KR101316519B1 - A Complex containing Oligo-Aspartic acids-PNA for PNA delivery - Google Patents

Abstract

The present invention relates to a complex for improving cell uptake and effective delivery of PNA into a cell and a method of using the same, and specifically, to forming a complex of oligo-aspartic acid (Asp) -PNA-cationic carrier (PEI or Lipofectamine) The present invention relates to an effective and excellent gene delivery system that increases cell uptake and transfection efficiency and acts as a splicing correction of mRNA.

Description

A complex containing oligo-aspartic acids-PNA for PNA delivery}

The present invention relates to a complex for improving cellular uptake and effective delivery of PNA into a cell and a method of using the same.

Although various gene therapy techniques have been developed as a substitute for conventional protein therapy, there are still important challenges to be solved. One of the major challenges of gene therapy is to achieve efficient influx of therapeutic genes and expression of genes through efficient cell and nuclear membrane passages using gene carriers with little cytotoxicity.

Gene delivery systems based on synthetic peptides can overcome the above problems associated with polymeric gene delivery systems by condensing DNA by causing leakage in low pH endosomal membranes and also promoting endosomal escape. For this reason, various synthetic peptides are in It has been developed to promote gene delivery in various cell lines in vitro . However, this too in Problems such as toxicity and serum instability exist in vivo applications.

Since protein transduction domains (PTDs) for protein delivery can enhance intracellular uptake and nuclear translocation, they have been proposed as an alternative to polymeric or synthetic peptide-based gene delivery systems.

Poly (L-lysine) (PLL) and polyethyleneimine (PEI) based gene carriers are typical examples of nonviral polymeric gene carriers. Among the cationic polymers, branched 25 kD PEI is one of the most widely studied compounds. Branched PEIs are readily protonated in the acidic environment of the endosome by containing primary and secondary amine groups. As a result, proton sponge activity leads to endosomal escape of DNA / PEI followed by endosomal expansion and membrane disruption.

In addition, condensed DNA with short cationic peptides has a problem of low stability due to a lower cooperative effect, as a result of which the complex is unstable in the bloodstream, in It is insufficient to protect DNA from metabolism in vitro . On the other hand, if the cationic peptide binds too strongly with a negatively charged gene, the gene may not be released from the complex.

That is, in the case of a viral vector, there is a problem such as an immune response or a self-replicating, and in the case of a general polymeric vector, there is a problem in that the biocompatibility is deteriorated, which results in high cell and biotoxicity and low nucleic acid delivery efficiency.

Therefore, there is a need for the development of a new type of gene delivery system that can enhance the level of gene expression by stabilizing nucleic acids in the extracellular space, while smoothing the influx of nucleic acids into cells and the release of nucleic acids from complexes.

The present inventors conjugate PNA and oligo aspartic acid (Asp) to design negatively charged oligo-aspartic acid (Asp) -PNA to form a complex with a cationic carrier, thereby increasing the cellular uptake and transfection efficiency of PNA, and further mRNA The inventors have discovered that the splicing correction function of and has completed the present invention.

An object of the present invention is to provide a PNA delivery complex consisting of a negatively charged oligo-aspartic acid (Asp) -PNA and a cationic carrier.

Another object of the present invention to provide a method for gene correction (gene correction) using the complex for PNA delivery.

In order to solve the above object, the present invention provides a complex for delivery of PNA consisting of a negatively charged oligo-aspartic acid (Asp) -PNA and a cationic carrier, and the complex is specifically composed of Asp (n) -PNA-PEI, At this time, n may be 5 to 12, and oligo-aspartic acid (Asp) forms a conjugate with PNA.

In addition, the cationic carrier may be, but not limited to, polyethyleneimine (PEI) or lipofectamine. Thus, the complex may be composed of Asp (n) -PNA-PEI or Asp (n) -PNA-Lipofectamine. At this time, it is preferable that the said PEI is 20-30 kD in molecular weight.

Such a complex of the present invention acts in the sub-nanomolar range and thus shows excellent effects even at low concentrations.

The complex of the present invention not only has high intracellular delivery, that is, transfection efficiency, of PNA, but also has excellent cell uptake. In addition, the complexes are gene correction in cells. In particular, it acts as a splicing correction.

At this time, the transfection efficiency of the complex is dependent on the number of oligo-aspartic acid (Asp) conjugated to PNA and the cation carrier capacity, respectively.

In another aspect, the present invention provides a method for correcting a gene using the Asp (n) -PNA-PEI / Lipofectamine complex, in particular, for splicing correction of mRNA of a target gene. Provide a method.

The present invention conjugates PNA with oligo aspartic acid (Asp) to design negatively charged oligo-aspartic acid (Asp) -PNA to form a complex with a cationic carrier, for example, Asp (n) -PNA-PEI / Lipofectamine complex. Increasing the cellular uptake and transfection efficiency of the PNA, splicing correction of mRNA can be used as an effective excellent gene delivery system.

1 shows the results of observing luciferase activity in HeLa Luc705 cells of free PNA and Asp ₍₁₂₎ -PNA / PEI complex.
2 shows the results of observing luciferase activity at various concentrations of free PNA and Asp _{(12), (8), and (4)} -PNA / PEI complexes.
3 shows the results of observing luciferase activity of PEI or Lipofectamine complexes in various nanomolar PNAs.
4 shows the results of an RT-PCR experiment confirming splicing restoration.
5 shows fluorescence microscopic observations of HeLa Luc705 cells transfected with free PNA and Asp-PNA / PEI complexes.
6 shows the results of confocal microscopy observation of the cellular uptake of Asp ₍₁₂₎ -PNA / PEI complex.
Figure 7 shows the MTT assay results of evaluation of cytotoxicity.

The terms used in the present invention are defined as follows.

"Nucleic acid" is meant to include any DNA or RNA, PNA, such as chromosomes, mitochondria, viruses, and / or bacterial nucleic acids present in tissue samples. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule. In particular, in the present invention, PNA.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role in protein coding or transcription or in the regulation of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

"Transfection" refers to a method of expressing genotype in a cell by directly introducing nucleic acid (DNA, PNA, etc.) into the culture animal cell. When the target is a plant cell, the cell wall is often removed and introduced into the protoplasm. In this way, various problems of medicine and biology, such as carcinogens, infections, immunity, development, and communication, can be accessed at the cellular level. The introduced nucleic acid is generally introduced by introducing the desired gene into a medium such as a plasmid. When the introduced gene was stabilized in the cells, it was often interrupted by the chromosome. Cells into which the nucleic acid is introduced are called transgenic plants. Since the transfection efficiency is very low, several methods have been developed to increase the efficiency. Among them, calcium phosphate coprecipitation and DEAE-textlan treatment. Electroporation, redistribution (fusion method with cells that make artificial membranes and DNA complexes called liposomes).

Luciferase is an enzyme that promotes the oxidation of luciferin and converts chemical energy into light energy and emits light. It measures the expression in real time continuously and in real time. It functions as a reporter gene that allows you to verify the effect. It can be obtained directly from insects such as firefly or glow-worm or by expression from microorganisms comprising recombinant DNA fragments encoding such enzymes.

The term "carrier" refers to a polymer material that is responsible for transporting a carrier when an active substance in an organism is present in combination with another substance or when a substance is transported through a cell membrane. Examples of carriers include, but are not limited to, buffers such as phosphate, citrate and other organic acids, antioxidants such as ascorbic acid, low molecular weight polypeptides (less than about 10 residues), proteins such as serum albumin, gelatin or immunoglobulins, polyvinylpyrrolidone Hydrophilic polymers such as glycine, glutamine, asparagine, arginine or lysine, amino acids such as monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, sodium and Salt-forming counterions such as, and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG) and PLURONICS®.

"Geneology" refers to the treatment of genetic diseases by correcting the gene that caused the mutation. That is, it is a method of treating a disease by transplanting a normal gene from the outside to the cell of the patient and changing the phenotype of the cell. Currently, gene therapy for human sperm, egg, and fertilized egg has raised ethical issues all over the world. However, in 1993, it started to enter the gene therapy era after designating guidelines on clinical research on gene therapy. Gene therapy requires the study of gene vectors and systems that introduce genes into the body.

"About" means 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4 for reference quantities, levels, values, numbers, frequencies, percentages, dimensions, sizes, quantities, weights, or lengths. , Amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length, varying by about 3, 2 or 1%.

Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to intracellular delivery of peptide nucleic acid (PNA).

In one aspect, the present invention relates to a PNA delivery complex consisting of negatively charged oligo-aspartic acid (Asp) -PNA and a cationic carrier.

Peptide nucleic acid (PNA) is a natural DNA analogue originally developed in 1991 by P. Nielsen, Denmark, and artificially synthesized. Structurally, unlike DNA, PNA is composed of polyamide, but it has complementary sequences because it has the same bases (adenine, guanine, cytosine, thymidine, and uridine) as natural DNA. The double helix structure can be combined with nucleic acids such as DNA or RNA. PNA has not only superior biological, chemical and thermal stability compared to DNA, but also strong affinity and excellent specificity for complementary sequences.

Many known peptide sequences are positively charged because they contain lysine or arginine residues. Positively charged amino acids are typically at the edge of most of these peptides and interact with the negatively charged plasma membrane.

However, in the present invention, the PNA charged by the negative charge was designed to be similar to DNA or RNA.

To this end, the present invention provides oligo-aspartic acid-PNA [Asp (n) -PNA]. Conjugation of oligo-aspartic acid at the end of the PNA sequence, wherein n means an integer of one to 5 to 12. At this time, the carboxyl anion (COO-) is present at the end of the oligo-aspartic acid (Asp), the Asp (n) -PNA is a negative charge.

The negatively charged PNA using asparagine acid has the advantage of using more cationic carriers than the positively charged peptides. That is, it can form a complex with a cationic carrier to deliver a large number of negatively charged PNAs.

As such, the combination of oligo-aspartic acid and PNA not only improves the poor solubility of PNA, but also forms a complex with a cationic carrier to enhance PNA delivery efficiency.

Therefore, the oligo-aspartic acid-PNA of the present invention enables the interaction between charges between the PNA and the cationic carrier through the carboxy group negative charge of aspartic acid, specifically, through covalent and electrostatic interactions by electron pairs, Negatively charged PNAs can be delivered into the cell.

Such cationic carriers are sometimes referred to as transfection reagents and are potentially very useful for (therapeutic) genes or drug delivery.

In the present invention, cationic carriers include, for example, poly (L-lysine) (PLL), polyethyleneimine (PEI), chitosan, PAMAM dendrimer, and poly (2-dimethylamino) ethyl methacrylate (pDMAEMA). It can be used as a carrier. In certain cases, PEI provides effective gene delivery without the use of endosomal disintegrants or targeting agents. Boussif O., et al., Proc Natl Acad Sci USA. Aug. 1, 1995, 92 (16) 7297-301.

In particular, preferably PEI or cationic lipid Lipofectamine can be used as the cationic carrier.

Thus, as a specific aspect of the present invention, the complex may be composed of "Asp (n) -PNA-PEI", wherein n is an integer of 5 to 12, for example n = 12 days. Can be.

The PEI is a synthetic cationic polymer, which forms a complex with negatively charged residues for cellular uptake purposes. In the present invention, to deliver the PNA into the cell, a complex is formed by electrostatic interaction between the cationic PEI and the oligo-aspartic acid-PNA. The PEI enhances cell uptake and promotes escape of the endosomes for nucleic acid delivery.

However, since the PEI has a problem in that cytotoxicity increases as molecular weight increases, it is preferable to select PEI having an appropriate molecular weight so as not to have too high cytotoxicity while being efficient in PNA delivery. For example, in the present invention, a PEI of 20-30 kD may be used, and in one embodiment, a PEI of about 25 kD is used.

Similarly, in another specific aspect of the present invention, the complex may be composed of "Asp (n) -PNA-Lipofectamine", wherein n is an integer of 5-12, for example n = 12 Can be.

Lipofectamine, which is used for transfection using liposomes, has a positive charge in the hydrophilic portion, and thus the negative charge oligo-aspartic acid-PNA Interact with each other to form a complex, through which PNA is delivered into the cell. In particular, the Asp ₍₁₂₎ -PNA-Lipofectamine of the present invention showed very good transfection efficiency.

As such, in order to deliver PNA to cells, unlike conventional methods, the use of a "cationic carrier" by charging the PNA of the present invention with "negative charge" with oligo-aspartic acid is a new effective PNA delivery method in the art. .

The composite of the present invention and the method using the same have the following advantages.

(1) "Asp (n) -PNA-PEI / Lipofectamine" of the present invention acts as a gene correction, in particular splicing correction of mRNA.

Gene correction is one of the gene therapy methods, which means that the nucleotide sequence of a defective gene is corrected, that is, only the mutated part is corrected. (splicing) Corrective action.

Primary RNA (transcript) synthesized by DNA polymerase in the nucleus by RNA polymerase contains both axons and introns, and the introns are removed from these precursor RNAs to encode the amino acid sequence of the protein or 5'-, The process of collecting only exons that are 3'-untranslated sites is called splicing. In the present invention, PNA transfection corrects a mutant portion generated during this splicing process.

In one embodiment of the present invention, the splicing correction effect of such mRNA was confirmed by measuring luciferase activity, for example, RT-PCR experiment. That is, the complex of the present invention promotes luciferase expression by correcting gene expression through splicing correction of luciferase mRNA.

Accordingly, the present invention improves the delivery efficiency of PNA using the Asp (n) -PNA-PEI / Lipofectamine complex from another aspect, and gene correction, particularly splicing of mRNA, using the delivered PNA. It relates to a method of doing splicing correction.

(2) The complex of the present invention operates in the sub-nanomolar range.

The Asp (n) -PNA-PEI / Lipofectamine complex of the present invention has the advantage of functioning at very low concentrations in the sub-nanomolar range. As shown in FIG. 3, the transfection efficiency into the cells is excellent even at low concentrations.

(3) The transfection efficiency of the complex of the present invention is characterized in that the number of oligo-aspartic acids (Asp) or / and the amount of the cationic carrier is dependent.

The transfection efficiency of the Asp (n) -PNA-PEI / Lipofectamine complex of the present invention is dependent on the number of oligo-aspartic acid (Asp) conjugated to the PNA and the dose of the cationic carrier PEI or Lipofectamine, respectively.

At this time, the number n of Asp acid is preferably 3 to 15, preferably 5 to 12, and in the embodiment of the present invention, 12 was used. In addition, the dose of the cationic carrier is preferably used in consideration of transfection efficiency and cytotoxicity. In one embodiment of the present invention, 25 kD of PEI and Lipofectamin 2000 ^™ were used.

As such, the present invention uses the Asp (n) -PNA-PEI / Lipofectamine complex and the same to achieve intracellular delivery of PNA cells with high transfection efficiency and cellular uptake, and furthermore, splicing correction of mRNA. It is effective. Thus, it can be used as an effective and excellent gene delivery system.

In addition, the concentration of the cationic carrier that can be used in the present invention is preferably used in a concentration of 1 ~ 2ug as a non-toxicity to the cell, in the embodiment of the present invention is the most when using the amount of PEI 1ug Good effect could be obtained.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Experiment Preparation and Experiment Method

1. Material Preparation

Polyethylene imines (branched, 25 kD and 2 kD) and 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Seoul, Korea). Lipofectamine ™ 2000 transfection reagent and TRIzol reagent were purchased from Invitrogen (Carlsbad, CA, USA). Luciferase 1000 Assay Kit and reporter lysis buffer were purchased from Promega (USA, Wisconsin, Madison). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) medium were purchased from GIBCO (Gaysburg, Maryland, USA), PNA and oligo-Asp-PNA (Asp (4) -PNA, Asp (8)- PNA, Asp (12) -PNA, PNA-Fam, Asp (12) -PNA-Fam) were synthesized by Panagene Co. (Korea, Daejeon).

2. Cell culture

HeLa pLuc 705 cells were cultured in a 37 ° C. incubator in a humid atmosphere containing 5% carbon dioxide using DMEM medium containing 10% FBS.

3. PNA Transfection

HeLa pLuc 705 cells were cultured in 96-well plates at 2 × 10 ⁴ cells and 1.2 × 10 ⁴ cells per well, respectively, 24 hours before transfection. PNA and oligo-aspartic acid-PNA / polymer complexes were prepared in various molar ratios or polymer weight ratios and incubated at room temperature for 30 minutes. Cells were treated with 20 μl / well culture containing PNA, oligo-Asp-PNA / 25kD PEI and oligo-Asp-PNA / 2kD PEI at the designed concentrations. And incubated in a 5% CO ₂ , 37 ℃ incubator for one day before analysis.

4. Luciferase assay

After 24 hours of PNA transfection, cells of 96-well plates were washed with PBS solution and reporter lysis buffer (Promega) was added to each well. After incubation for 30 minutes at room temperature, cells were obtained and transferred to fine centrifuge tubes. After vortexing for 15 seconds, cells were separated by centrifugation at 12000 rpm for 20 minutes. Luciferase activity was measured in a relative light unit (RLU) using a luminometer. Final luciferase values were reported as RLU / Well.

5. RT - PCR

Sequence of primers used:

luciferase sense: 5′-TTGATATGTGGATTTCGAGTCGTC-3 ’(SEQ ID NO: 2)

luciferase antisense: 5'-AGCCACGCTTTCATACTGCT-3 '(SEQ ID NO: 3)

GAPDH sense: 5'-AACTTTGGCATTGTGGAAGG-3 '(SEQ ID NO: 4)

GAPDH antisense: 5'-ACACATTGGGGG TAGGAACA-3 '(SEQ ID NO: 5)

Total RNA was isolated with Trizol reagent according to the manufacturer's design. The concentration and purity of total RNA were calculated by absorbances of 260nm and 280nm. One-strand cDNA was synthesized using Omniscript reverse transcriptase with 2 μg of total RNA and 1 μM of oligo-dT15 primers (Qiagen, Valencia, CA, USA).

Quantification using Peltier thermal cycler (MJ Research, Massachusetts, Watertown, MJ Research) and Taq PCR Master Mix kit (Qiagen) containing 20 pmol primer and cDNA diluted 1:25 according to manufacturer's protocol PCR amplification was done. PCR reactions initiated denaturation at 94 ° C for 3 minutes, 40 seconds at 94 ° C, 40 seconds at 60 ° C and 1 minute at 72 ° C, and final expansion at 72 ° C. extention).

PCR products were analyzed on 2% agarose gel in TBE buffer and visualized by ethidium bromide staining. Gel images were captured with a DP001-IPC device (Marne-La-Vallee, Vilber Lourmat, France).

6. Fluorescence imaging  ( nuclear staining )

HeLa pLuc705 cells were re-plated at 5 × 10 ³ cells / well in 96-well plates. The next day, the cells were cultured for one day by transfection of the Fam-PNA / PEI (25kD) complex with the culture. After incubation, the cells were washed twice with PBS, and then fixed with 10% formalin for 5 minutes. After washing with PBS again, the nuclei were stained with 10 μg / ml Hoechst 33342 for 5 minutes in the dark. Stained cell images were captured by fluorescence microscope (IX51) equipped with DP Controller (Olympus Optical, Tokyo).

7. Confocal imaging

Exponentially expanded cells were plated on 8-well Lab-Tek chambered coverglass (Nunc) at 5 × 10 ⁴ cells / well density the day before transfection. For PNA / PEI transfection 1 uM Fam-PNA was prepared in 100 ul / well DMEM culture with or without PEI, treated and cultured in cells.

After 4 hours of incubation, cells were supplemented with 100 μl / well of fresh culture (DMEM with 10% FBS) and then incubated for another 16 hours. The following day, cells were washed twice with PBS and fixed with 10% formalin for 5 minutes, and then stained with 10 μg / ml Hoechst 33342.

Cells on the slides were fixed with a mounting solution (Burlingame, Vectashield mounting medium for fluorescence, Vectro Lab. Cat no.H1400, USA) and covered with a cover glass. Cells using a Deltavison RT confocal system equipped with an argon laser (excitation and absorbance wavelengths, 528 nm and UV, respectively; Applied Precision, Issaquah, WA, USA) connected to an Olympus IX71microscope (oil immersion 60 × 1.4 NA objective; Olympus) The images were analyzed. An image software package (Applied Precision) was used for image acquisition and processing.

8. Cell Viability Assay Cell viability assays )

MTT assay was used to assess cell viability. HeLa pLuc 705 cells were seeded in 96-well plates at a density of 1.2 × 10 ⁴ cells / well and incubated for one day. After 24 hours of incubation, free PNA, oligo-aspartic acid-PNA-PEI / Lipofectamine complex, cationic carrier PEI, and Lipofectamine were treated with cells and cultured at 20 ° C. for one day.

After incubation, 2 mg / ml MTT solution was added. Cells were then incubated at 37 ° C. for 4 hours, MTT-containing medium was removed, and DMSO added to dissolve formazan crystals formed by living cells. Absorbance was measured at 570 nm using a microplate reader (VersaMax, Molecular Devices, Sunnyvale, California, USA).

< Example  1>

Oligo-aspartic acid ( Asp ) - PNA  And PNA Of PEI  Composite fabrication

As the PNA sequence, CCTCCTACCTCAGTTACA (SEQ ID NO: 1) was used. The PNA was used to screen luciferase expression of PNA delivered for splicing correction in HeLa pLuc 705 cells.

Oligo-aspartic acid-PNA [Asp (n) -PNA] was devised that is complementary to the aberrant splice site in the luciferase reporter gene. Oligo-aspartic acid was conjugated to the end of the PNA sequence.

As a result, the binding of oligo-aspartic acid and PNA improved the poorly soluble solubility of PNA and allowed the formation of a complex with a cationic carrier. That is, the carboxyl anion of the oligo-aspartic acid enabled the interaction between charges between the PNA and the cation carrier.

That is, Asp (n) -PNA of the present invention forms a complex with PEI or Lipofectamine.

< Example  2>

Oligo-aspartic acid- PNA Of PEI  By complex Luciferase  Upregulation of expression ( Upregulating )

Since luciferase activity is observed in the splicing correction, this luciferase activity measurement is indicative of functional activity by the delivered PNA. Data is shown in micrograms of whole cell protein per well.

free PNA and Asp (n) -PNA / PEI complexes were mixed with DMEM and added to HeLa Luc705 cells. Carriers and conditions were examined at various PEI concentration ratios (2kD, 25kD) for transfection with Asp ₍₁₂₎ -PNA.

As a result, as shown in FIG. 1, the high molecular weight (MW) PEI polymer showed the excellent transfection effect. This means that transfection efficiency increases with increasing PEI size (MW). However, if MW is increased to increase transfection efficiency, there is a problem of increasing cytotoxicity. Thus, taking this into account, we experimented with 25 kD PEI for a balance between delivery efficiency and cytotoxicity.

As shown in FIG. 1, luciferase activity was dose dependent for Asp ₍₁₂₎ -PNA / 25kD PEI complex and no change for 2kD PEI (low molecular weight).

In addition, luciferase activity was dependent on the number of aspartic acid conjugated. 2 clearly shows the relationship between aspartic acid and PEI polymers.

That is, the number of aspartic acid affected luciferase activity by increasing the interaction with the PEI polymer. It can be seen that not only the amount of PEI but also the number of bound aspartic acid promotes luciferase activity.

In addition, it was observed that the Asp (n) -PNA / PEI complex of the present invention is effective in the sub-nanomolar range (FIG. 3). However, this was not the case with free PNA and Asp (n) -PNA. Lipofectamine complexes also worked in the sub-nanomolar range.

Asp ₍₁₂₎ -PNA markedly enhanced luciferase activity by binding PEI to Lipofectamine. Asp ₍₄₎ , ₍₈₎ -PNA also enhanced luciferase activity by binding to high concentrations of PEI or Lipofectamine. In particular, the Asp ₍₁₂₎ -PNA / Lipofectamine complex showed a surprising transfection efficiency at 5 nM.

These results show that both PNA modification and cationic carriers are important. Transfection efficiency was dependent on the amount of aspartic acid bound to the amount of PEI. In other words, the negative charge of Asp influences the formation of the complex between PEI and Asp (n) -PNA, and the complex has a decisive effect on luciferase activity.

< Example  3>

Oligo-aspartic acid- PNA Of PEI  By complex Splicing  correction

Increased luciferase activity directly reflected the level of calibrated luciferase mRNA. RT-PCR analysis was used to identify quantitative differences in correctly spliced mRNA.

RT-PCR results are shown in FIG. 5.

Luciferase mRNA bands corrected by Asp (n) -PNA / PEI complex were identified. The corrected mRNA bands show the response of the PNA to the abnormal splicing position of the luciferase pre-mRNA. This means a relative increase in luciferase activity during PNA transfection.

< Example  4>

Oligo-aspartic acid- PNA Of PEI  Cellular uptake of the complex ( Cellular uptake )

Absorption of the Asp (n) -PNA / PEI complex was confirmed by a fluorescence microscope capable of recognizing the fluorescently labeled Asp ₍₁₂₎ -PNA / PEI complex (Asp (12) -PNA-Fam / PEI complex).

As shown in FIG. 5, the PNA fluorescence signal was not observed in free PNA-Fam, whereas Asp (12) -PNA-Fam / PEI complexes showed visible cell uptake, and cytosol and nuclear nuclei ( nuclear fraction). The PNA fluorescence signal is a clear evidence of this cell uptake, but the method could not confirm the location of the cellular fluorescence signal in the cell.

However, fluorescence emission sites in Asp ₍₁₂₎ -PNA-Fam cells were observed by confocal fluorescence microscopy. The PNA fluorescence signal was located in the cell nucleus (FIG. 6), indicating that PNA was delivered to the nucleus.

And delivery of PNA reaffirmed the dose-dependent effect of PEI (FIG. 6). The fluorescence data provided by confocal studies showed that the PEI polymer carried PNA into the nucleus. However, it was unclear whether Asp ₍₁₂₎ -PNA-Fam was present with PEI in the nucleus.

Nevertheless, the fluorescence data may explain the pathway of PNA uptake into the cell, and the modified mRNA band through splicing correction and the activity of luciferase increased by the absorbed PNA can also be described. have.

< Example  5>

PNA  And oligo-aspartic acid- PNA Of PEI  Cytotoxicity of the complex

PEI is very effective for polymer-based DNA and siRNA delivery, but is highly cytotoxic and molecular weight (MW) dependent.

MTT assays were used to determine the cytotoxicity of PNA and Asp (n) -PNA / PEI complexes. And PEI and Lipofectamine results were compared in HeLa pLuc 705 cells to optimize the range of cell delivery for toxicity (FIG. 7).

The Asp (n) -PNA / PEI complex showed higher toxicity than the free PNA and Asp (n) -PNA / Lipofectamine complexes. This result is caused by the toxicity of PEI.

Comparison of PEI and Lipofectamine showed that PEI was much more toxic, and the higher the concentration of PEI, the more toxic it was (FIG. 7 b). It is the cytotoxicity of PEI itself that cationic polymers have by having a high cationic charge density. In addition, as shown in FIG. 4A, it was found that PNA was more toxic at higher concentrations. That is, PNA and Asp (n) -PNA / PEI complexes are cytotoxic due to high concentrations of PEI.

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

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Claims (12)

PNA delivery complex consisting of a negatively charged oligo-aspartic acid (Asp) -PNA and a cationic carrier. The method of claim 1,
The cationic carrier is polyethyleneimine (PEI) or lipofectamine (Lipofectamine), characterized in that the PNA delivery complex. The method of claim 1,
The oligo-aspartic acid (Asp) is a complex for PNA delivery, characterized in that forming a conjugate with PNA. 3. The method of claim 2,
The complex is composed of Asp (n) -PNA-PEI, wherein n is a complex for PNA delivery, characterized in that 5 to 12. 5. The method of claim 4,
The PEI complex for delivering PNA, characterized in that the molecular weight of 20 ~ 30kD. 3. The method of claim 2,
The complex is composed of Asp (n) -PNA-lipopeptamine, wherein n is a complex for PNA delivery, characterized in that 5 to 12. The method of claim 1,
The complex is a PNA delivery complex, characterized in that the splicing correction (splicing correction) action in the cell. The method of claim 1,
The complex is PNA delivery complex, characterized in that acting in the sub-nanomolar range. The method of claim 1,
Transfection efficiency of the complex is a complex for PNA delivery, characterized in that dependent on the number of oligo-aspartic acid (Asp) conjugated to the PNA. The method of claim 1,
The transfection efficiency of the complex is PNA delivery complex, characterized in that the capacity of the cation carrier. A method for genetically correcting an in vitro cell comprising introducing the complex for delivery of PNA according to claim 1 or 2 into a cell in vitro. 12. The method of claim 11,
The gene correction is a splicing correction of mRNA.

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