KR20110132086A - Peptide gene carrier and method for transfecting gene into adult stem cell using the same - Google Patents

Abstract

PURPOSE: A transfection method of a gene into adult stem cells is provided to minimize the number of necessary peptides for intracellular gene delivery. CONSTITUTION: A peptide gene carrier contains linear saturated carboxylic acid of C10-C20 and 5-20 alginine residues. The linear saturated carboxylic acid is palmitic acid. A method for carrying a gene into adult stem cells comprises: a step of forming a polynucleotide complex and peptide gene carrier; and a step of applying the complex into the adult stem cells. The linear saturated carboxylic acid is palmitic acid. The adult stem cell is mesenchymal stem cells. The surface phenotype of the mesenchymal stem cells is CD105+, CD166+, CD29+, CD44+, CD14-, CD34-, and CD45-.

Description

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

The present invention relates to a peptide gene transporter and a method for gene delivery to adult stem cells using the same.

Recently, many efforts have been made to use genetic manipulations in human mesenchymal stem cells, particularly in human mesenchymal stem cells, to use them as gene therapy methods. However, in most animal cells, macromolecules cannot easily enter macromolecules into cells, and human mesenchymal stem cells are no exception. In particular, adult stem cells are very sensitive and have low gene transfer efficiency, and are known to cause cytotoxicity easily by gene transfer methods.

Gene delivery methods currently used in animal cells can be divided into a method using a virus and a method using a non-virus. Viral methods use viral vectors, which have high delivery efficiency but involve side effects such as inducing immune responses and activating tumorigenicity. Non-viral delivery methods include electric shock methods for delivering genes by electric shock, liposome methods using fusion ability to cell membranes, and polymers using cationic polymers. Most of these nonviral delivery methods are known to be less safe than viral vectors, but are safer than viral vectors. However, these non-viral methods also cause high cytotoxicity in vivo and are not suitable for treatment.

Various stem cells have attempted gene transfer using the above viral and non-viral delivery methods, but the non-viral methods are mainly studied because the stem cells used for treatment cannot use viral methods. However, stem cells have generally been found to be very low in gene transfer efficiency compared to other animal cells, and are currently used non-viral methods and show about 10% gene transfer efficiency in stem cells. It is known.

One of the methods used to solve this problem is to use a specific protein carrier in nature as a non-viral gene transfer method. Protein transporters are known to be composed of short peptides that can effectively transport macromolecules such as proteins and nucleic acids through cell membranes and into cells. Representative protein carriers include Tat and VP22 proteins (Lindgren et al., TIPS, 21:99 (2000)), and various protein carriers are currently known (Korean Patent Publication No. 10-2008-0076622).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and 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 have made extensive efforts to develop a method for efficiently and safely delivering external genes to adult stem cells. As a result, a single C ₁₀ -C ₂₀ linear saturated carboxylic acid or alkanoic acid is used for short arginine peptides. The present invention has been completed by finding that the gene can be efficiently transferred to adult stem cells without cytotoxicity when used in combination with).

Accordingly, an object of the present invention is to provide a peptide gene transporter that can efficiently transfer genes to adult stem cells.

Another object of the present invention is to provide a method for delivering a gene to adult stem cells.

Still other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a peptide gene carrier comprising one C ₁₀ -C ₂₀ linear saturated carboxylic acid and 10 to 20 arginine residues.

The present inventors have made intensive studies to develop a method for efficiently and safely delivering foreign genes to adult stem cells. As a result, the C ₁₀ -C ₂₀ is applied to a short arginine peptide. The present invention has been completed by finding that the use of a single linear saturated carboxylic acid can be used to efficiently transfer genes to adult stem cells having low gene transfer efficiency without cytotoxicity.

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 in conjunction with a carrier or a delivery method does not mean only a structural gene, but should be broadly interpreted as referring to conventional DNA and RNA molecules.

The gene carrier of the present invention includes an arginine residue, which is a positively charged basic amino acid with 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 carrier and the gene of the present invention.

In a preferred embodiment of the invention, the number of arginines in the peptide gene transporter is determined by various factors, such as the size of the gene to be delivered, the delivery conditions employed, etc., but typically 5-20 is preferred, with less than 5 There is a problem that the efficiency of the gene is delivered into the cell is lowered, when there are more than 20 genes enter the cell there is a problem that the efficiency of delivery to the nucleus is lowered. According to a more preferred embodiment of the present invention, the number of arginines in the peptide gene delivery medium is 10-17, most preferably 15.

In addition, the gene carrier of the present invention is characterized by using one of C ₁₀ -C ₂₀ linear saturated carboxylic acid (alkaline carboxylic acid or alkanoic acid) to oligo arginine. The long hydrocarbon chain of C ₁₀ -C ₂₀ linear saturated carboxylic acid is very hydrophobic, so when used in connection with oligoarginine, a small number of arginines can efficiently enter genes into cells. As a result, the efficiency of gene transfer into the nucleus can also be enhanced. The linear saturated carboxylic acid of C ₁₀ -C ₂₀ may be linked to the amine group included in the side chain of the oligoarginine, or may be linked to the N terminal of the oligoarginine.

In one embodiment of the present invention, C ₁₀ -C ₂₀ linear saturated carboxylic acid linked to the oligo arginine is palmitic acid.

In the gene carrier of the present invention, the number of C ₁₀ -C ₂₀ linear saturated carboxylic acids linked to the oligo arginine is one.

In the present invention, it is most preferable to bind only one C ₁₀ -C ₂₀ linear saturated carboxylic acid, and in this case, the N / P charge ratio of the complex, the amount of polynucleotide used, and the complex formation time are optimized so that gene transfer is It is characterized by efficient gene transfer to adult stem cells, which are known to be very difficult, and without causing cytotoxicity. On the other hand, according to the known technology, a specific factor (eg, nuclear localization signal (NLS)) is required to stably transfer genes into the cell nucleus, particularly into the nucleus of eukaryotic cells. However, in the present invention, only a single C ₁₀ -C ₂₀ linear saturated carboxylic acid and a peptide containing 5 to 20 arginine residues enable stable delivery of the gene into the cell nucleus. This is probably because the NLS contains a large number of arginine so that the oligoarginine used in the present invention can transfer the gene to the nucleus in place of the role of NLS (Kim et al. IJP, 335: 70 (2007)). .

According to another aspect of the invention, the invention provides a complex of (a) a peptide gene carrier comprising one C ₁₀ -C ₂₀ linear saturated carboxylic acid and 10 to 20 arginine residues and a polynucleotide to be delivered. Making a step; And (b) provides a method for delivering a gene to adult stem cells comprising applying the complex to the adult stem cells.

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

Adult stem cells of the present invention refers to cells that are present in extremely small amounts in the body and provide the minimum cells necessary to maintain a healthy state at all times, and neural stem cells, mesenchymal stem cells, cartilage stem cells, hematopoietic stem cells and liver Stem cells, and the like. Adult stem cells are very small in each tissue of the body and very sensitive to be difficult to cultivate in vitro, difficult to transfer genes, and even if the gene is delivered, the cytotoxicity is characterized.

Embryonic stem cells, on the other hand, are stem cells derived from fertilized eggs, which are the beginnings of life, and have stem cells that are capable of differentiating into all kinds of cells that make up our bodies. However, since the sensitivity is much less sensitive than adult stem cells, such as no chromosome abnormality, gene transfer is easier than adult stem cells and more stable in cytotoxicity. However, embryonic stem cells are also known to have poor gene transfer compared to normal cells.

The present invention combines oligoarginine with one C ₁₀ -C ₂₀ linear saturated carboxylic acid to minimize the number of peptides required for gene transfer, thereby efficiently transferring genes into the nucleus of adult stems, which is extremely difficult to deliver. It is characterized by the achievement of gene transfer without causing any cytotoxicity to extremely sensitive adult stem cells.

In the method of the present invention, a binder is required to provide a surrounding environment necessary to promote binding between the gene carrier and the polynucleotide to be delivered, which binder is a buffer solution having a pKa value of 6.5-8.5. This is preferred. On the other hand, according to the known technique, for proper binding between the gene carrier and the gene, a special material is required or a complicated process is required. However, in the present invention, the binding step is performed through a simple method of incubating the gene carrier 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 present 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 adult stem cells, and most preferably human mesenchymal stem cells.

In one embodiment of the present invention, the mesenchymal stem cells are characterized in that the surface phenotype (surface phenotype) of CD105 +, CD166 +, CD29 +, CD44 +, CD14-, CD34-, CD45-.

In a preferred embodiment of the present invention, the step (a) is a mixture of the peptide gene carrier and the polynucleotide such that the ratio of the positive charge of the peptide and the negative charge of the polynucleotide (N / P charge ratio) is 3: 1 Is performed. In the peptide carrier of the present invention, when the N / P charge ratio of the peptide and the DNA is 3: 1, the gene of the GFP is most effectively expressed, and in this case, there is an advantage of showing little cytotoxicity.

The polynucleotide to be delivered of the present invention may be used without limitation as long as it is a polynucleotide having a negative charge such as antisense oligonucleotide, aptamer, siRNA, shRNA, miRNA, ribozyme, plasmid, vector, gene, recombinant nucleotide, 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.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention provides a peptide gene transporter and a method for gene delivery to adult stem cells using the same.

(Ii) The peptide gene carrier of the present invention binds one C ₁₀ -C ₂₀ linear saturated carboxylic acid to oligoarginine to minimize the number of peptides required for intracellular gene transfer, thereby transferring the gene into the cell as well as in the nucleus. Its characteristic is that it maximizes the transmission efficiency at the same time.

(iii) In addition, the present invention optimizes the N / P charge ratio of the complex, the amount of polynucleotides used, and the complex formation time to efficiently transfer genes to adult stem cells that are difficult to deliver, as well as to induce cytotoxicity. Do not.

1 is a graph measuring the expression level of the GFP gene delivered by the peptide carrier in human mesenchymal stem cells.
Figure 2 is a graph measuring the expression level of GFP gene by the amount of various DNA delivered by the peptide carrier in human mesenchymal stem cells.
Figure 3 is a graph measuring the effect of the complex formation time on the expression level of the GFP gene delivered by the peptide carrier in human mesenchymal stem cells.
Figure 4 is a graph showing the results of measuring the cell survival rate according to the peptide carrier concentration in human mesenchymal stem cells.
Figure 5 is a photograph observing the change in cell morphology after delivery of the GFP gene using baculovirus, liposomes, polymers, peptide transporters in human mesenchymal stem cells.
Figure 6 is a graph measuring the amount of EPO production after delivery of the EPO gene using a peptide transporter to human mesenchymal stem cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not to be construed as limiting the scope of the present invention. It will be self-evident.

Example

Example 1. Gene Delivery Synthesis

In order to prepare an arginine peptide (R15) including palmitic acid, which is a gene carrier used in the present invention, it was prepared by ordering to Peptron Co., Ltd. (Daeseong Metropolitan City Yuseong-gu), and the prepared compound was confirmed to have purity of 90% or more through HPLC. It was used after. The chemical formula of the synthesized gene carrier is as follows.

CH3 (CH2) 14CO- (R) ₁₅

Example 2 Complex Formation of Genes and Gene Carriers

PEGFP plasmid containing GFP (green fluorescent protein) gene (Clonetech, USA) and the carrier peptide synthesized in Example 1 are mixed with phosphate-buffered saline (PBS) in order to form a complex of gene and gene in the present invention. I was. At this time, the amount of the peptide and the amount of DNA to be mixed was quantified according to the N / P charge ratio, and in the present invention was applied by applying a variety of N / P charge ratio. Subsequently, the phosphate-buffered saline mixture was mixed vigorously for 1 minute using a vortex, and the reaction was continued for 1 hour at room temperature, and then the mixing process was terminated.

Example 3 Gene Delivery to Human Mesenchymal Stem Cells by Gene Delivery

Gene delivery in human mesenchymal stem cells by the gene carrier of the present invention was carried out as follows. Mesenchymal stem cells (Lonza, Basel, Switzerland) were dispensed 16-18 hours before the start of the experiment. When the cell density reached about 80-90%, the mixture containing peptide carriers and genes did not contain fetal bovine serum. It was added to cells in DMEM medium. It was then incubated for 4 hours in an incubator fed with 5% carbon dioxide at 37 ℃. Then, cells washed with PBS were further cultured in the culture medium for 48 hours in freshly prepared DMEM medium containing 10% fetal calf serum. After 48 hours of incubation, to confirm the effect of gene transfer. Fluorescence was measured using FACS.

Example 4. Gene Delivery Efficiency Measurement

Fluorescence of proteins expressed by the GFP gene delivered by the carrier of the present invention in cells treated as in Example 3 was quantified using FACS Calibur (Becton Dickinson, UK). The results are shown in FIGS. 1, 2 and 3.

1 is a result of quantifying the expression level of the delivered GFP gene according to the N / P charge ratio of the carrier and DNA in human mesenchymal stem cells. As a result of using two carriers in which palmitic acid is linked to the arginine peptide and the arginine peptide, the expression of GFP gene was very low in the arginine peptide carrier, but the expression efficiency of the GFP gene was significantly increased in the carrier of the arginine peptide including palmitic acid. In particular, peptide carriers including palmitic acid showed the most effective expression of GFP genes when the N / P charge ratio of peptide and DNA was 3: 1.

Figure 2 shows that as the result of optimizing the amount of DNA used to maximize the delivery efficiency by the peptide carrier as the amount of DNA administered to the cell while the N / P charge ratio of the peptide and DNA is maintained 3: 1 Gene expression efficiency was increased, but in the case of 6 ug / ml gene expression efficiency was high, but cytotoxicity appeared cell survival rate was reduced to 50% 3 ug / ml was used in the present invention.

Figure 3 is a graph of the effect of the conditions of complex formation of peptides and DNA on the gene transfer efficiency is generally known that the longer the reaction time of the peptide and DNA increases the size of the complex formed. In the case of the present invention, it was observed that the delivery efficiency was best when the peptide and the complex were passed at room temperature for 60 minutes.

Accordingly, the present invention is to optimize the N / P charge ratio of the complex, the formation time of the complex, the amount of DNA used, and the like, which are the conditions for the delivery of the arginine-linked arginine transporter, to use the palmitic acid-linked arginine peptide transporter in human mesenchymal stem cells. The delivery system was completed.

Example  5. Measurement of Cytotoxicity by Gene Carriers

In order to measure cell viability after treating the cells with arginine peptides including palmitic acid, the pathogen of the present invention, tetrazodium bromide (MTT) analysis was performed. 5% carbon dioxide was supplied for 48 hours in the experimental group (1 x 10 ⁴ cells / ml) treated with the carrier having various N / P charge ratios (0.5, 1.0, 3.0 and 6.0) and the standard group without the carrier. After incubation in a 37 ° C. incubator, the medium was changed to a medium containing 200 μg / ml MTT per well, and further incubated for 4 hours in the incubator. After 4 hours, the medium was removed, and 50 μl of DMSO was added to each well, followed by incubation with shaking for 15 minutes. After incubation using a multi-scanner (SpectraMax 250, USA), cytotoxicity was measured and the results are shown in FIG.

Referring to FIG. 4, as a result of treating various concentrations of carriers, the experimental group treated with a carrier having an N / P charge ratio of 3: 1 showed that the survival rate of mesenchymal stem cells was almost the same as that of the standard group even after 48 hours of incubation. . However, as the N / P charge ratio increased to 6: 1, cell viability decreased rapidly. This result shows that the peptide transporter used in the present invention can be used safely as a gene transporter.

In order to elucidate the safety of the carrier of the present invention, after the gene was transferred to mesenchymal stem cells by a gene transfer method that has been used in the past, the change in cell shape was compared with the case of using the peptide carrier. That is, the cell morphology was observed 48 hours after delivery of the GFP gene using baculovirus, a commercialized liposome, a commercialized polymer (Jet-PEI), and a peptide of the present invention, and the results are shown in FIG. 5. It was.

Comparing the cell populations with baculovirus, liposomes or polymers with the standard group without any treatment, the shape of the cells became thinner and longer, and the number of cells was also very small. In contrast, it can be observed that the cell group using the peptide carrier of the present invention has almost no difference in cell shape or number from the standard group. This result shows that the peptide transporter of the present invention is much safer in human mesenchymal stem cells than in gene, virus, liposome, and polymer gene transfer methods.

Example  6. EPO  Human by gene transfer Mesenchymal stem  In a cell EPO  production

As a specific example of the practical use using the delivery system of the present invention, after delivering EPO (erythropoietin) gene to human mesenchymal stem cells, it was confirmed that EPO was produced by these genes. In other words, after forming a complex of the EPO gene and the peptide by the above method, and putting it into human mesenchymal stem cells, ELISA analysis is performed on whether the EPO expressed from human mesenchymal stem cells is secreted into the cell culture medium for about 1 month. It was confirmed by the method. ELISA analysis was performed using an ELISA assay kit (ELISA assay kit, R & D system, USA). The result is shown in FIG.

Figure 6 shows the results of measuring the expression of EPO in the cell culture after delivery of the EPO gene using the carrier of the present invention to human mesenchymal stem cells. EPO was successfully produced for 27 days after 48 hours of EPO gene delivery. These results show that peptides can be used to deliver specific genes to human mesenchymal stem cells, which can be used as a therapeutic agent for certain diseases.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (8)

Peptide gene carrier comprising one C ₁₀ -C ₂₀ linear saturated carboxylic acid and 5 to 20 arginine residues.
The peptide gene carrier of claim 1, wherein the C ₁₀ -C ₂₀ linear saturated carboxylic acid is palmitic acid.
The peptide gene carrier according to claim 1, wherein the number of arginine residues in the peptide gene carrier is 15.
Gene delivery to adult stem cells comprising the following steps:
(a) forming a complex of a peptide gene carrier comprising one C ₁₀ -C ₂₀ linear saturated carboxylic acid and 5 to 20 arginine residues and a polynucleotide of interest; And
(b) applying the complex to adult stem cells.
The method of claim 4, wherein the C ₁₀ -C ₂₀ linear saturated carboxylic acid is palmitic acid.
The method of claim 4, wherein the adult stem cells are mesenchymal stem cells.
8. The method of claim 6, wherein the mesenchymal stem cells have a surface phenotype of CD105 +, CD166 +, CD29 +, CD44 +, CD14-, CD34-, and CD45-.
The method of claim 4, wherein step (a) is performed by mixing the peptide gene carrier and the polynucleotide such that the ratio of the positive charge of the peptide and the negative charge (N / P charge ratio) of the polynucleotide is 3: 1. Method for delivering a gene, characterized in that.

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