KR102713418B1 - Cell membrane penetrating peptide and intracellular delivery carrier comprising thereof - Google Patents

Abstract

The present invention relates to a cell membrane penetrating peptide and an intracellular delivery vehicle comprising the same. The intracellular delivery vehicle of the present invention has an advantage in that it can efficiently deliver a substance into a cell even at a low concentration, compared to a cell penetrating peptide derived from a virus.

Description

{CELL MEMBRANE PENETRATING PEPTIDE AND INTRACELLULAR DELIVERY CARRIER COMPRISING THEREOF}

The present invention relates to a cell membrane penetrating peptide and an intracellular delivery vehicle comprising the same.

The nucleocapsid protein (hereinafter referred to as 'NC') of HIV (human immunodeficiency virus) plays an important functional role in the viral life cycle as well as a structural role in viral entity formation. The functions are as follows. First, the NC peptide is involved in the genomic encapsidation of the virus. This function is derived from two zinc finger domains consisting of a unique CCHC motif. The domains are highly conserved in all retroviruses and are known to be essential for HIV RNA packaging and infectious virus production. Second, the NC peptide is known to promote tRNA primer annealing and strand transfer during the reverse transcription (RT) reaction of the virus, which suggests that the NC peptide plays an important function in viral replication. Third, the NC peptide has a nucleic acid chaperone activity necessary for the viral life cycle. Recently, it has been reported that NC peptides also play a role when viral DNA is inserted into the host cell chromosome.

Meanwhile, Cell Penetrating Peptides (CPPs) are membrane-penetrating peptides composed of about 10-30 short peptides, and are mostly derived from protein-transduction domains or membrane-translocating sequences. Unlike the general intracellular influx pathways of external substances, CPPs are known to move into cells without damaging the cell membrane, and can even deliver DNA and proteins, which are known to not pass through the cell membrane, into cells. The best-known peptide as CPP is the Tat peptide, which is derived from the human immunodeficiency virus (HIV) and is currently being used in various applications such as cell therapy and diagnostic reagents. In addition, penetratin, derived from the DNA-binding domain of the homeodomain transcription factor, is used to deliver useful proteins to the skin. Transportan is a peptide created by fusing a neuropeptide called galanin and a peptide isolated from wasp venom called mastoparan. It is used as a cell death-inducing peptide by combining it with a peptide that induces cell death.

While studying the physiological activity of the HIV NC peptide, the present inventors confirmed that a variant of the linker peptide among the NC peptides has cell membrane penetration activity, thereby confirming that it can be used as a drug delivery system capable of intracellular substance delivery, thereby completing the present invention.

KR 10-1964863 B1

One aspect of the present invention is to provide a cell-penetrating composition comprising a polypeptide in which the amino acid sequence represented by SEQ ID NO: 3 (ACP5: ARAPRKKG) is repeated one to seven times.

Another aspect of the present invention is to provide an intracellular delivery vehicle comprising a polypeptide in which the amino acid sequence represented by SEQ ID NO: 3 (ACP5: ARAPRKKG) is repeated 1 to 7 times, and a cargo to be transported into the cell is linked to the N-terminus or C-terminus of the polypeptide.

Another aspect of the present invention is to provide a composition for cargo delivery into cells or tissues, comprising the intracellular delivery agent as an effective ingredient.

One aspect of the present invention provides a cell-penetrating composition comprising a polypeptide in which the amino acid sequence represented by SEQ ID NO: 3 (ACP5: ARAPRKKG) is repeated one to seven times.

The term "peptide" or "polypeptide", as used herein, means a linear molecule formed by amino acid residues being linked to each other by peptide bonds, and consisting of 4 to 70 amino acid residues, preferably 4 to 40, more preferably 4 to 30, and most preferably 4 to 20.

The polypeptide represented by the above sequence number 3 represents the addition of alanine to the linker domain present in the NC peptide of HIV (Human immunodeficiency virus) (SEQ ID NO: 3). The inventors of the present invention confirmed through experiments that the polypeptide including the above sequence has excellent cell membrane permeability, and through this, confirmed that it can be used as a drug delivery system capable of intracellular substance delivery.

In the present invention, the amino acid sequence represented by the above SEQ ID NO: 3 is not only an amino acid sequence represented by the SEQ ID NO: 3, but also an amino acid sequence having 40% or more, preferably 60% or more, more preferably 80% or more, still more preferably 90% or more, still more preferably 95% or more, and most preferably 99% or more homology with the above sequence, and if it is an amino acid sequence that substantially exhibits the effect of improving solubility, it includes without limitation, and if it is an amino acid sequence having such homology and having a biological activity substantially identical to or corresponding to the amino acid of SEQ ID NO: 3, it is obvious that even if it has an amino acid sequence in which a part of the sequence is deleted, modified, substituted, or added, it is also included in the scope of the present invention.

In one specific embodiment of the present invention, the polypeptide may be one in which the amino acid sequence represented by SEQ ID NO: 3 is repeated one to seven times, specifically once or twice, and more specifically, may be the amino acid sequence represented by SEQ ID NO: 3 or the amino acid sequence represented by SEQ ID NO: 2. As described above, the present inventors not only confirmed the cell membrane permeability of a polypeptide including the amino acid sequence represented by SEQ ID NO: 3, but also confirmed that a polypeptide including an amino acid sequence (SEQ ID NO: 2) in which the amino acid sequence represented by SEQ ID NO: 3 is repeated twice also has excellent cell membrane permeability.

The above "NC peptide" refers to a polypeptide that constitutes the nucleocapsid of a retrovirus, and the polypeptide is known to strongly bind to the genomic RNA of the retrovirus to form a ribonucleoprotein core complex.

As used herein, the term "cell penetrating peptide (CPP)" refers to a peptide having the ability to transport a cargo of a transport target into a cell in vitro and/or in vivo , and a cell penetrating peptide is a peptide having an amino acid sequence that can pass through the cell membrane of the phospholipid bilayer membrane itself.

Another aspect of the present invention provides an intracellular delivery agent comprising a polypeptide in which an amino acid sequence represented by SEQ ID NO: 3 (ACP5: ARAPRKKG) is repeated 1 to 7 times, and a cargo to be transported into the cell is linked to the N-terminus or C-terminus of the polypeptide.

In one specific embodiment of the present invention, the polypeptide may be one in which the amino acid sequence represented by SEQ ID NO: 3 is repeated one to seven times, specifically one or two times, and more specifically, may be the amino acid sequence represented by SEQ ID NO: 3 or the amino acid sequence represented by SEQ ID NO: 2.

As used herein, the term “intracellular delivery vehicle” refers to a delivery vehicle that can penetrate cell membranes and enter tissues.

According to one specific example of the present invention, the cargo of the intracellular transport target may be a chemical substance, a polypeptide, a nucleic acid, a carbohydrate, or a lipid.

As used herein, the term "cargo" refers to a chemical, small molecule, polypeptide, nucleic acid, etc. that can be transported into a cell by binding to an NC peptide that acts as a cell membrane permeable peptide.

The substances that can be bound to the polypeptide of the present invention, that is, substances that can be cargo, are diverse and include, but are not limited to, proteins (polypeptides), nucleic acids (polynucleotides), chemical substances (drugs), etc.

For example, but not limited to, drugs, contrast agents (e.g., T1 contrast agents, T2 contrast agents such as superparamagnetic substances, radioisotopes, etc.), fluorescent markers, dyes, etc. The polypeptide is a polymer of amino acids consisting of two or more residues and includes peptides and proteins. The polypeptide can be, but is not limited to, proteins involved in cell immortality (e.g., SV40 Large T antigen and telomerase), anti-apoptotic proteins (e.g., mutant p53 and BclxL), antibodies, oncogenes (e.g., ras, myc, HPV E6/E7 and adenovirus Ela), cell cycle regulatory proteins (e.g., cyclins and cyclin-dependent kinases) or enzymes (e.g., green fluorescent protein, beta-galactosidase and chloramphenicol acetyltransferase). Additionally, the nucleic acid can be, for example, RNA, DNA or cDNA, and the sequence of the nucleic acid can be a coding region sequence or a noncoding region sequence (for example, an antisense oligonucleotide or siRNA). The nucleotides as the nucleic acid cargo can be standard nucleotides (for example, adenosine, cytosine, guanine, thymine, inosine and uracil) or analogs (for example, phosphorothioate nucleotides). For example, the nucleic acid cargo can be an antisense sequence or RNAi composed of phosphorothioate nucleotides.

According to one specific example of the present invention, a substance bound to a polypeptide in which the amino acid sequence represented by SEQ ID NO: 3 (ACP5: ARAPRKKG) is repeated 1 to 7 times penetrates the cell membrane with very high efficiency and remains in the cytoplasm and nucleus within the cell.

Another aspect of the present invention provides a composition for cargo delivery into a cell or tissue, comprising the intracellular delivery agent as an active ingredient.

According to one specific example of the present invention, the cargo to be transported within the cell may be a chemical substance, a polypeptide, a nucleic acid, a carbohydrate or a lipid.

Another aspect of the present invention provides a method for delivering a cargo of an intracellular transport target into a cell or tissue, comprising the step of contacting an intracellular transporter, in which a cargo of an intracellular transport target is linked to a polypeptide terminus in which an amino acid sequence represented by the above SEQ ID NO: 3 (ACP5: ARAPRKKG) is repeated 1 to 7 times, with the cell or tissue.

In one specific embodiment of the present invention, the polypeptide may be one in which the amino acid sequence represented by SEQ ID NO: 3 is repeated one to seven times, specifically one or two times, and more specifically, may be the amino acid sequence represented by SEQ ID NO: 3 or the amino acid sequence represented by SEQ ID NO: 2.

Since the method of the present invention utilizes the intracellular transporters, the common content between the two is omitted to avoid excessive complexity of this specification.

Meanwhile, when a polypeptide having an amino acid sequence represented by SEQ ID NO: 3 (ACP5: ARAPRKKG) repeated 1 to 7 times, to which a cargo is bound, is given an opportunity to contact a cell membrane in vitro or in vivo, the cargo bound to the polypeptide is transported into the cell. There are no conditions specifically required for the contact between the intracellular transporter and the cell membrane, such as limited time, temperature, and concentration, and the contact can be carried out under general conditions applied to cell membrane penetration in the art.

The intracellular delivery vehicle of the present invention has the advantage of being able to efficiently deliver substances into cells even at low concentrations, compared to peptides derived from existing viruses.

Figure 1 shows the results comparing the cell membrane permeation activities of FITC-ACP peptide, FITC-ACP4 peptide, FITC-ACP5 peptide, and FITC-Tat peptide.
Figure 2 shows the results showing the skin tissue penetration activity of ACP-FITC peptide, ACP4-FITC, ACP5-FITC, and Tat-FITC.

Below, one or more specific examples are described in more detail by way of examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

Experimental Example 1: Comparison of cell penetrating activity of cell penetrating peptides of the present invention

After treating HeLa cells with FITC-ACP peptide, FITC-ACP4 peptide, FITC-ACP5 peptide, and FITC-Tat peptide, the degree of cell membrane penetration of each peptide was confirmed.

Specifically, HeLa cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 100 U/㎖ penicillin/streptomycin. HeLa cells were cultured in a humidified incubator with 5% CO2 at 37℃. The cultured HeLa cells were seeded at a density of 1x10 ⁵ cells/well in a 12-well plate containing glass and cultured for 24 h. Then, the cells were treated with FITC-ACP peptide (3 μM, 3 h), FITC-ACP4 peptide (30 μM, 3 h /60 μM, 3 h), FITC-ACP5 peptide (60 μM, 2 h /120 μM, 2 h), and FITC-Tat peptide (60 μM, 3 h), and then the cells were washed three times with PBS. The washed cells were fixed with 3.7% formaldehyde for 20 min and treated with phosphate-buffered saline (PBS) containing 0.2% Triton X-100 to increase cell membrane permeability. Next, the cells were blocked with 3% BS for 1 h and reacted with tubulin antibody (Santa Cruz Biotechnology, sc-5286) at room temperature for 2 h, and then washed three times with PBS. After washing, the cells were treated with Cy3 secondary antibody and reacted for 1 h at room temperature, washed twice with PBS, and then stained with DAPI (4', 6-diamidino-2-phenylindol) for 10 min. The glass with the cells attached was removed, placed on a slide glass, and observed using a confocal laser scanning microscope (LSM 700, Zeiss, Germany). The sequences of the ACP peptide, ACP 4 peptide, and ACP5 peptide used in the experiment are as follows.

name Sequence number Amino acid sequence ACP peptide Sequence number 1 MQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQAN ACP4 peptide Sequence number 2 ARAPRKKGARAPRKKG ACP5 peptide Sequence number 3 ARAPRKKG

As a result, as shown in Fig. 1, when FITC-ACP4 was treated at 30 mM and 60 mM, fluorescence was observed within the cell, and when FITC-ACP5 was treated at 120 mM, fluorescence was observed within the cell. On the other hand, when FITC-Tat was treated at 60 mM, a weak fluorescence signal was observed, indicating that it barely penetrated into the cell.

Through this, it can be seen that the cell-penetrating peptide of the present invention has excellent cell membrane penetration ability even at low concentrations and can be usefully utilized for intracellular substance delivery.

Experimental Example 2: Comparison of Tissue Penetration Activity of Cell Penetrating Peptides of the Present Invention

After treating skin tissue with FITC-ACP peptide, FITC-ACP4 peptide, FITC-ACP5 peptide, and FITC-Tat peptide, the degree of tissue penetration of each peptide was confirmed.

Gauze was placed in a 6-well plate, and 3 ml of RPMI medium was added to each well. A 0.5 cm x 0.5 cm piece of skin tissue isolated from a hairless mouse (Orient Bio Co., Ltd., Korea) was gently placed on the gauze, and a Whatman paper was placed on top of the mouse skin tissue. FITC-ACP, FITC-ACP4, FITC-ACP5, and FITC-Tat were then treated on the Whatman paper (represented as ACP-FITC, ACP4-FITC, ACP5-FITC, and Tat-FITC, respectively) and reacted for 24 hours. The mouse skin tissue was fixed with 4% paraformaldehyde for 1 hour and immersed in 30% sucrose until the skin tissue sank. The skin tissue was frozen with OCT compound (OCT compound: Leica, Germany), and skin tissue section slides were created using a cryostat. After staining with DAPI for 10 minutes, observations were made using a confocal laser scanning microscope.

As a result, as confirmed in Fig. 2, when FITC-ACP4 and FITC-ACP5 were treated, strong fluorescence signals were observed in the epidermal and dermal layers of skin tissue, confirming deep penetration into the skin tissue. On the other hand, when FITC-Tat was treated, weak fluorescence signals were observed, confirming that it hardly penetrated into the skin tissue.

Through this, it can be seen that the cell-penetrating peptide of the present invention has excellent cell membrane penetration ability even at low concentrations and can be usefully utilized for intracellular substance delivery.

The present invention has been described with reference to preferred embodiments thereof. Those skilled in the art will appreciate that the present invention may be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated by the claims, not the foregoing description, and all differences within the scope equivalent thereto should be interpreted as being included in the present invention.

<110> Avixgen Inc. <120> CELL MEMBRANE PENETRATING PEPTIDE AND INTRACELLULAR DELIVERY CARRIER COMPRISING THEREOF <130> BPN200235 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 55 <212> PRT <213> Artificial Sequence <220> <223> ACP Peptide <40 0> 1 Met Gln Arg Gly Asn Phe Arg Asn Gln Arg Lys Ile Val Lys Cys Phe 1 5 10 15 Asn Cys Gly Lys Glu Gly His Thr Ala Arg Asn Cys Arg Ala Pro Arg 20 25 30 Lys Lys Gly Cys Trp Lys Cys Gly Lys Glu Gly His Gln Met Lys Asp 35 40 45 Cys Thr Glu Arg Gln Ala Asn 50 55 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> ACP4 Peptide <400> 2 Ala Arg Ala Pro Arg Lys Lys Gly Ala Arg Ala Pro Arg Ly s Lys Gly 1 5 10 15 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ACP5 Peptide <400> 3 Ala Arg Ala Pro Arg Lys Lys Gly 1 5

Claims (7)

A cell-penetrating composition comprising a polypeptide in which the amino acid sequence represented by sequence number 3 is repeated 1 to 7 times.
A cell-penetrating composition in claim 1, wherein the polypeptide has an amino acid sequence represented by sequence number 3 repeated one or two times.
It consists of a polypeptide in which the amino acid sequence represented by sequence number 3 is repeated one to seven times,
An intracellular transporter in which a cargo to be transported within a cell is linked to the N-terminus or C-terminus of the above polypeptide.
In the third paragraph, the polypeptide is an intracellular delivery agent in which the amino acid sequence represented by sequence number 3 is repeated once or twice.
An intracellular delivery vehicle in claim 3, wherein the cargo to be transported within the cell is a chemical substance, a polypeptide, a nucleic acid, a carbohydrate or a lipid.
A composition for cargo delivery into cells or tissues, comprising the intracellular delivery agent of claim 3 as an active ingredient.
A composition in claim 6, wherein the cargo to be transported within the cell is a chemical substance, a polypeptide, a nucleic acid, a carbohydrate or a lipid.