pressure can be adjusted, and the position of the gene to be irradiated with the ultrasonic wave and Gene packaging method characterized in that the frequency is adjustable according to the length.
The method according to claim 1,

If the solution is irradiated with ultrasonic waves corresponding to the ultrasonic irradiation conditions,

Non-linear radiation is generated due to the difference in density and acoustic impedance between the gene contained in the solution and the fluid around the gene,

Packaging the gene by modifying the shape of the gene such that the gene is wound or folded by the nonlinear radioactive force to reduce its volume.
In the gene packaging device implemented in the ultrasonic stimulation system,

A solution receiving portion containing a solution containing a gene;

Ultrasonic determination unit for determining the ultrasonic irradiation conditions including the intensity and time of the ultrasonic wave to be irradiated according to the amount and length of the gene contained in the solution; And

And an ultrasonic output unit for irradiating the ultrasonic wave corresponding to the ultrasonic irradiation condition on the solution so that the gene contained in the solution is wound or folded due to a radiation force.
The method according to claim 5,

The gene packaging device,

When the ultrasonic wave corresponding to the ultrasonic irradiation condition is irradiated to the solution by the ultrasonic output unit,

Non-linear radiation is generated due to the difference in density and acoustic impedance between the gene contained in the solution and the fluid around the gene,

And a gene packaging unit for packaging the gene by modifying the shape of the gene such that the gene is wound or folded due to the nonlinear radiation force to reduce the volume.