Patent Application
20210388029
The content of the electronically submitted sequence listing, file name: 4061-1006-C_SubSeqList20210903.txt; size: 9,000 bytes; and date of creation: Sep. 3, 2021, filed herewith, is incorporated herein by reference in its entirety.
The present invention relates to a cell membrane penetrating peptide and an intracellular delivery carrier including the same.
The nucleocapsid protein (hereinafter referred to as ‘NC’) of the human immunodeficiency virus (HIV) plays a functional role in the viral life cycle as well as the structural role for viral growth. The functions are as follows. First, NC peptides are involved in the genomic encapsulation of viruses. This function results from two zinc finger domains consisting of unique CCHC motifs. The domains are known to be highly conserved in all retroviruses and are essential for HIV RNA packaging and infectious virus production. Second, NC peptides are known to promote tRNA primer annealing and strand transfer during viral reverse transcription (RT), suggesting that NC peptides play an important role in viral replication. Third, NC peptides have the nucleic acid chaperone activity necessary for the viral life cycle. Recently, it has been reported that NC peptides play a predetermined role even when the viral DNA is inserted into the host cell chromosome.
Meanwhile, cell penetrating peptides (CPP) are cell membrane penetrating peptides composed of about 10-30 short peptides. Most of them are derived from protein-transduction domain or membrane-translocating sequence. Unlike the general intracellular entry pathway of foreign substances, CPP is known to be capable of transferring DNA and proteins that are known to be unable to pass through cell membranes into cells without damaging the cell membrane. The best-known peptides for CPP are the Tat peptides, which are derived from Human Immunodeficiency Virus (HIV) and are currently being used in a variety of applications, such as cell therapy and diagnostic reagents. In addition, Penetratin, derived from the DNA-binding domain of a homeodomain transcription factor, is also being used to deliver proteins useful to the human body to the skin. The transportan is a peptide made by fusing some of the peptides isolated from the venom of the wasp called mastoparan with a neuropeptide called galanin and is used as a cell death-inducing peptide by binding with a peptide inducing cell death.
The present inventors have confirmed that NC peptides have cell membrane penetration activity while studying the physiological activity of HIV NC peptides so that NC peptides can be used as a drug delivery carrier capable of delivering intracellular substances, thereby completing the present invention.
An object of the present invention is to provide a cell penetrating peptide including an amino acid sequence represented by the following Formula I:
in which Xaa1 is an amino acid selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn and Gln,
in which Xaa2 is an amino acid selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Arg, His, Lys, Asn, Ser, Thr and Gln,
in which Xaa3 and Xaa4 are individually an amino acid selected from the group consisting of Asp, Glu, Arg, His, Lys, Ser, Thr, Asn, Gln and Gly,
in which Xaa5 is an amino acid selected from the group consisting of Asp, Glu, Arg, His, Lys, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp and Pro,
in which Xaa6 and Xaa7 are individually an amino acid selected from the group consisting of Ser, Thr, Asn, Gln, Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp,
in which Xaa8 is an amino acid of Lys, Ala or Arg, and
in which Xaa9 is an amino acid selected from the group consisting of Asp, Glu, Ser, Thr, Asn and Gln.
Another object of the present invention is to provide an intracellular delivery carrier in which a cargo of an object to be delivered into a cell is conjugated with the end of the cell penetrating peptide.
Still another object of the present invention is to provide a method for delivering a cargo of an object to be delivered into a cell, the method including contacting the intracellular delivery carrier with a cell.
Yet another object of the present invention is to provide an intracellular delivery carrier in which a cargo of an object to be delivered into a cell is conjugated with the end of an NC peptide of a retrovirus.
Yet another object of the present invention is to provide a method for delivering a cargo of an object to be delivered into a cell, the method including contacting an intracellular delivery carrier in which a cargo of an object to be delivered into a cell is conjugated with the end of an NC peptide of retrovirus with a cell.
In order to achieve the objects, an aspect of the present invention provides a cell penetrating peptide including an amino acid sequence represented by the following Formula I:
in which Xaa1 is an amino acid selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn and Gln,
in which Xaa2 is an amino acid selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Arg, His, Lys, Asn, Ser, Thr and Gln,
in which Xaa3 and Xaa4 are individually an amino acid selected from the group consisting of Asp, Glu, Arg, His, Lys, Ser, Thr, Asn, Gln and Gly,
in which Xaa5 is an amino acid selected from the group consisting of Asp, Glu, Arg, His, Lys, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp and Pro,
in which Xaa6 and Xaa7 are individually an amino acid selected from the group consisting of Ser, Thr, Asn, Gln, Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp,
in which Xaa8 is an amino acid of Lys, Ala or Arg, and
in which Xaa9 is an amino acid selected from the group consisting of Asp, Glu, Ser, Thr, Asn and Gln.
Another aspect of the present invention provides an intracellular delivery carrier in which a cargo of an object to be delivered into a cell is conjugated with the end of the cell penetrating peptide.
As used herein, the term “intracellular delivery carrier” refers to a carrier capable of penetrating through the cell membrane and penetrating into the tissue.
As used herein, the term “peptide” or “polypeptide” refers to a linear molecule formed by linking amino acid residues together through peptide bonds and includes 4-70 amino acid residues, preferably 4-40 amino acid residues, more preferably 4-30 amino acid residues, and most preferably 4-20 amino acid residues.
The polypeptide represented by the above Formula I refers to the consensus sequence (Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-His-Xaa-Xaa-Xaa-Xaa-Cys, SEQ ID NO:24) of the zinc finger domain present in the NC peptide of various retroviruses (for example, human immunodeficiency virus (HIV), murine leukemia virus (MLU), simian immunodeficiency virus (SIV), rous sarcoma virus (RSV), Feline immunodeficiency virus (FIV), and equine immunodeficiency virus (EIV)). The present inventors have confirmed through experiments that the polypeptides including the above sequences are excellent in cell membrane penetration activity and that they can be used as a drug delivery system capable of delivering intracellular substances.
According to an embodiment of the present invention, the Xaa1 is an amino acid selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp as a hydrophobic amino acid and Ser, Thr, Asn and Gln as a polar amino acid. More preferably, the Xaa1 is an amino acid selected from the group consisting of Phe, Trp, Tyr, Ala and Gln.
According to an embodiment, the Xaa2 is an amino acid selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp as a hydrophobic amino acid, Arg, His and Lys as a basic amino acid and Ser, Thr, Asn and Gln as a polar amino acid. More preferably, the Xaa2 is an amino acid selected from the group consisting of Asn, Thr, Lys, Leu and Tyr.
Further, according to an embodiment, the Xaa3 and Xaa4 are individually an amino acid selected from the group consisting of Asp, Glu, Arg, His, Lys, Ser, Thr, Asn, Gln and Gly as a hydrophilic amino acid. More preferably, the Xaa3 is an amino acid selected from the group consisting of Gly, Asp and Lys. More preferably, the Xaa4 is an amino acid selected from the group consisting of Arg, Ser, Gly and Glu.
Further, according to an embodiment of the present invention, the Xaa5 is an amino acid selected from the group consisting of Asp, Glu, Arg, His, Lys, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp and Pro except for the polar amino acid. More preferably, the Xaa5 is an amino acid selected from the group consisting of Pro, Glu, Lys, Ile and Met.
Further, according to an embodiment, the Xaa6 and Xaa7 are individually an amino acid selected from the group consisting Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp as a hydrophobic amino acid and Ser, Thr, Asn and Gln as a polar amino acid. More preferably, the Xaa6 is an amino acid selected from the group consisting of Thr, Asn, Gln, Met, Tyr and Trp. More preferably, the Xaa7 is an amino acid selected from the group consisting of Ala, Met and Gln.
Further, according to an embodiment, the Xaa5 is an amino acid of Lys, Ala or Arg. The Xaa9 is an amino acid selected from the group consisting of Asp and Glu as an acidic amino acid and Ser, Thr, Asn and Gln as a polar amino acid. More preferably, the Xaa9 is Asp, Glu, Asn or Gln.
According to an embodiment of the present invention, the peptide including the amino acid sequence represented by the Formula I is a polypeptide including a zinc finger domain of a retrovirus, in which the polypeptide may be selected from the group consisting of polypeptide having the amino acid sequences represented by SEQ ID NO: 17 to SEQ ID NO: 23.
Another aspect of the present invention provides an intracellular delivery carrier in which a cargo of an object to be delivered into a cell is conjugated with the end of an NC peptide of a retrovirus.
As used herein, the term “NC peptide” refers to a polypeptide that constitutes the nucleocapsid of a retrovirus. It is known that the polypeptide binds strongly to the retroviral genomic RNA to form the ribonucleoprotein core complex.
As used herein, the term “cell penetrating peptide (CPP)” refers to a peptide having the ability to carry a cargo of an object to be delivered into a cell in vitro and/or in vivo. The cell penetrating peptide means a peptide having an amino acid sequence that can pass through the cell membrane of a phospholipid bilayer itself.
According to an embodiment of the present invention, the retrovirus may be selected from the group consisting of human immunodeficiency virus (HIV), murine leukemia virus (MLU), simian immunodeficiency virus (SIV) and rous sarcoma virus (RSV) and may preferably be HIV.
As used herein, the term “cargo” refers to a chemical substance, a small molecule, a polypeptide, a nucleic acid and the like, which can be conjugated with NC peptides that act as a cell membrane penetrating peptide to be carried into cells.
In the present invention, the substance that can be conjugated with the end of the NC peptide of retrovirus or the peptide represented by the Formula I, that is, a substance that can be a cargo, may be various. For example, the substance includes a protein (polypeptide), a nucleic acid (polynucleotide), a chemical substance (drug), and the like, but is not limited thereto.
For example, it can be a drug, a contrast agent (e.g., T1 contrast agent, T2 contrast agent such as a superparamagnetic substance, a radioisotope, etc.), a fluorescent marker, a dyeing agent, and the like, but is not limited thereto. The polypeptide is a polymer of amino acids composed of two or more residues and includes peptides and proteins. Polypeptides may be, for example, proteins that are involved in cell immortalization (e.g., SV40 large T antigen and telomerase), anti-apoptotic proteins (e.g., mutant p53 and BclxL), antibodies, cancer genes (e.g., ras, myc, HPV E6/E7 and adenoviridae Ela), cell cycle regulating proteins (e.g., cyclin and cyclin-dependent phosphorylase) or enzymes (e.g., green fluorescent protein, beta-galactosidase and chloramphenicol acetyltransferase), but is not limited thereto. Also, the nucleic acid may be, for example, RNA, DNA or cDNA, and the sequence of the nucleic acid may be an encoding site sequence or a non-coding site sequence (e.g., an antisense oligonucleotide or a siRNA). Nucleotides as nucleic acid cargo may be standard nucleotides (e.g., adenosine, cytosine, guanine, thymine, inosine and uracil) or analogs (e.g., phosphorothioate nucleotides). For example, the nucleic acid cargo may be an antisense sequence consisting of a phosphorothioate nucleotide or RNAi.
According to an embodiment of the present invention, the substance conjugated with the NC peptide or the peptide represented by the Formula I of the present invention penetrates the cell membrane with very high efficiency and remains in the cytoplasm and the nucleus in the cell.
According to an embodiment of the present invention, the NC peptide may have one or more zinc finger domains and preferably two zinc finger domains. According to an embodiment, the NC peptide may be selected from the group consisting of polypeptides having the amino acid sequences represented by SEQ ID NO: 12 to SEQ ID NO: 15.
Another aspect of the present invention provides a method for delivering a cargo of an object to be delivered into a cell, the method including contacting the intracellular delivery carrier in which a cargo of an object to be delivered into a cell is conjugated with the end of a peptide including an amino acid sequence represented by the Formula I or an intracellular delivery carrier in which a cargo of an object to be delivered into a cell is conjugated with the end of an NC peptide of retrovirus with a cell.
Since the methods of the present invention utilize intracellular delivery carriers, the description common to both is omitted in order to avoid the excessive complexity of the present application.
Meanwhile, when the peptide containing the amino acid sequence represented by the Formula I to which a cargo is conjugated or the NC peptide to which a cargo is conjugated contacts the cell membrane in vitro or in vivo, the cargoes conjugated with peptides are delivered into the cell. There is no particular requirement for the contact between the intracellular delivery carrier and the cell membrane, for example, a limited time, temperature and concentration. It can be carried out under the general conditions applicable to cell membrane penetration in the art.
The intracellular delivery carrier including the peptide including the amino acid sequence represented by the Formula I of the present invention or the intracellular delivery carrier including the NC peptide has an advantage of efficiently transferring substances into cells even at a low concentration thereof compared with the existing peptide derived from the virus.
Hereinafter, one or more embodiments are described in more detail by way of Examples. However, these Examples are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these Examples.
1. Construction of a Recombinant Vector for NC Peptide Expression
A recombinant vector was prepared as follows to confirm the expression of a recombinant protein conjugated with NC peptide and EGFP and to purify the protein. Polymerase chain reaction (PCR) was performed using a primer including restriction enzyme recognition sequences in order to add restriction enzyme recognition sequences at the N-terminus of the NC peptide gene to allow NdeI which is a restriction enzyme (New England Biolabs; NEB, USA) to act on and at the C-terminus of the NC peptide gene to allow BspEI to act on. The primer sequences (SEQ ID NO: 1 and SEQ ID NO: 2) used in PCR and the PCR conditions, respectively, are shown in Tables 1 and 2 below.
Next, in order to add restriction enzyme recognition sequences at the N-terminus of enhanced green fluorescent protein (EGFP) gene to allow BspEI to act on and at the C-terminus of the EGFP gene to allow HindIII to act on, PCR was carried out under the same conditions as in Table 2 as described above. However, the primers of SEQ ID NO: 3 and SEQ ID NO: 4 described in Table 3 below were used as the primers.
The NC peptide gene and the EGFP gene to which the restriction enzyme recognition sequence was added were reacted at 16° C. for 12 hours, resulting in the ligation. PCR was performed on the ligated NC peptide-EGFP gene (hereinafter referred to as ‘NC-EGFP gene’) (using primers of SEQ ID NOS: 1, 2, 3 and 4) to obtain the entire sequence of NC-EFGP gene. The conditions of NC-EGFP gene ligation reaction are shown in Table 4 below.
)
)
Then, pET21a (Novagen, USA) vector was cut with NdeI and HindIII restriction enzymes. Then, vector fragments were isolated according to the manufacturer's protocol using a PCR purification kit (Qiagen, USA). The restriction enzyme reaction was performed using NEBuffer #2 at 37° C. for 2 hours. The isolated pET21a vector fragment was ligated with the NC-EGFP gene, and then the recombination vector was isolated using the PCR Purification Kit. The recombinant vector into which the NC-EGFP gene was inserted was named pET21a NC-EGFP.
In order to replicate the pET21a NC-EGFP vector, the vector was transformed into E. coli DH5α and shake-cultured at 37° C. until the OD was 0.5 to 0.6 in LB liquid medium. After the culture was completed, the culture solution was centrifuged to collect the E. coli pellet, and the pET21a NC-EGFP vector was isolated from the E. coli pellet collected according to the manufacturer's protocol using a plasmid extraction kit (Qiagen). The isolated pET21a NC-EGFP vector was identified by quantifying the concentration using the UV method.
2. Expression, Isolation and Purification of NC-EGFP
In order to confirm the protein expression of the pET21a NC-EGFP vector prepared as described above, the following experiment was conducted.
PET21a NC-EGFP vector was transformed into BL21 (DE3) (Thermo Fisher, USA), plated on LB plate and cultured at 37° C. for 12 hours. Colonies formed after 12 hours were inoculated into LB liquid medium and further cultured at 37° C. After about 12 hours, the culture solution having reached an OD of 0.5 to 0.6 was inoculated into 250 ml of LB liquid medium and cultured at 37° C. for 3 hours to 4 hours to have an OD of 0.5 to 0.6. When the OD of the culture solution reached 0.5 to 0.6, 0.5 mM isopropyl β-D-thiogalactoside (IPTG) was added to the culture solution. Then, the culture was performed at 25° C. for 24 hours. After 24 hours, the culture solution was centrifuged to obtain BL21 pellet. The obtained pellet was suspended in a dissolution buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole and pH 8.0), and then the E. coli was disrupted (amplitude 40%) using an ultrasonic wave crusher.
The E. coli debris was centrifuged and separated into supernatant and precipitate, and the supernatant was filled into a tube using a 0.45 μm filter. The supernatant filled in the tube was placed in a column packed with Ni-NTA (nitrilotriacetic acid) resin to bind the protein and the resin to each other. In order to remove foreign proteins that did not bind to the resin, the resin was then washed with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole and pH 8.0). The final protein was obtained in a gradient mobile phase using an imidazole-added buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole and pH 8.0). Next, in order to remove imidazole, the obtained protein was placed in a membrane tube, and buffer exchange by osmotic action was performed using a buffer (20 mM NaH2PO4, 300 mM NaCl and pH 7.2) not containing imidazole. Finally, the concentration of the protein dissolved in the buffer not containing imidazole was measured by Bradford assay to identify NC-EGFP protein expression. The results confirmed that even if other sequences were conjugated with the NC gene sequence, it did not affect the NC peptide expression.
3. Analysis of Cell Membrane Penetration Activity of NC Peptides
3-1. Cell Culture
HeLa, RAW 264.7 and 293 FT cells were cultured in DMEM medium supplemented with 10% FBS and 100 U/ml penicillin/streptomycin. MDCK cells were cultured in EMEM medium supplemented with 10% FBS and 100 U/ml penicillin/streptomycin (Hyclone, Logan, Utah, USA). THP-1 and CEM cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 100 U/ml penicillin/streptomycin (Hyclone, Logan, Utah, USA). All cells were cultured in a humidified thermostat in 5% CO2 at 37 C.
3-2. Analysis of Cell Membrane Penetration Activity of NC Peptide—Fluorescence-Activated Cell Sorting (FACS)
Various types of cells cultured in the Example 3-1 as described above were seeded into 6-well plates at a density of 1×106 cells/well, and cultured for 24 hours to attach the cells to the plates. Then, cells were treated with the FITC-conjugated NC peptide (hereinafter referred to as “FITC-NC peptide”), the EGFP-conjugated NC peptide (hereinafter referred to as “NC-EGFP peptide”) and the EGFP-conjugated Tat peptide (hereinafter referred to as “Tat-EGFP peptide”), respectively, at different peptide concentrations (0.5, 1.0, 2.5, 5.0 and 10 μM) or different peptide treatment time. The peptides were prepared by chempeptide (China) and Life tein (USA). The cells were then washed three times with PBS, and the cells were separated with trypsin-EDTA and suspended in PBS containing 2% FBS/0.1% bovine serum albumin (BSA). The suspension was centrifuged at 2,000 rpm for 2 minutes to obtain cell pellets, and the cells obtained were fixed with 3.7% formaldehyde for 20 minutes. Then, cell pellets were obtained by centrifugation at 2,000 rpm for 2 minutes, washed twice with PBS and then suspended in PBS containing 2% FBS and 0.1% BSA. Cells were analyzed by flow cytometry using a fluorescence activated cell sorter (FACS Calibur, Beckon Dickinson, Calif., USA).
3-3. Analysis of Cell Membrane Penetration Activity of NC Peptide—Immunostaining
HeLa cells were seeded into a 12-well plate having glass at a density of 1×105 cells/well, and then cultured for 24 hours to attach the cells to the glass. Then, cells were treated with FITC-NC peptides at 3 mM concentration for 3 hours. After 3 hours, the cells were washed three times with PBS, the cells were fixed with 3.7% formaldehyde for 20 minutes, and the cells were treated with PBS containing 0.2% Triton X-100 to increase the cell membrane penetration activity. Then, the cells were blocked with 3% BSA for 1 hour, reacted with lamin A/C antibody (Sigma-Aldrich, USA) at room temperature for 2 hours, and washed three times with PBS. Next, the cells were treated with Cy3-conjugated secondary antibody (Jackson ImmunoResearch, USA) at room temperature for 1 hour, washed twice with PBS, and then stained with DAPI (4′,6-diamidino-2-phenylindol) for 10 minutes. The HeLa cell-attached glass was removed and placed on a slide glass. Then, the cells were observed with a confocal laser scanning microscope (LSM 700, Zeiss, Germany).
In addition, the following method was used to compare the penetration activity of NC peptides with other cell penetrating peptides.
The HeLa cells were seeded into a 12-well plate containing glass at a density of 1×105 cells/well, and then cultured for 24 hours to attach the cells to the glass. Thereafter, the FITC-NC peptide, the trans-activating transcriptional activator peptide (FITC-Tat peptide), the FITC-MA11 peptide, the protein transduction domain-ys peptide (FITC-PTD-ys peptide), the translocation motif peptide (FITC-TLM peptide) and FITC-TD1 peptide were treated with the HeLa cells for 3 hours. After 3 hours, the cells were washed 3 times with PBS and the cells were fixed with 3.7% formaldehyde for 20 minutes. The cells were treated with PBS containing 0.2% Triton X-100 to increase cell membrane penetration activity and blocked with 3% BSA for 1 hour. Then, the cells were reacted with tubulin antibody (Santa Cruz Biotechnology, USA) at room temperature for 2 hours and washed three times with PBS. The cells were treated with Cy3 secondary antibody. They were reacted at room temperature for 1 hour, washed twice with PBS, and stained with DAPI for 10 minutes. The HeLa cell-attached glass was removed and placed on a slide glass. Then, the cells were observed with a confocal laser scanning microscope.
Further, in order to identify a change in cell membrane penetration activity by FITC-NC peptide, the HeLa cells were treated with hexapeptide, FITC-hexapeptide, FITC-NC-hexapeptide, FITC-13NC35 peptide and FITC-29NC50 peptide for 3 hours in the same manner as above and observed with a confocal laser scanning microscope. Meanwhile, the amino acid sequences of the hexapeptide, PTD-ys peptide, TLM peptide, TD1 peptide, NC-hexapeptide, 13NC35 peptide and 29NC50 peptide used are shown in Table 5 below.
3-4. Analysis of Cell Membrane Penetration Activity of NC Peptide—Fluorometer
RAW264.7 cells were seeded into 24-well plates at a density of 5×104 cells/well and cultured for 24 hours to attach the cells to the plates. Then, each of the Tat-EGFP peptide and the NC-EGFP peptide was treated at different concentrations for 1 hour. After 1 hour, the cells were washed three times with PBS, and 100 ml of radio-immunoprecipitation assay (RIPA) buffer was added to dissolve the cells at 4° C. for 30 minutes. 100 ml of the cell lysate was transferred to a 96-well plate for fluorescence analysis, and GFP fluorescence was measured using a fluorescence analyzer (Synergy MX, BIOTEK, USA).
Further, NC peptides of FITC-conjugated Human immunodeficiency virus (HIV), Murine leukemia virus (MLU), Simian immunodeficiency virus (SIV) and RSV (Rous sarcoma virus) (hereinafter referred to as FITC-HIV-NC, FITC-MLUNC, FITC—SIV—NC and FITC—RSV-NC, respectively) and Tat peptide were treated with RAW264.7 cells at a concentration of 1.0 μM for 1 hour. Then, the fluorescence signal of the cells was measured.
3-5. Change in RNA Transduction Efficiency by NC Peptide
MT4 cells were seeded into 24-well plates at a density of 2×105 cells/well and cultured in a humidified thermostat in 5% CO2 at 37° C. for 24 hours. 2 μl of NC peptides and 40 nM siGLO (Green transfection indicator, Dharmacon, D-001630-01-05) having different concentrations were mixed and reacted at room temperature for 30 minutes and then treated to MT4 cells. After further culture for 24 hours, MT4 cells were washed three times with PBS and analyzed by flow cytometry using a fluorescence activated cell sorter. Further, the cells were observed with a fluorescence inverted microscope (Olympus).
4. In Vivo Tissue Penetration of NC Peptide and Distribution of NC Peptide in Tissue Cells
The FITC-HIV-NC peptide and FITC-Tat peptide having a concentration of 3.2 mg/100 μl, respectively, were injected into mouse tail vein using a 30 gauge syringe. The kidney, liver, lung, heart and spleen of the mice were removed 2 hours after the injection of the peptide and fixed with 4% paraformaldehyde for 1 hour. They were placed in 30% sucrose until the tissue was subsided. Then, each tissue was frozen using an OCT compound (Leica, Germany), and a tissue section slide was prepared with a freezing sectional device. Tissue section slides were stained with DAPI for 10 minutes according to methods known in the art and observed with a confocal laser scanning microscope.
In addition, 1 μl of a 100 μM stock of FITC-HIV-NC peptide was intravitreally injected, and 2 μl of a 100 μM stock of FITC-HIV-NC peptide was subretinally injected. After 24 hours, mouse eye tissue was extracted. Eye tissue section slides were prepared in the same manner as described above. Slides were stained with DAPI for 10 min and observed with a confocal laser scanning microscope.
Experiment Result
1. Identification of Cell Membrane Penetration Activity of Various Retroviral NC Peptides
In order to confirm whether the degree of cell membrane penetration activity was varied depending on the degree of similarity of NC peptide sequence, RAW264.7 Cells were treated with 1.0 μM FITC-conjugated NC peptide of HIV, SIV, RSV and MLV and Tat peptide for 1 hour. As a result, as shown in
Meanwhile, the amino acid sequences of the NC peptides of HIV, SIV, RSV and MLV used above are shown in Table 6 below.
2. Identification of Cell Membrane Penetration Activity of HIV-NC Peptide
In order to examine the cell membrane penetration activity of HIV-NC peptide (SEQ ID NO: 12) having the highest cell membrane penetration activity in the above experiment according to its concentrations, RAW264.7 macrophages were treated with FITC-HIV-NC peptide at a concentration of 0.5, 1, 2.5, 5.0 and 10 μM for 1 hour. The fluorescence intensity of the cells was measured using a fluorescence microscope (Leica Micoscope Systems, Germany). As shown in
Further, in order to confirm the degree of cell membrane penetration of FITC-HIV-NC peptides according to the treatment time, the peptides were treated with RAW 264.7 cells at a concentration of 2.5 μm for 20 minutes, 40 minutes and 60 minutes. As a result, it was confirmed by fluorescence microscopy and FACS analysis that as shown in
In order to verify the cell membrane penetration activity of HIV-NC peptides, HeLa cells were immunostained and observed with a confocal laser scanning microscope. As a result, it was confirmed that as shown in
3. Identification of Cell Membrane Penetration Activity of HIV-NC Peptides in Various Cells
In order to identify whether the cell membrane penetration activity of HIV-NC peptide varies depending on kinds of cells, RAW264.7, THP-1, CEM, MDCK and 293FT cells were treated with FITC-HIV-NC peptide at a concentration of 1.0, 2.5 and 5.0 μM for 1 hour or 2 hours. As a result, it was confirmed by fluorescence microscopy and FACS analysis that as shown in
4. Comparison of Cell Membrane Penetration Activity Between HIV-NC and Tat Peptides
In order to confirm the degree of cell membrane penetration activity of HIV-NC peptides, a comparative experiment carried out with a Tat peptide known as a conventional cell penetrating protein. RAW264.7 cells were treated with green fluorescent protein (GFP)-conjugated HIV-NC peptide (hereinafter referred to as GFP-HIV-NC peptide) at 0.5, 1.0, 2.5 and 5.0 μM and GFP-conjugated Tat peptide at 1.0, 2.5, 5.0 and 10 μM, respectively, for 1 hour. After 1 hour, the fluorescence of the peptides was confirmed. As a result, it was confirmed that as shown in
Further, as a result of the fluorescence analysis, it was confirmed that as shown in
5. Comparison of Cell Membrane Penetration Activity of HIV-NC Peptides and Other Cell Penetrating Peptides
The HeLa cells were treated with FITC-HIV-NC peptides, FITC-Tat peptides, FITC-MA11 peptides, FITC-PTD-ys peptides, FITC-TLM peptides and FITC-TD1 peptides, and then the penetration activity of each peptide was confirmed.
As a result, it was confirmed that as shown in
6. Identification of the Cell Membrane Penetration Activity of Zinc Finger Domains in NC Peptides
Experimental result 1 confirmed that several retroviral NC peptides had cell membrane penetration activity. As shown in
In order to confirm whether the cell membrane penetration activity is caused by the zinc finger domain which is a consensus sequence in several NC peptides, FITC was conjugated with a peptide containing the first zinc finger domain consisting of the 13th amino acid to the 35th amino acid of the HIV-NC peptide (13NC35 peptide, SEQ ID NO: 10), and, FITC was conjugated with a peptide containing the second zinc finger domain consisting of the 29th amino acid to the 50th amino acid of the HIV-NC peptide (29NC50 peptide, SEQ ID NO: 11) so that the cell membrane penetration activity was confirmed in HeLa cells, respectively.
As a result, it was confirmed that as shown in
Meanwhile, the amino acid sequences of the zinc finger domains of NC peptides of HIV, SIV, RSV and MLV are shown in Table 7 below.
7. In Vivo Tissue Penetration of HIV-NC Peptide and its Distribution in Tissue Cells
In order to confirm whether the NC peptides penetrate into tissues in vivo and are uniformly distributed in tissue cells, HIV-NC peptides were injected to the mouse tail vein. As a result, it was confirmed that as shown in
Further, HIV-NC peptides were injected through the intravitreal and subretinal injection routes. As a result, it was confirmed that as shown in
8. Confirmation of Cell Membrane Penetration Activity of Polypeptide by HIV-NC Peptide
In order to confirm whether HIV-NC peptides using a polypeptide as a cargo can have cell membrane penetration activity, hexapeptide was conjugated to HIV-NC peptide, and cells were treated with the peptides to observe the fluorescence signal. As a result, it was confirmed that as shown in
9. Confirmation of Cell Membrane Penetration Activity of Polynucleotide by HIV-NC Peptide
In order to confirm whether HIV-NC peptides using a polynucleotide as a cargo can have cell membrane penetration activity, siGLO RNA was conjugated to HIV-NC peptide, and cells were treated with the peptides to observe the fluorescence signal. As a result, it was confirmed that as shown in
Hereinabove, the present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that the present invention may be carried out as modified embodiments without departing from the spirit and scope of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. It should be construed that 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 included in the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2016-0172548 | Dec 2016 | KR | national |
| 10-2017-0173642 | Dec 2017 | KR | national |
This application is a continuation of U.S. application Ser. No. 16/469,632, which is a 371 of PCT/KR2017/014897, filed on Dec. 15, 2017 which claims the benefit of Korean Patent Application No. 10-2016-0172548, filed Dec. 16, 2016 and Korean Patent Application No. 10-2017-0173642, filed Dec. 15, 2017, the contents of each of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16469632 | Jun 2019 | US |
| Child | 17303250 | US |
