KR20030078115A - Peptide gene carrier and method for transfecting gene into cell using the same - Google Patents

Abstract

The present invention relates to a peptide gene transfer medium and a gene transfer method using the same, and more particularly, to a peptide gene transfer capable of effectively transferring the gene into a cell, particularly into a cell nucleus by combining a gene with a peptide including a basic amino acid arginine. The present invention relates to a medium and a gene delivery method using the same.

The present invention can not only bind the peptide gene transfer medium including the basic amino acid arginine to the gene to be delivered in a simple manner, but also safely transfer the gene into the cell without causing cell toxicity. Therefore, the present invention can be effectively used for gene therapy from the medical point of view as well as to effectively express the foreign genes to the desired cells.

Description

Peptide Gene Carrier and Method for Transfecting Gene into Cell Using the Same}

The present invention relates to a peptide gene transfer medium and a gene transfer method using the same, and more particularly, to a peptide gene transfer capable of effectively transferring the gene into a cell, particularly into a cell nucleus by combining a gene with a peptide including a basic amino acid arginine. It relates to a medium and a method for gene delivery using the same.

There are many ways to express and transfer external genes into cells, which can be broadly classified into the following two types.

The first type uses a viral vector, in which foreign genes are loaded into the viral vector and introduced into the cell to express the gene in the cell. These methods are widely used for the purpose of regulating cellular action by specific genes or for expressing therapeutic proteins. However, this method has several problems. That is, the limitation of the gene size that can be delivered, the generation of viral particles by the virus used, the generation of an immune response when used in tissues, the instability of the expression rate of the delivered gene, and the limitation of the range of use available only in selective cells. There is a problem such as.

The second type is a method of transferring a gene without using a virus, and this method is generally known to be less effective than the method using a virus. However, the method is commonly used because it can overcome the disadvantage of the method using the virus. . These methods can be further divided into physical and chemical delivery methods.

Physical methods include microinjection methods that inject genes directly into cells, electroporation methods that deliver genes by electrical shock, or particle bombardment methods that deliver genes using gene guns. Etc. These physical methods have very low gene transfer efficiency and are used in limited cases where viruses or chemical methods cannot be used.

In chemical delivery, specific cationic lipids, peptides, proteins, liposomes, and the like are used as carriers to deliver desired genes into cells. Currently, such chemical delivery methods are widely used not only for gene delivery but also for delivering medically useful substances.

Recently, it has been found that a specific protein can effectively enter a cell through a cell membrane, and studies are being actively conducted to deliver a useful substance into a cell by using the protein as a vehicle. Representative proteins include Tat protein, ANTP and VP22 protein (Lindgren et al. TIPS 21:99 (2000)). These proteins have the ability to deliver molecules many times larger than they can easily through the cell membrane and into the cell. Dozens of peptides have been identified that play an important role in these proteins, particularly across cell membranes. These peptides are located at specific sites in the amino acid sequence of the protein, which is called the protein transduction domain (PTD). Although the mechanism by which the peptides in the PTD region pass through the cell membrane is not known in detail, genetically or chemically linking these peptides to useful materials has shown that effective materials are effectively delivered into the cells. A representative example is the Tat- β -galactosidase fusion protein made by genetic engineering of Tat protein and β -galactosidase enzyme. When the Tat- β -galactosidase fusion protein was administered to the abdominal cavity of the mouse, the fusion protein was delivered to cells of various tissues, and the activity of β -galactosidase was detected in various tissues of the mouse including brain tissue ( Schwarze et al. Science 285: 1569 (1999).

The PTD regions of these proteins have some things in common, and one of them is that most of the peptides in the PTD region consist mainly of basic and hydrophilic peptides. Therefore, it has been suggested that positive charges probably play an important role in the process of crossing cell membranes (Vladmir et al. PNAS 98: 8786 (2001)). Peptides in the PTD region of the Tat protein were also found to finally reach the nucleus within the cell rather than through the cell membrane and staying in the cytoplasm (Vives et al . JBC. 272: 16010 (1997)). Recently, peptides in this PTD region have been reported to recognize specific sites in the structure of ribonucleic acid and thus to specifically bind to ribonucleic acid (Harada et al. PNAS 94: 11887 (1997)).

However, conventional gene delivery media and methods of delivery require time and economic sacrifice because the process of linkage between genes and genes is very difficult and requires complicated procedures (Luo et al . Nature Biotech 18:33 (2000)). Most of the peptides used to induce intracellular toxicity (Fawell et al. PNAS 91: 664 (1994)) and the structure of the delivery medium has a problem that the synthesis is not simple.

Thus, (a) simpler structures or simpler synthesis, (b) simpler methods of binding to the gene of interest, and (c) no adverse effects such as inducing toxicity in cells Gene delivery media and methods of delivery are required.

Throughout this specification, a number of articles are referenced and their citations are indicated in parentheses. The disclosure of the cited article is incorporated herein by reference in its entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors completed the present invention by confirming that the use of a peptide containing the basic amino acid arginine not only improves the efficiency of gene transfer into cells, but also makes it possible to transfer genes into cells without adversely affecting the cells in a simpler way. Was done.

Accordingly, it is an object of the present invention to provide a peptide gene delivery medium.

Another object of the present invention to provide a gene delivery method using a peptide gene delivery medium.

Figures 1a to 1d is a photograph of observing the expression level of the GFP gene delivered by the arginine peptide in normal cells 293 cells by fluorescence microscopy.

Figure 2a to 2d is a photograph observing the expression level of the GFP gene delivered by the arginine peptide in cancer cells HeLa cells with a fluorescence microscope.

Figure 3 is a graph showing the results of measuring the cell survival rate according to the arginine peptide concentration in 293 cells that are normal cells.

Figure 4 is a graph showing the results of measuring the cell survival rate according to the concentration of arginine peptide in cancer cells HeLa cells.

According to one aspect of the present invention, there is provided a peptide gene delivery medium comprising a plurality of arginine residues.

As used herein, the term "gene carrier" refers to a substance that carries a gene to be delivered into a cell. As used herein, the term "gene" used with a delivery medium or delivery method does not mean only a structural gene, but broadly interpreted as meaning a conventional DNA molecule.

The gene transfer medium of the present invention includes an arginine residue, which is a positively charged basic amino acid having a large charge capable of electrostatically binding to a negatively charged gene. The pKa value of the amino group located in the R group of the arginine included in the peptide of the present invention is 12. This high pKa is required because it is necessary to form a complex that is easy for gene transfer through the electrostatic interaction between the gene delivery medium and the gene of the present invention.

In a preferred embodiment of the invention, the number of arginines in the peptide gene delivery medium is determined by various factors, such as the size of the gene to be delivered, the delivery conditions employed, etc., but typically 5-15 is preferred, with less than 5 In this case, there is a problem in that the efficiency of transferring the gene into the cell is lowered, and in the case of more than 15 genes, there is a problem in that the efficiency of transferring the gene into the cell is reduced. According to a more preferred embodiment of the present invention, the number of arginine in the peptide gene transfer medium is 8-14, most preferably 12.

The peptide gene delivery medium of the present invention can form a complex very simply with a gene, and the complex thus formed can be easily delivered into cells (eg, mammalian cells).

On the other hand, according to the known technology, a special factor (eg, nuclear localization signal (NLS)) is required to stably transfer a gene into the cell nucleus, particularly into the nucleus of a eukaryotic cell. However, in the present invention, only a peptide containing an arginine residue, which is a basic amino acid, enables stable delivery of a gene into the cell nucleus.

According to another aspect of the present invention, the present invention provides a mixture of (a) a peptide gene delivery medium comprising a plurality of arginine residues, a polynucleotide to be delivered and a binder which enables the binding of the peptide gene delivery medium to a polynucleotide. Manufacturing step; And (b) provides a gene delivery method using a peptide gene delivery medium comprising applying the mixture to the cell.

The overlapping content between the method of the present invention and the peptide gene transfer medium is omitted in order to avoid excessive complexity due to duplication herein.

In the method of the present invention, a binder is required to provide a surrounding environment necessary to promote the binding between the gene transfer medium and the polynucleotide to be delivered, and the binder is a buffer having a pKa value of 6.5-8.5. Solution is preferred. On the other hand, according to known techniques, for proper binding between the gene delivery medium and the gene, a special material is required or a complicated process is required. However, in the present invention, the binding step is performed by a simple method of incubating the gene transfer medium and the polynucleotide of the delivery target in a buffer solution having a suitable pKa value at a suitable temperature for a period of time.

According to a more preferred embodiment of the invention, the binder is phosphate buffer, HEPES buffer, MOPS buffer, various cell cultures without serum, such as DMEM (Dulbecco's modified minimum essential medium), Ham's F12, CMRL 1066, RPMI 1640, IMDM (Iscove's modified Dulbecco's medium) and the like, most preferably phosphate buffered saline.

Cells to which the present invention can be applied include various eukaryotic cells (monocytic eukaryotic cells, animal cells and plant cells), preferably animal cells, more preferably mammalian cells and most preferably human cells. Such cells include both normal cells and transformed cells (eg tumor cells) and the like.

The present invention can be used for the treatment of ex vivo and in vivo genes by carrying genes into cells. Ex vivo gene therapy consists of removing the cells of interest from the patient's body, transferring the genes by ex vivo method, and then re-injecting them into the patient's body.

The method of the present invention can be applied to transient gene expression or stable gene expression of foreign genes, the difference in gene expression pattern is also important for gene therapy. For example, transient gene expression is used when expression of a gene is only required for a short time or when the prolonged effect of foreign protein expression is unknown. Stable gene expression, on the other hand, is used when a patient has a fatal genetic defect such as cancer.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example 1 Synthesis of Peptides

A peptide consisting of 12 residues of arginine, which is a basic amino acid, was synthesized by Peptron Co., Ltd. (Daejeon, South Korea) and used commercially.

Only 95% purity of the synthesized peptide was selected and used as the gene transfer medium of the present invention.

Example 2: Gene Delivery by Arginine Peptides

Gene delivery by the gene delivery medium of the present invention was carried out as follows: Cells were used as two kinds of cells, namely 293 cells (ATCC, United States) and normal cells, HeLa cells (ATCC, United States), cancer cells, Again, the experiment was separated into a standard group and three treatment groups. Treatment groups were divided into cells treated with peptides and genes, cells treated with genes only, and groups treated with peptides only. 0.5 μg of plasmid pEGFP-N3 (Clontech, USA) and an arginine peptide (10 μM) containing the GFP (Green fluorescent protein) gene were mixed in 300 μl of phosphate-buffered saline (PBS). Subsequently, the mixture was allowed to react on ice for 10 minutes and then continued at room temperature for 50 minutes, after which the mixing process was terminated. The cells were dispensed 16 hours before the experiment, and when the density of the cells was about 80-90%, DMEM (Dulbecco's modified minimum essential medium, Gibco-BRL, U.S.A.) containing 10% fetal bovine serum was mixed with peptides and genes. ) Was added to each cell in the medium. Then, the cells were incubated for 2 hours in an incubator at 37 ° C. supplied with 95% oxygen and 5% carbon dioxide. After 2 hours of incubation, the cells were washed again with PBS solution, and peptides or genes that did not enter the cells were removed. The washed cells were then placed in freshly prepared DMEM medium and further cultured in the incubator for 24 hours. After incubation for 24 hours, the fluorescence emitted by the cells was measured using a fluorescence microscope to confirm the degree of gene transfer.

Example 3: Observation by Fluorescence Microscopy

Fluorescence of the protein expressed by the GFP gene delivered by the peptide of the present invention in cells treated as in Example 2 was measured using a fluorescence microscope (Nikon, Diaphot 300). The results are the same as in FIGS. 1A-1D and FIGS.

Figures 1a to 1d is a photograph observing the expression level of the GFP gene delivered by the arginine peptide in normal cells 293 cells by fluorescence microscopy. 1a shows that the GFP gene is expressed and shows bright fluorescence as a result of fluorescence microscopy observation of a cultured cell group in which the peptide and the GFP gene (11: 1, w / w) were put together. However, no fluorescence could be observed in the cell group treated with the gene alone (FIG. 1B), the cell group treated with the peptide alone (FIG. 1C), and the cell-only standard group (FIG. 1D).

On the other hand, Figures 2a to 2d is a photograph of observing the expression level of the GFP gene delivered by the arginine peptide in cancer cells HeLa cells with a fluorescence microscope. FIG. 2a shows that the GFP gene is expressed and shows bright fluorescence when the peptide and the GFP gene (11: 1, w / w) are put together and observed with a cultured cell group. However, no fluorescence was observed in the cell group treated with the gene alone (FIG. 2B), the cell group treated with the peptide alone (FIG. 2C), and the cell-only standard group (FIG. 2D).

Therefore, it can be seen that the peptide of the present invention can act as a gene transfer medium to carry the desired genetic material into the cell.

Example 4: Determination of Cytotoxicity of Arginine Peptides

The cell viability was measured after incubating the group treated with the arginine peptide of the present invention (control) and the group without treatment (standard group) for a predetermined time, and MTT (tetrazolium bromide) analysis was used to measure cell viability. First, arginine peptides with a concentration of 0-50 ?? M were treated to cells (10 ⁴ / ml) and then cultured in microplate wells at 37 ° C incubator with 95% oxygen and 5% carbon dioxide for 24 hours. Then, 25 μl of MTT solution (5 mg / ml, Sigma) was added and reacted again at 37 ° C. for 2 hours. After the reaction, 100 ml of extract (solution of 20% sodium dodecyl sulfate dissolved in 50% N , N -dimethyl formamide) was added thereto and reacted again at 37 ° C. for 15 hours. Cell viability was then measured using a multi scanner (ICN Titerteck, United States) (Hansen J. Immunol. Meth. 119: 203 (1989)). The results are shown in FIG. 3 and FIG. 4.

3 and 4 are graphs showing the results of measuring cell viability according to the concentration of arginine peptides in 293 cells, which are normal cells, and HeLa cells, which are cancer cells. 3 and 4 show that the treatment of arginine peptides of various concentrations (0-50 ?? M) showed that the survival rate of 293 cells and HeLa cells was hardly reduced even when the peptides of 50 ?? M peptide were treated for 24 hours. give. In other words, the cell viability of the two different cells was almost the same as the standard group without the peptide. As a result, it can be seen that arginine peptide can be safely used as a gene carrier.

On the other hand, gene transfer efficiency was also analyzed for 293 cells and other cells other than HeLa cells, that is, COS cells (ATCC, United States) and Jurkat cells (ATCC, United States) using the arginine peptide of the present invention. And delivery efficiency almost identical to that of HeLa cells.

In addition, the plasmid pcDNA3.1 / His / LacZ (Invitrogen, USA) including the β -galactosidase gene in addition to the plasmid pEGFP-N3 containing the GFP gene was tested for gene transfer efficiency by the arginine peptide of the present invention. As a result, almost the same result as plasmid pEGFP-N3 was obtained.

The present invention provides a peptide gene delivery medium. The present invention also provides a gene delivery method using a peptide gene delivery medium. The present invention can not only bind the peptide gene transfer medium including the basic amino acid arginine to the gene to be delivered in a simple manner, but also safely transfer the gene into the cell without causing cell toxicity. Therefore, the present invention can be effectively used for gene therapy from the medical point of view as well as to effectively express the foreign genes to the desired cells.

Claims (10)

Peptide gene delivery medium comprising a plurality of arginine residues. The peptide gene transfer medium of claim 1, wherein the number of arginines in the peptide gene transfer medium is 5 to 14. The peptide gene delivery medium of claim 2, wherein the number of arginines in the peptide gene delivery medium is 12. Gene delivery method using a peptide gene delivery medium comprising the following steps: (a) preparing a mixture of a peptide gene delivery medium comprising a plurality of arginine residues, a polynucleotide to be delivered and a binder that enables binding of the peptide gene delivery medium to a polynucleotide; And (b) applying said mixture to cells. The method of claim 4, wherein the number of arginines in the peptide gene delivery medium is 5 to 14. 5. 6. The method of claim 5, wherein the number of arginines in the peptide gene delivery medium is 12. The method of claim 4, wherein the binder is a buffer solution having a pKa value of 6.5-8.5. 8. The method of claim 7, wherein the binder is a phosphate buffer, a cell culture without serum, a HEPES buffer, or a MOPS buffer. The method of claim 8, wherein the binder is a phosphate buffer. The method of claim 4, wherein the cell is a normal animal cell or a tumor animal cell.