KR20230100212A - A Composition for Nonviral-based high-efficiency Nucleic Acid Transfection and Use Thereof - Google Patents

Abstract

The present invention relates to a non-viral composition for high-efficiency nucleic acid transfection in which a fusion peptide of a nuclear localization signal peptide and a polyarginine peptide is linked to a cationic liposome, and uses thereof. "Peptide-assisted Nanolipoplex (P-NLP)", a complex of plasmid DNA and a non-viral delivery system designed in the present invention, has significantly improved permeability to cell membranes and nuclear membranes and delivers plasmid DNA to target cells. Efficiency can be significantly increased.

Description

A Composition for Nonviral-based high-efficiency Nucleic Acid Transfection and Use Thereof

The present invention relates to a non-viral high-efficiency transfection technology capable of efficiently delivering a large nucleic acid such as CRISPR-Cas9 into the nuclear membrane by using the synergistic effect of peptide-mediated nanolipoplex penetrating the cell membrane and the nuclear membrane.

Gene therapy is a new treatment alternative that can fundamentally treat diseases at the molecular level through the regulation of genes involved in diseases. This is a revolutionary treatment that replaces genetically defective genes with normal ones or introduces genes with new functions. For the application of effective gene therapy, a vector system, which is a carrier capable of delivering useful genes into target cells, is essential.

Vector systems widely used through many studies are typically divided into viral and non-viral vectors. Viral vectors are based on the propagation mechanism of viruses and include adenoviruses, retroviruses, adeno-associated viruses, lentiviruses, and the like. Although this mediates high gene delivery efficiency, it has limitations in its use in in vivo gene therapy due to the characteristics of immunogenicity that may occur due to viruses, the possibility of potential oncogenes, and the size of the gene to be inserted.

On the other hand, non-viral vectors refer to lipids, polymers, nanoparticles, etc. obtained from biomaterials or chemical synthesis. This has the advantage of low risk of immunogenicity, no restriction on the size of the gene to be delivered, and easy production, but has a limitation in that the efficiency of delivery into cells and nuclei is lower than that of viral vectors.

Among non-viral vectors, cationic liposomes having a phospholipid bilayer structure are currently widely used industrially. Since it has a positive charge, it can form a complex with a negatively charged gene and electrostatic attraction through physical mixing to deliver the gene to the target cell. However, when the size of the gene to be transported is large, the size of the complex also increases, and the specificity of the cationic liposome to cover the negative charge of the gene decreases, so the efficiency of intracellular penetration may decrease. In addition, since liposomes are relatively poor in delivery ability to the nuclear membrane compared to cell membranes, it is not appropriate to use only liposomes as a carrier in the case of plasmid DNA to be delivered into the nucleus.

Republic of Korea Patent No. 10-1671114

In the present invention, a peptide-based synergist capable of overcoming the limitations of cationic liposomes was devised for high-efficiency delivery of large-sized plasmid DNA into cells and nuclei.

It was designed as a fusion of a nuclear localization signal peptide that enhances delivery into the nucleus and a polyarginine peptide that adds a positive charge to help penetration into cells.

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

In order to solve the above problems, the present inventors have developed a highly efficient non-viral based gene delivery system capable of delivering large nucleic acid molecules into target cells and nuclei with high efficiency.

As a result, when the combination of the nuclear localization signal peptide and the polyarginine peptide is applied to a large-sized plasmid DNA and cationic liposome complex to be delivered, the permeability to the cell membrane and nuclear membrane is greatly improved, so that the efficiency of plasmid DNA delivery to the target cell is improved. The present invention was completed by finding that it rises remarkably.

The present inventors confirmed the synergistic effect by applying a fusion peptide in which a nuclear localization signal peptide and a polyarginine peptide are linked to previously used cationic liposomes for high-efficiency delivery of large-sized plasmid DNA into cells and nuclei.

When large-sized plasmid DNA is transported to cells and nuclei using cationic liposomes, the size of the complex increases due to electrostatic attraction and it is not sufficient to cover the negative charge of the nucleic acid molecule, making it difficult for endocytosis. In addition, since cationic liposomes have poor delivery ability into the nucleus, the delivery efficiency of large plasmid DNA is reduced.

In the present invention, in order to overcome the limitations of cationic liposomes, a fusion peptide linking a nuclear localization signal peptide derived from SV40 (Simian virus 40) T antigen and a polyarginine peptide was designed as a synergist.

Specifically, according to one embodiment of the present invention, a non-viral based high-efficiency nucleic acid transfection composition in which a fusion peptide in which a nuclear localization signal peptide and a polyarginine peptide are fused is linked to cationic liposomes is provided.

The nuclear localization signal peptide binds to the importin protein and mediates the transport of the target protein from the cytoplasm to the nucleus. and form self-assembling complexes.

The SV40-R11 fusion peptide increases the positive charge specific gravity of the plasmid DNA and the cationic liposome complex, thereby increasing cell penetration efficiency and forming a condensed complex to increase the stability of the plasmid DNA to be delivered to the nucleus. Since it contains a nuclear localization signal peptide, it has a function of transporting plasmid DNA into the nucleus. In addition, the fusion peptide has excellent biocompatibility and is suitable for use as a synergist for cationic liposomes.

The complex of plasmid DNA and non-viral delivery system designed in the present invention was named "Peptide-assisted Nanolipoplex (P-NLP)".

The cationic liposome is characterized by forming a complex with plasmid DNA.

The fusion peptide in which the nuclear localization signal peptide and the polyarginine peptide are fused is characterized in that they are connected to each other by a linker having SEQ ID NO: 1 sequence: GCG.

Another embodiment of the present invention provides a composition for performing any one or more of cancer cell research, detection, and treatment, including the non-viral-based composition for high-efficiency nucleic acid transfection.

Another embodiment of the present invention provides a method for delivering a non-viral-based high-efficiency nucleic acid transfusion composition to cells, comprising the step of contacting the non-viral-based high-efficiency nucleic acid transfusion composition with the cell.

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

(a) The present invention provides a non-viral-based composition for highly efficient nucleic acid transfection in which a fusion peptide in which a nuclear localization signal peptide and a polyarginine peptide are fused is linked to cationic liposomes.

(b) "Peptide-assisted Nanolipoplex (P-NLP)", which is a complex of plasmid DNA and a non-viral delivery system designed in the present invention, has greatly improved permeability to cell membranes and nuclear membranes, so that it can enter target cells. The efficiency of plasmid DNA transfer can be significantly increased.

(c) "Peptide-assisted Nanolipoplex (P-NLP)", a complex of plasmid DNA and non-viral delivery system designed in the present invention, is safe as a delivery vehicle for transfection of plasmid DNA. did

(d) It was confirmed that the "Peptide-assisted Nanolipoplex (P-NLP)", a complex of plasmid DNA and a non-viral delivery system designed in the present invention, can efficiently deliver large sized plasmid DNA. did

1 is a gel retardation analysis result for the P-NLP complex.
Figure 2a is a graph showing the gene transfer efficiency of P-NLP to HEK293T cells.
Figure 2b is a graph showing the efficiency of P-NLP gene transfer to MCF-7 cells.
Figure 2c is a graph showing the efficiency of P-NLP gene transfer to MDA-MB-231 cells.
Figure 2d is a graph showing the efficiency of P-NLP gene transfer to HeLa cells.
3 is a graph showing the biocompatibility of P-NLP.

According to one embodiment of the present invention, a non-viral based high-efficiency nucleic acid transfection composition in which a fusion peptide in which a nuclear localization signal peptide and a polyarginine peptide are fused is linked to cationic liposomes is provided.

The nuclear localization signal peptide binds to the importin protein and mediates the transport of the target protein from the cytoplasm to the nucleus. and form self-assembling complexes.

The SV40-R11 fusion peptide increases the positive charge specific gravity of the plasmid DNA and the cationic liposome complex, thereby increasing cell penetration efficiency and forming a condensed complex to increase the stability of the plasmid DNA to be delivered to the nucleus. Since it contains a nuclear localization signal peptide, it has a function of transporting plasmid DNA into the nucleus. In addition, the fusion peptide has excellent biocompatibility and is suitable for use as a synergist for cationic liposomes.

The complex of plasmid DNA and non-viral delivery system designed in the present invention was named "Peptide-assisted Nanolipoplex (P-NLP)".

The cationic liposome is characterized by forming a complex with plasmid DNA.

The fusion peptide in which the nuclear localization signal peptide and the polyarginine peptide are fused is characterized in that they are connected to each other by a linker having SEQ ID NO: 1 sequence: GCG.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experiment method

Manufacture of P-NLPs

Plasmid DNA was prepared by extracting pU6-(BbsI)_CBh-Cas9-T2A-mCherry (Addgene) with EndoFree® Plasmid Maxi Kit (Qiagen) to 1 μg/μL with autoclaved tertiary distilled water.

The SV40-R11 fusion peptide (LifeTein) was synthesized to have a purity of 95% or higher and dissolved in tertiary distilled water to a concentration of 0.5 mg/mL. It was synthesized by adding a linker sequence (GCG) between the fusion peptide sequences.

The structure of the SV40-R11 fusion peptide largely consists of a linker sequence between a nuclear localization signal peptide and a polyarginine peptide. It has the function of self-assembling structure formation and cell membrane penetration through In the P-NLP formation, lipoplexes are prepared by first mixing plasmid DNA and Lipofectamine ^TM 2000 (Lipo2000), then the ratio of amine groups (N) of the fusion peptide and phosphate groups (P) of the plasmid DNA is calculated and appropriate The final P-NLP was prepared by adding fusion peptides to lipoplexes according to the ratio and incubating them at room temperature for 20 minutes.

gel retardation assay

After mixing plasmid DNA and Lipofectamine ^TM 2000 and incubating at room temperature for 5 minutes, the N/P ratio of the fusion peptide was 1(1Х), 5(5Х), 10(10Х), 20(20Х), 30(30Х) ), 40 (40Х), 50 (50Х), and 100 (100Х) were added and incubated for 20 minutes at room temperature. A 1% (w/v) agarose gel was prepared in 1Х TAE buffer containing 10,000Х TopRed Nucleic Acid Gel Stain (GenomicBase), and 6Х loading buffer (Enzynomics, Inc.) was added to each sample before loading onto the gel. did After electrophoresis was performed for 30 minutes using a Mupid-2plus electrophoresis system (Optima Inc.), the results were analyzed using a gel recording system LSG 1000 (iNtRON biotechnology).

cell culture

Human embryonic kidney cells (HEK293T), human cervical cancer cells (HeLa), and breast cancer cells (MDA-MB-231, MCF-7) were cultured in 10% fetal bovine serum (Corning) and 1% penicillin-streptomycin (Life Technologies). DMEM (Dulbecco's modified eagle's medium, Corning) containing 37 ℃, 5% CO ₂ It was cultured under standard conditions. Subculture was performed at 2-3 day intervals depending on the density of the cells.

Fluorescence measurement

The efficiency of expression of red fluorescent protein encoded in plasmid DNA by delivery of P-NLP to various cells was analyzed by fluorescence measurement. Each of the cells cultured at a density of 80% or more was seeded in a 24-well plate at 1.2Х10 ⁵ cells/well in a total amount of 0.5 mL, and then cultured for one day under standard conditions.

After replacing the medium with Opti-MEM (Gibco), cells were treated with P-NLP formed by setting the N/P ratio of the fusion peptide to 5Х and cultured for one day under standard conditions. After replacing Opti-MEM with fresh DMEM medium, the cells were cultured for one day under standard conditions. After removing the cell medium, 100 μL of cell lysis buffer was added and stirred at 4°C.

40 μL of the cell lysate was transferred to a 384-well plate and the fluorescence intensity was measured at an absorption wavelength of 580 nanometers and an emission wavelength of 610 nanometers using a plate reader (Varioskan LUX, Thermo Fisher Scientific). For total protein quantification, the cell lysate was diluted and subjected to bicychoninic acid test (Pierce ^TM BCA Protein Assay Kit, Thermo Fisher Scientific). Four measurements are presented as individual points and mean ± standard deviation.

WST-1 exam

The biocompatibility of P-NLP was evaluated in normal cells, human neonatal dermal fibroblasts (HDFn). Cells cultured at a density of 80% or more were dispensed in a 96-well plate at 1.0Х10 ⁴ cells/well in a total amount of 0.1 mL, and cultured for one day under standard conditions.

After replacing the medium with Opti-MEM (Gibco), the cells were treated with P-NLP formed at N/P ratios of 1Х, 2.5Х, and 5Х of the fusion peptide, respectively, and cultured for one day under standard conditions. After replacing Opti-MEM with fresh DMEM medium, the cells were cultured for one day under standard conditions. CCK reagent (DonginLS) was quickly added at 10% of the total volume, and then incubated for 1 hour under standard conditions while blocking light. Absorbance was measured at a wavelength of 450 nm using a plate reader (Epoch 2, BioTek). Three measurements are presented as individual points and mean ± standard deviation.

Experiment result

P-NLP formation assay

The P-NLP complex formed by the interaction of plasmid DNA, Lipofectamine ^TM 2000, and the SV40-R11 fusion peptide was confirmed by gel retardation analysis (FIG. 1).

In the conventional lipoplex to which the fusion peptide was not added, it was confirmed that a complete complex was not formed, as the plasmid DNA band clearly remained at the bottom of the loaded well. On the other hand, in P-NLP in which the fusion peptide was added at the set N/P ratio, a delay in the plasmid DNA band was observed as the N/P ratio increased. In addition, in P-NLP with an N/P ratio of 5Х, no plasmid DNA band was observed in the well, confirming that the complex was completely formed.

Evaluation of delivery efficiency of P-NLP

Quantitative delivery efficiency evaluation of P-NLP was performed by fluorescence analysis. Fluorescence intensity was measured based on the expression of red fluorescent protein (mCherry) encoded in the plasmid DNA when the plasmid DNA in the P-NLP was delivered into cells and nuclei. The fluorescence intensity measured under each condition was divided by the total amount of protein, and the corresponding value in the control group in which only plasmid DNA was delivered was normalized to 1 (FIGS. 2a to 2d). In the four types of cells, P-NLP showed a statistically significant increase in normalized fluorescence intensity compared to lipoplexes formed by conventional Lipofectamine ^TM 2000. This increased by about 3.1 times in HEK293T, about 4.4 times in MCF-7, about 2 times in MDA-MB-231, and about 2.9 times in HeLa, respectively. Through this, it was confirmed that large-sized plasmid DNA can be efficiently delivered when the SV40-R11 fusion peptide, which improves cell membrane and nuclear membrane penetration efficiency, is applied to existing Lipofectamine ^TM 2000 as a synergistic agent.

Biocompatibility evaluation of P-NLP

The biocompatibility of P-NLP for normal cells was evaluated by the WST-1 test. The absorbance values in the control group delivered with only plasmid DNA were normalized to 100% (FIG. 3).

All experimental groups of P-NLP to which various peptide ratios were added showed cell viability of 80% or more. Through this, it was confirmed that the P-NLP of the present invention has safety as a delivery vehicle for transfection of plasmid DNA.

Although one embodiment of the present invention has been described above, those skilled in the art can add, change, delete, or add components within the scope not departing from the spirit of the present invention described in the claims. The present invention can be variously modified and changed by the like, and this will also be said to be included within the scope of the present invention.

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Claims (7)

A composition for high-efficiency non-viral nucleic acid transfection in which a fusion peptide of a nuclear localization signal peptide and a polyarginine peptide is linked to a cationic liposome.
According to claim 1,
The nuclear localization signal peptide is a non-viral based high-efficiency nucleic acid transfusion composition, characterized in that derived from the SV40 (Simian virus 40) T antigen.
According to claim 1,
The polyarginine peptide is a non-viral based composition for high-efficiency nucleic acid transfection, characterized in that 11 cationic arginine amino acids are linked [R (arginine) 11].
According to claim 1,
The cationic liposome is a non-viral-based high-efficiency nucleic acid transfusion composition, characterized in that the complex is formed with plasmid DNA.
According to claim 1,
A fusion peptide in which a nuclear localization signal peptide and a polyarginine peptide are fused are linked to each other by a linker having SEQ ID NO: 1 sequence: GCG.
A composition for performing any one or more of research, detection, and treatment of cancer cells, comprising the non-viral-based composition for high-efficiency nucleic acid transfection according to any one of claims 1 to 5.
A method for delivering a non-viral-based composition for high-efficiency nucleic acid transfection to cells, comprising the step of contacting the composition for transfusion with a non-viral-based high-efficiency nucleic acid according to any one of claims 1 to 5.

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