WO2024117321A1 - Cell membrane penetrating peptide, and intracellular carrier including same - Google Patents

Cell membrane penetrating peptide, and intracellular carrier including same Download PDF

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WO2024117321A1
WO2024117321A1 PCT/KR2022/019332 KR2022019332W WO2024117321A1 WO 2024117321 A1 WO2024117321 A1 WO 2024117321A1 KR 2022019332 W KR2022019332 W KR 2022019332W WO 2024117321 A1 WO2024117321 A1 WO 2024117321A1
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polypeptide
amino acid
peptide
seq
cell
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PCT/KR2022/019332
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French (fr)
Korean (ko)
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백이용
성보경
구혜정
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㈜에빅스젠
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  • the present invention relates to cell membrane-penetrating peptides and intracellular delivery vehicles containing the same.
  • the nucleocapsid protein (hereinafter referred to as 'NC') of human immunodeficiency virus (HIV) not only plays a structural role in virus ontogeny, but also plays an important functional role in the viral life cycle of the virus. If we look at its functions, they are as follows. First, the NC peptide is involved in the genomic encapsidation of the virus. This function results from two zinc finger domains consisting of a unique CCHC motif, which is highly conserved in all retroviruses and is 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 viral reverse transcription (RT), which suggests that the NC peptide plays an important function in viral replication. You can see that it does. Third, the NC peptide has nucleic acid chaperone activity necessary for the virus life cycle. Recently, it has been reported that the NC peptide plays a role even when viral DNA is inserted into the host cell chromosome.
  • CPP Cell Penetrating Peptides
  • Tat peptide which is derived from the Human Immuno-deficiency Virus (HIV) and is currently being used in various fields such as cell therapy and diagnostic reagents.
  • penetratin derived from the DNA-binding domain of a homeodomain transcription factor, is used to deliver proteins useful to the human body to the skin.
  • Transportan is a peptide made by fusing a neuropeptide called galanin and some of the peptides isolated from wasp venom called mastoparan. It is used as a cell death-inducing peptide by combining with a peptide that induces cell death. It is becoming.
  • One aspect of the present invention aims to provide a cell-penetrating composition
  • 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 includes a polypeptide in which the amino acid sequence represented by SEQ ID NO: 3 (ACP5: ARAPRKKG) is repeated one to seven times, and a cargo for intracellular transport is attached to the N-terminus or C-terminus of the polypeptide.
  • ACP5: ARAPRKKG amino acid sequence represented by SEQ ID NO: 3
  • the purpose is to provide an intracellular carrier to which (cargo) is bound.
  • Another aspect of the present invention aims to provide a composition for cargo delivery within cells or tissues containing the intracellular delivery vehicle as an active ingredient.
  • One aspect of the present invention provides a cell-penetrating composition
  • a cell-penetrating composition comprising a polypeptide in which the amino acid sequence represented by SEQ ID NO: 3 is repeated 1 to 7 times.
  • the polypeptide may be one in which the amino acid sequence represented by SEQ ID NO: 3 is repeated once or twice.
  • another aspect of the present invention includes a polypeptide in which the amino acid sequence shown in SEQ ID NO: 3 is repeated one to seven times, and a cargo for intracellular transport is attached to the N-terminus or C-terminus of the polypeptide. Provides an intracellular transporter to which is bound.
  • the polypeptide may be one in which the amino acid sequence represented by SEQ ID NO: 3 is repeated once or twice.
  • the cargo to be transported within the cell may be a chemical substance, polypeptide, nucleic acid, carbohydrate, or lipid.
  • Another aspect of the present invention provides a composition for cargo delivery into cells or tissues, including an intracellular carrier as an active ingredient.
  • the cargo to be transported within the cell may be a chemical substance, polypeptide, nucleic acid, carbohydrate, or lipid.
  • the intracellular delivery vehicle of the present invention has the advantage of being able to efficiently deliver substances into cells even at low concentrations.
  • Figure 1 shows the results of comparing the cell membrane penetration activities of FITC-ACP peptide, FITC-ACP4 peptide, FITC-ACP5 peptide, and FITC-Tat peptide.
  • Figure 2 shows results showing the skin tissue penetration activity of ACP-FITC peptide, ACP4-FITC, ACP5-FITC and Tat-FITC.
  • One aspect of the present invention provides a cell-penetrating composition
  • 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.
  • peptide or “polypeptide” refers to a linear molecule formed by combining amino acid residues with peptide bonds, and contains 4-70 amino acid residues, preferably 4-40 amino acid residues, more preferably 4-40 amino acid residues. It consists of 4-30 pieces, most preferably 4-20 pieces.
  • the polypeptide represented by SEQ ID NO: 3 represents the addition of alanine to the linker domain present in the NC peptide of HIV (Human immunodeficiency virus) (SEQ ID NO: 3), and through experiments, the present inventors discovered a polypeptide containing the above sequence. It was confirmed that it has excellent cell membrane permeability, and through this, it was confirmed that it can be used as a drug delivery system capable of transferring substances into cells.
  • the amino acid sequence represented by SEQ ID NO: 3 is not only the amino acid sequence represented by SEQ ID NO: 3, but also is at least 40%, preferably at least 60%, more preferably at least 80%, and even more preferably at least 40% of the sequence.
  • the polypeptide may be one in which the amino acid sequence represented by SEQ ID NO. 3 is repeated once to seven times, specifically once or twice, and more specifically, the amino acid sequence represented by SEQ ID NO. 3. It may be a sequence or an amino acid sequence represented by SEQ ID NO: 2. As described above, the present inventors not only confirmed the cell membrane penetration ability of the polypeptide containing the amino acid sequence shown in SEQ ID NO: 3, but also confirmed the amino acid sequence (SEQ ID NO: 2) in which the amino acid sequence shown in SEQ ID NO: 3 is repeated twice. It was confirmed that the containing polypeptide also had excellent cell membrane penetration ability.
  • NC peptide refers to a polypeptide constituting the nucleocapsid of a retrovirus.
  • the polypeptide binds strongly to the genomic RNA of the retrovirus, forming a ribonucleoprotein core complex. It is known to form.
  • cell penetrating peptide refers to the ability to transport cargo of a transport target into cells in vitro and/or in vivo.
  • a cell-penetrating peptide is a peptide that has an amino acid sequence that can penetrate the cell membrane of the phospholipid bilayer.
  • Another aspect of the present invention includes a polypeptide in which the amino acid sequence represented by SEQ ID NO: 3 (ACP5: ARAPRKKG) is repeated one to seven times, and a cargo for intracellular transport is attached to the N-terminus or C-terminus of the polypeptide. Provides an intracellular carrier to which (cargo) is bound.
  • the polypeptide may be one in which the amino acid sequence represented by SEQ ID NO. 3 is repeated once to seven times, specifically once or twice, and more specifically, the amino acid sequence represented by SEQ ID NO. 3. It may be a sequence or an amino acid sequence represented by SEQ ID NO: 2.
  • intracellular carrier refers to a carrier that can penetrate the cell membrane and into tissues.
  • the cargo to be transported within the cell may be a chemical substance, polypeptide, nucleic acid, carbohydrate, or lipid.
  • the term “cargo” refers to chemicals, small molecules, polypeptides, nucleic acids, etc. that can be transported into cells by binding to NC peptides that act as cell membrane penetrating peptides.
  • Substances that can be bound to the polypeptide of the present invention are diverse and include, for example, proteins (polypeptides), nucleic acids (polynucleotides), chemical substances (drugs), etc. It is not limited to this.
  • polypeptide is a polymer of amino acids composed of two or more residues and includes peptides and proteins.
  • Polypeptides include, for example, 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.
  • the nucleic acid may be, for example, RNA, DNA, or cDNA, and the sequence of the nucleic acid may be a coding region sequence or a non-coding region sequence (e.g., an antisense oligonucleotide or siRNA).
  • cell cycle regulatory proteins e.g., cyclins and cyclin-dependent phosphatases
  • enzymes e.g., green fluorescent protein, beta-galactosidase, and chloramphenicol acetyltransferase
  • the nucleic acid may be, for example, RNA, DNA, or cDNA, and the sequence of the nucleic acid may be a coding region sequence or a non-coding region sequence (e.g., an antisense oligonucleotide or siRNA).
  • Nucleotides as nucleic acid cargo can be standard nucleotides (e.g., adenosine, cytosine, guanine, thymine, inosine, and uracil) or analogs (e.g., phosphorothioate nucleotides).
  • the nucleic acid cargo can be an antisense sequence composed of phosphorothioate nucleotides or RNAi.
  • Another aspect of the present invention provides a composition for cargo delivery into cells or tissues containing the intracellular delivery vehicle as an active ingredient.
  • the cargo to be transported within the cell may be a chemical substance, polypeptide, nucleic acid, carbohydrate, or lipid.
  • Another aspect of the present invention is to provide an intracellular transporter in which the cargo of the intracellular transport target is bound to the end of a polypeptide in which the amino acid sequence shown in SEQ ID NO: 3 (ACP5: ARAPRKKG) is repeated one to seven times to a cell or tissue.
  • ACP5: ARAPRKKG the amino acid sequence shown in SEQ ID NO: 3
  • a method of delivering cargo of an intracellular transport target into a cell or tissue including the step of contacting it.
  • the polypeptide may be one in which the amino acid sequence represented by SEQ ID NO. 3 is repeated once to seven times, specifically once or twice, and more specifically, the amino acid sequence represented by SEQ ID NO. 3. It may be a sequence or an amino acid sequence represented by SEQ ID NO: 2.
  • the description of common content between the two is omitted to avoid excessive complexity of the specification.
  • HeLa cells were cultured in DMEM medium supplemented with 10% FBS (fetal bovine serum) and 100 U/ml penicillin/streptomycin. HeLa cells were cultured in a humidified thermostat at 37°C and 5% CO2. Cultured HeLa cells were distributed at a density of 1x10 5 cells/well in a 12-well plate containing glass and cultured for 24 hours.
  • FBS fetal bovine serum
  • FITC-ACP peptide (3 ⁇ M, 3 h), FITC-ACP4 peptide (30 ⁇ M, 3 h /60 ⁇ M, 3 h), FITC-ACP5 peptide (60 ⁇ M, 2 h /120 ⁇ M, 2 h) and FITC-Tat peptide (60 ⁇ M, 3 h), respectively.
  • the cells were washed three times with PBS. Washed cells were fixed with 3.7% formaldehyde for 20 minutes and treated with PBS containing 0.2% Triton X-100 to increase cell membrane permeability.
  • the cells were treated with 3% BS and blocked for 1 hour, reacted with a tubulin antibody (Santa Cruz Biotechnology, sc-5286) at room temperature for 2 hours, and then washed three times with PBS. After washing, the cells were treated with Cy3 secondary antibody and reacted at room temperature for 1 hour, washed twice with PBS, and stained with DAPI (4', 6-diamidino-2-phenylindol) for 10 minutes. The glass with attached cells was removed, placed on a glass slide, and observed using a confocal laser scanning microscope (LSM 700, Zeiss, Germany). The sequences of ACP peptide, ACP 4 peptide, and ACP5 peptide used in the experiment are as follows.
  • the cell-penetrating peptide of the present invention has excellent cell membrane penetrating ability even at low concentrations and can be usefully used for intracellular material transfer.
  • FITC-ACP FITC-ACP4, FITC-ACP5, and FITC-Tat were treated on Whatman paper (indicated as ACP-FITC, ACP4-FITC, ACP5-FITC, and Tat-FITC, respectively) and reacted for 24 hours.
  • Mouse skin tissue was fixed with 4% paraformaldehyde for 1 hour and soaked in 30% sucrose until the skin tissue settled. Skin tissue was frozen with OCT compound (Leica, Germany), and skin tissue section slides were produced using a cryosection machine. After staining with DAPI for 10 minutes, it was observed with a confocal laser scanning microscope.
  • the cell-penetrating peptide of the present invention has excellent cell membrane penetrating ability even at low concentrations and can be usefully used for intracellular material transfer.

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Abstract

The present invention relates to a cell membrane penetrating peptide and an intracellular carrier including same. The intracellular carrier according to the present invention has the advantage of being able to efficiently deliver a substance into a cell even at a low concentration, when compared to a cell penetrating peptide derived from a virus.

Description

세포막 투과 펩티드 및 이를 포함하는 세포내 전달체Cell membrane penetrating peptides and intracellular transporters containing the same
본 발명은 세포막 투과 펩티드 및 이를 포함하는 세포내 전달체에 관한 것이다.The present invention relates to cell membrane-penetrating peptides and intracellular delivery vehicles containing the same.
HIV(human immunodeficiency virus)의 뉴클레오캡시드 단백질(nucleocapsid, 이하, 'NC'라 명명함)은 바이러스 개체형성을 위한 구조적 역할뿐만 아니라, 바이러스의 생활주기(viral life cycle)에서 기능적으로도 중요한 역할을 하는데, 그 기능을 살펴보면 다음과 같다. 첫째, NC 펩티드는 바이러스의 유전적 포막(genomic encapsidation)에 관여한다. 이러한 기능은 독특한 CCHC 모티프로 구성되는 두 개의 징크핑거 도메인으로부터 기인하는데, 상기 도메인은 모든 레트로바이러스(retrovirus)에서 높은 보존성을 나타내며, HIV RNA 포장(packaging)과 감염성 바이러스 생산에 필수적인 것으로 알려져 있다. 둘째, NC 펩티드는 바이러스의 역전사 반응(reverse transcription, RT)동안 tRNA 프라이머 어닐링(annealing)과 가닥 이동(strand transfer)을 촉진한다고 알려져 있으며, 이로부터 NC 펩티드가 바이러스 복제(viral replication)에 중요한 기능을 한다는 것을 알 수 있다. 셋째, NC 펩티드는 바이러스의 생활주기에 필요한 핵산 샤페론(chaperone) 활성을 가지며. 최근에는 바이러스 DNA가 숙주세포 염색체에 삽입될 때에도 NC 펩티드가 소정의 역할을 담당한다고 보고되고 있다.The nucleocapsid protein (hereinafter referred to as 'NC') of human immunodeficiency virus (HIV) not only plays a structural role in virus ontogeny, but also plays an important functional role in the viral life cycle of the virus. If we look at its functions, they are as follows. First, the NC peptide is involved in the genomic encapsidation of the virus. This function results from two zinc finger domains consisting of a unique CCHC motif, which is highly conserved in all retroviruses and is 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 viral reverse transcription (RT), which suggests that the NC peptide plays an important function in viral replication. You can see that it does. Third, the NC peptide has nucleic acid chaperone activity necessary for the virus life cycle. Recently, it has been reported that the NC peptide plays a role even when viral DNA is inserted into the host cell chromosome.
한편, 세포 투과 펩티드(Cell Penetrating Peptides: CPP)는 약 10-30개 정도의 짧은 펩티드로 이루어진 세포막 투과성 펩티드로 대부분 단백질-투과 도메인(protein-transduction domain)이나 막-이동 시퀀스(membrane-translocating sequence)로부터 유도된다. 외부 물질의 일반적인 세포 내 유입 경로와는 달리 CPP는 세포막을 손상시키지 않으면서 세포 내로 이동하고, 세포막을 통과하지 못하는 것으로 알려져 있는 DNA나 단백질까지도 세포 내로 전달시킬 수 있다고 알려져 있다. CPP로 가장 잘 알려진 펩티드는 Tat 펩티드가 있으며, 이는 인간 면역 결핍 바이러스(Human Immuno-deficiency Virus: HIV)로부터 유도되어 현재 세포 치료제나 진단시약 같은 다양한 방면에서 응용되어 사용되고 있다. 또한, 호메오도메인 전사 인자의 DNA-결합 도메인으로부터 유도된 페네트라틴(penetratin)은 인체에 유용한 단백질을 피부에 전달하는 용도로 이용되고 있다. 트랜스포탄(transportan)은 갈라닌(galanin)이라는 뉴로펩티드와 마스토파란(mastoparan)이라는 말벌의 독으로부터 분리된 펩티드 중 일부를 융합시켜 만든 펩티드로 세포사를 유도시키는 펩티드와 결합시켜 세포사 유도 펩티드로 이용되고 있다. Meanwhile, Cell Penetrating Peptides (CPP) are cell membrane penetrating peptides composed of about 10-30 short peptides, most of which have a protein-transduction domain or membrane-translocating sequence. It is derived from Unlike the general intracellular entry route for foreign substances, CPP is known to move into cells without damaging the cell membrane, and can even deliver DNA or proteins that are known not to pass through the cell membrane into cells. The most well-known peptide as CPP is the Tat peptide, which is derived from the Human Immuno-deficiency Virus (HIV) and is currently being used in various fields such as cell therapy and diagnostic reagents. Additionally, penetratin, derived from the DNA-binding domain of a homeodomain transcription factor, is used to deliver proteins useful to the human body to the skin. Transportan is a peptide made by fusing a neuropeptide called galanin and some of the peptides isolated from wasp venom called mastoparan. It is used as a cell death-inducing peptide by combining with a peptide that induces cell death. It is becoming.
본 발명자들은 HIV NC 펩티드의 생리학적 활성을 연구하던 중, NC 펩티드 중 링커펩티드의 변이체가 세포막 투과 활성을 가지고 있다는 것을 확인하였으며, 이를 통해 세포 내 물질 전달이 가능한 약물 전달 시스템으로 이용할 수 있다는 점을 확인함으로써 본 발명을 완성하였다.While studying the physiological activity of HIV NC peptides, the present inventors confirmed that a linker peptide variant among NC peptides had cell membrane penetration activity, and that it could be used as a drug delivery system capable of transporting substances into cells. By confirming this, the present invention was completed.
본 발명의 일 양상은 서열번호 3 (ACP5: ARAPRKKG) 으로 표시되는 아미노산 서열이 한 번 내지 7번 반복되는 폴리펩티드를 포함하는 세포 투과용 조성물을 제공하는 것을 목적으로 한다.One aspect of the present invention aims 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.
본 발명의 다른 일 양상은 서열번호 3 (ACP5: ARAPRKKG) 으로 표시되는 아미노산 서열이 한 번 내지 7번 반복되는 폴리펩티드를 포함하고, 상기 폴리펩티드의 N-말단 또는 C-말단에 세포 내 운반 대상의 카고(cargo)가 결합되어 있는 세포내 전달체를 제공하는 것을 목적으로 한다.Another aspect of the present invention includes a polypeptide in which the amino acid sequence represented by SEQ ID NO: 3 (ACP5: ARAPRKKG) is repeated one to seven times, and a cargo for intracellular transport is attached to the N-terminus or C-terminus of the polypeptide. The purpose is to provide an intracellular carrier to which (cargo) is bound.
본 발명의 다른 일 양상은 상기 세포내 전달체를 유효성분으로 포함하는 세포 또는 조직 내 카고 전달용 조성물을 제공하는 것을 목적으로 한다.Another aspect of the present invention aims to provide a composition for cargo delivery within cells or tissues containing the intracellular delivery vehicle as an active ingredient.
본 발명의 일 양상은 서열번호 3으로 표시되는 아미노산 서열이 1 내지 7번 반복되는 폴리펩티드를 포함하는 세포 투과용 조성물을 제공한다.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 is repeated 1 to 7 times.
본 발명의 일 구체예로 상기 폴리펩티드는 서열번호 3으로 표시되는 아미노산 서열이 하나 또는 두 번 반복되는 것일 수 있다. In one embodiment of the present invention, the polypeptide may be one in which the amino acid sequence represented by SEQ ID NO: 3 is repeated once or twice.
또한, 본 발명의 다른 일 양상은 서열번호 3으로 표시되는 아미노산 서열이 한 번 내지 7번 반복되는 폴리펩티드를 포함하고, 상기 폴리펩티드의 N-말단 또는 C-말단에 세포 내 운반 대상의 카고(cargo)가 결합되어 있는 세포내 전달체를 제공한다.In addition, another aspect of the present invention includes a polypeptide in which the amino acid sequence shown in SEQ ID NO: 3 is repeated one to seven times, and a cargo for intracellular transport is attached to the N-terminus or C-terminus of the polypeptide. Provides an intracellular transporter to which is bound.
본 발명의 일 구체예로, 상기 폴리펩티드는 서열번호 3으로 표시되는 아미노산 서열이 한 번 또는 두 번 반복되는 것일 수 있다.In one embodiment of the present invention, the polypeptide may be one in which the amino acid sequence represented by SEQ ID NO: 3 is repeated once or twice.
본 발명의 일 구체예로, 상기 세포 내 운반 대상의 카고는 화학물질, 폴리펩티드, 핵산, 탄수화물 또는 지질인 것일 수 있다.In one embodiment of the present invention, the cargo to be transported within the cell may be a chemical substance, polypeptide, nucleic acid, carbohydrate, or lipid.
본 발명의 다른 일 양상은 세포내 전달체를 유효성분으로 포함하는 세포 또는 조직 내 카고 전달용 조성물을 제공한다.Another aspect of the present invention provides a composition for cargo delivery into cells or tissues, including an intracellular carrier as an active ingredient.
본 발명의 일 구체예로, 상기 세포 내 운반 대상의 카고는 화학물질, 폴리펩티드, 핵산, 탄수화물 또는 지질일 수 있다.In one embodiment of the present invention, the cargo to be transported within the cell may be a chemical substance, polypeptide, nucleic acid, carbohydrate, or lipid.
본 발명의 세포내 전달체는 기존의 바이러스로부터 유래된 펩티드와 비교할 때, 낮은 농도로도 효율적으로 물질을 세포 내로 전달할 수 있는 장점이 있다.Compared to peptides derived from existing viruses, the intracellular delivery vehicle of the present invention has the advantage of being able to efficiently deliver substances into cells even at low concentrations.
도 1은 FITC-ACP 펩티드, FITC-ACP4 펩티드, FITC-ACP5 펩티드 및 FITC-Tat 펩티드의 세포막 투과 활성을 비교한 결과를 보여준다.Figure 1 shows the results of comparing the cell membrane penetration activities of FITC-ACP peptide, FITC-ACP4 peptide, FITC-ACP5 peptide, and FITC-Tat peptide.
도 2는 ACP-FITC 펩티드, ACP4-FITC, ACP5-FITC 및 Tat-FITC 의 피부 조직 투과 활성을 나타내는 결과를 보여준다.Figure 2 shows results showing the skin tissue penetration activity of ACP-FITC peptide, ACP4-FITC, ACP5-FITC and Tat-FITC.
본 발명의 일 양상은 서열번호 3 (ACP5: ARAPRKKG)으로 표시되는 아미노산 서열이 한 번 내지 7번 반복되는 폴리펩티드를 포함하는 세포 투과용 조성물을 제공한다.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.
본 명세서에서 용어, "펩티드" 또는 "폴리펩티드"는 펩타이드 결합에 의해 아미노산 잔기들이 서로 결합되어 형성된 선형의 분자를 의미하며, 아미노산 잔기 4-70개, 바람직하게는 4-40개, 보다 바람직하게는 4-30개, 가장 바람직하게는 4-20개로 이루어져 있다.As used herein, the term “peptide” or “polypeptide” refers to a linear molecule formed by combining amino acid residues with peptide bonds, and contains 4-70 amino acid residues, preferably 4-40 amino acid residues, more preferably 4-40 amino acid residues. It consists of 4-30 pieces, most preferably 4-20 pieces.
상기 서열번호 3으로 표시되는 폴리펩티드는 HIV(Human immunodeficiency virus)의 NC 펩티드 내에 존재하는 링커 도메인에 알라닌을 추가한 것 (서열번호 3)을 나타내는 것으로, 본 발명자들은 실험을 통해 상기 서열을 포함하는 폴리펩티드가 세포막 투과능이 우수함을 확인하였으며, 이를 통해 세포 내 물질 전달이 가능한 약물 전달 시스템으로 이용할 수 있다는 점을 확인하였다. The polypeptide represented by SEQ ID NO: 3 represents the addition of alanine to the linker domain present in the NC peptide of HIV (Human immunodeficiency virus) (SEQ ID NO: 3), and through experiments, the present inventors discovered a polypeptide containing the above sequence. It was confirmed that it has excellent cell membrane permeability, and through this, it was confirmed that it can be used as a drug delivery system capable of transferring substances into cells.
본 발명에서 상기 서열번호 3으로 표시되는 아미노산 서열은 서열번호 3으로 표시되는 아미노산 서열뿐만 아니라 상기 서열과 40% 이상, 바람직하게는 60% 이상, 더욱 바람직하게는 80% 이상, 보다 더욱 바람직하게는 90% 이상, 더욱 더 바람직하게는 95% 이상, 가장 바람직하게는 99% 이상의 상동성을 나타내는 아미노산 서열로서 실질적으로 용해도 개선의 효과를 발현하는 아미노산 서열이라면 제한없이 포함하며, 이러한 상동성을 갖는 서열로서 실질적으로 서열번호 3의 아미노산과 동일하거나 상응하는 생물학적 활성을 갖는 아미노산 서열이라면, 일부 서열이 결실, 변형, 치환 또는 부가된 아미노산 서열을 갖는 경우도 본 발명의 범주에 포함됨은 자명하다.In the present invention, the amino acid sequence represented by SEQ ID NO: 3 is not only the amino acid sequence represented by SEQ ID NO: 3, but also is at least 40%, preferably at least 60%, more preferably at least 80%, and even more preferably at least 40% of the sequence. An amino acid sequence showing a homology of at least 90%, more preferably at least 95%, and most preferably at least 99%, including without limitation any amino acid sequence that substantially exhibits the effect of improving solubility, and a sequence having such homology. As long as it is an amino acid sequence that is substantially the same as or has a biological activity corresponding to the amino acid of SEQ ID NO: 3, it is obvious that even if some of the sequences have amino acid sequences deleted, modified, substituted, or added, they are included within the scope of the present invention.
본 발명의 일 구체예에서 상기 폴리펩티드는 서열번호 3으로 표시되는 아미노산 서열이 한 번 내지 7번, 구체적으로는 한 번 또는 두 번 반복되는 것일 수 있고, 더욱 구체적으로는 서열번호 3으로 표시되는 아미노산 서열 또는 서열번호 2로 표시되는 아미노산 서열일 수 있다. 본 발명자들은 전술한 바와 같이 서열번호 3으로 표시되는 아미노산 서열을 포함하는 폴리펩티드의 세포막 투과능을 확인하였을 뿐만 아니라, 서열번호 3으로 표시되는 아미노산 서열이 두 번 반복되는 아미노산 서열 (서열번호 2)을 포함하는 폴리펩티드 또한 우수한 세포막 투과능이 있음을 확인하였다. In one embodiment of the present invention, the polypeptide may be one in which the amino acid sequence represented by SEQ ID NO. 3 is repeated once to seven times, specifically once or twice, and more specifically, the amino acid sequence represented by SEQ ID NO. 3. It may be a sequence or an amino acid sequence represented by SEQ ID NO: 2. As described above, the present inventors not only confirmed the cell membrane penetration ability of the polypeptide containing the amino acid sequence shown in SEQ ID NO: 3, but also confirmed the amino acid sequence (SEQ ID NO: 2) in which the amino acid sequence shown in SEQ ID NO: 3 is repeated twice. It was confirmed that the containing polypeptide also had excellent cell membrane penetration ability.
상기 "NC 펩티드"는 레트로바이러스(retrovirus)의 뉴클레오캡시드(nucleocapsid)를 구성하는 폴리펩티드를 의미하는 것으로, 상기 폴리펩티드는 레트로바이러스의 게놈 RNA에 강하게 결합하여 리보 핵산단백질 코어 복합체(ribonucleoprotein core complex)를 형성하는 것으로 알려져 있다.The "NC peptide" refers to a polypeptide constituting the nucleocapsid of a retrovirus. The polypeptide binds strongly to the genomic RNA of the retrovirus, forming a ribonucleoprotein core complex. It is known to form.
본 명세서에서 용어, "세포 투과 펩티드(cell penetrating peptide, CPP)"는 인 비트로(in vitro) 및/또는 인 비보(in vivo) 상에서 운반 대상의 카고(cargo)를 세포 내로 운반할 수 있는 능력을 가진 펩티드를 의미하는 것으로, 세포 투과 펩티드는 그 자체로 인지질 이중막의 세포막을 통과할 수 있는 아미노산 서열을 가지는 펩티드이다.As used herein, the term “cell penetrating peptide (CPP)” refers to the ability to transport cargo of a transport target into cells in vitro and/or in vivo. A cell-penetrating peptide is a peptide that has an amino acid sequence that can penetrate the cell membrane of the phospholipid bilayer.
본 발명의 다른 일 양상은 서열번호 3 (ACP5: ARAPRKKG) 으로 표시되는 아미노산 서열이 한 번 내지 7번 반복되는 폴리펩티드를 포함하고, 상기 폴리펩티드의 N-말단 또는 C-말단에 세포 내 운반 대상의 카고(cargo)가 결합되어 있는 세포내 전달체를 제공한다.Another aspect of the present invention includes a polypeptide in which the amino acid sequence represented by SEQ ID NO: 3 (ACP5: ARAPRKKG) is repeated one to seven times, and a cargo for intracellular transport is attached to the N-terminus or C-terminus of the polypeptide. Provides an intracellular carrier to which (cargo) is bound.
본 발명의 일 구체예에서 상기 폴리펩티드는 서열번호 3으로 표시되는 아미노산 서열이 한 번 내지 7번, 구체적으로는 한 번 또는 두 번 반복되는 것일 수 있고, 더욱 구체적으로는 서열번호 3으로 표시되는 아미노산 서열 또는 서열번호 2로 표시되는 아미노산 서열일 수 있다. In one embodiment of the present invention, the polypeptide may be one in which the amino acid sequence represented by SEQ ID NO. 3 is repeated once to seven times, specifically once or twice, and more specifically, the amino acid sequence represented by SEQ ID NO. 3. It may be a sequence or an amino acid sequence represented by SEQ ID NO: 2.
본 명세서에서 용어, "세포내 전달체"는 세포막을 투과하여 조직까지 침투할 수 있는 전달체를 의미한다.As used herein, the term “intracellular carrier” refers to a carrier that can penetrate the cell membrane and into tissues.
본 발명의 일 구체예에 따르면, 상기 세포 내 운반 대상의 카고는 화학물질, 폴리펩티드, 핵산, 탄수화물 또는 지질일 수 있다. According to one embodiment of the present invention, the cargo to be transported within the cell may be a chemical substance, polypeptide, nucleic acid, carbohydrate, or lipid.
본 명세서에서 용어, "카고(cargo)"는 세포막 투과 펩티드로서 작용하는 NC 펩티드에 결합하여 세포 내로 운반될 수 있는 화학물질, 작은 분자, 폴리펩티드, 핵산 등을 의미한다.As used herein, the term “cargo” refers to chemicals, small molecules, polypeptides, nucleic acids, etc. that can be transported into cells by binding to NC peptides that act as cell membrane penetrating peptides.
본 발명의 폴리펩티드에 결합될 수 있는 물질, 즉 카고(cargo)가 될 수 있는 물질은 다양하며, 예를 들어, 단백질(폴리펩티드), 핵산(폴리뉴클레오티드), 화학 물질(약물) 등을 포함하나, 이에 한정되는 것은 아니다.Substances that can be bound to the polypeptide of the present invention, that is, substances that can be cargo, are diverse and include, for example, proteins (polypeptides), nucleic acids (polynucleotides), chemical substances (drugs), etc. It is not limited to this.
예를 들어, 약물, 조영제(예를 들어, T1 조영제, 초상자성 물질과 같은 T2 조영제, 방사성 동위 원소 등), 형광 마커, 염색 물질 등이 될 수 있으나, 이에 한정되지는 않는다. 상기 폴리펩티드는 둘 또는 그 이상의 잔기로 구성되는 아미노산의 중합체로 펩티드 및 단백질을 포함한다. 폴리펩티드는 예를 들어, 세포 불멸에 관여하는 단백질(예를 들어, SV40 라지 T 항원 및 텔로머라아제), 항-아폽토틱 단백질(예를 들어, 돌연변이 p53 및 BclxL), 항체, 암유전자(예를 들어, ras, myc, HPV E6/E7 및 아데노바이러스 Ela), 세포 주기 조절 단백질(예를 들어, 사이클린 및 사이클린 의존성 인산화효소) 또는 효소(예를 들어, 녹색 형광 단백질, 베타-갈락토시다아제 및 클로람페니콜 아세틸 트랜스퍼라아제)가 될 수 있으나, 이에 한정하지는 않는다. 또한, 핵산은 예를 들어, RNA, DNA 또는 cDNA가 될 수 있으며, 핵산의 시퀀스는 암호화 부위 서열 또는 비암호화 부위 서열(예를 들어, 안티센스 올리고뉴클레오티드 또는 siRNA)이 될 수 있다. 핵산 카고로서의 뉴클레오티드는 표준 뉴클레오티드(예를 들어, 아데노신, 시토신, 구아닌, 티민, 이노신 및 우라실) 또는 아날로그(예를 들어, 포스포로티오에이트 뉴클레오티드)일 수 있다. 예를 들어, 핵산 카고는 포스포로티오에이트 뉴클레오티드로 구성된 안티센스 시퀀스 또는 RNAi일 수 있다.For example, it may be a drug, a contrast agent (e.g., a T1 contrast agent, a T2 contrast agent such as a superparamagnetic material, a radioactive isotope, etc.), a fluorescent marker, a dye, etc., 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 include, for example, 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. (e.g., ras, myc, HPV E6/E7, and adenovirus Ela), cell cycle regulatory proteins (e.g., cyclins and cyclin-dependent phosphatases), or enzymes (e.g., green fluorescent protein, beta-galactosidase, and chloramphenicol acetyltransferase), but is not limited thereto. Additionally, the nucleic acid may be, for example, RNA, DNA, or cDNA, and the sequence of the nucleic acid may be a coding region sequence or a non-coding region sequence (e.g., an antisense oligonucleotide or siRNA). Nucleotides as nucleic acid cargo can be standard nucleotides (e.g., adenosine, cytosine, guanine, thymine, inosine, and uracil) or analogs (e.g., phosphorothioate nucleotides). For example, the nucleic acid cargo can be an antisense sequence composed of phosphorothioate nucleotides or RNAi.
본 발명의 일 구체예에 따르면, 서열번호 3 (ACP5: ARAPRKKG) 으로 표시되는 아미노산 서열이 한 번 내지 7번 반복되는 폴리펩티드에 결합된 물질은 세포막을 매우 높은 효율로 투과하고, 세포 내에서 세포질 및 핵에 잔류하게 된다.According to one embodiment 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 one to seven times penetrates the cell membrane with very high efficiency, and enters the cytoplasm and within the cell. It remains in the nucleus.
본 발명의 다른 일 양상은 상기 세포내 전달체를 유효성분으로 포함하는 세포 또는 조직 내 카고 전달용 조성물을 제공한다. Another aspect of the present invention provides a composition for cargo delivery into cells or tissues containing the intracellular delivery vehicle as an active ingredient.
본 발명의 일 구체예에 따르면 상기 세포 내 운반 대상의 카고는 화학물질, 폴리펩티드, 핵산, 탄수화물 또는 지질일 수 있다. According to one embodiment of the present invention, the cargo to be transported within the cell may be a chemical substance, polypeptide, nucleic acid, carbohydrate, or lipid.
본 발명의 또 다른 양상은 상기 서열번호 3 (ACP5: ARAPRKKG) 으로 표시되는 아미노산 서열이 한 번 내지 7번 반복되는 폴리펩티드 말단에 세포 내 운반 대상의 카고가 결합되어 있는 세포내 전달체를 세포 또는 조직에 접촉시키는 단계를 포함하는 세포 내 운반 대상의 카고를 세포 또는 조직 내로 전달하는 방법을 제공한다.Another aspect of the present invention is to provide an intracellular transporter in which the cargo of the intracellular transport target is bound to the end of a polypeptide in which the amino acid sequence shown in SEQ ID NO: 3 (ACP5: ARAPRKKG) is repeated one to seven times to a cell or tissue. Provided is a method of delivering cargo of an intracellular transport target into a cell or tissue, including the step of contacting it.
본 발명의 일 구체예에서 상기 폴리펩티드는 서열번호 3으로 표시되는 아미노산 서열이 한 번 내지 7번, 구체적으로는 한 번 또는 두 번 반복되는 것일 수 있고, 더욱 구체적으로는 서열번호 3으로 표시되는 아미노산 서열 또는 서열번호 2로 표시되는 아미노산 서열일 수 있다. In one embodiment of the present invention, the polypeptide may be one in which the amino acid sequence represented by SEQ ID NO. 3 is repeated once to seven times, specifically once or twice, and more specifically, the amino acid sequence represented by SEQ ID NO. 3. It may be a sequence or an amino acid sequence represented by SEQ ID NO: 2.
본 발명의 방법은 상기 세포 내 전달체들을 이용하기 때문에, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.Since the method of the present invention uses the intracellular delivery vehicles, the description of common content between the two is omitted to avoid excessive complexity of the specification.
한편, 카고가 결합되어 있는 서열번호 3 (ACP5: ARAPRKKG) 으로 표시되는 아미노산 서열이 한 번 내지 7번 반복되는 폴리펩티드가 인 비트로 또는 인 비보에서 세포막과 접촉할 수 있는 기회가 부여되면 폴리펩티드와 결합된 카고는 세포 내로 운반된다. 상기 세포내 전달체와 세포막의 접촉에서 특별하게 요구되는 조건, 예를 들어, 제한적인 시간, 온도 및 농도 등의 조건은 없으며, 당업계에서 세포막 투과에 적용되는 일반적인 조건으로 실시될 수 있다.On the other hand, when a polypeptide in which the amino acid sequence represented by SEQ ID NO. 3 (ACP5: ARAPRKKG) to which the cargo is bound is repeated once to seven times is given the opportunity to contact the cell membrane in vitro or in vivo, the polypeptide bound to the polypeptide is bound to the polypeptide. Cargo is transported into the cell. There are no special conditions required for contact between the intracellular carrier and the cell membrane, such as restrictive conditions such as time, temperature, and concentration, and can be carried out under general conditions applied to cell membrane permeation in the art.
이하 하나 이상의 구체예를 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, one or more specific examples will be described in more detail through 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.
실험예 1: 본 발명의 세포 투과 펩티드의 세포 투과 활성 비교Experimental Example 1: Comparison of cell penetrating activity of cell penetrating peptides of the present invention
FITC-ACP 펩티드, FITC-ACP4 펩티드, FITC-ACP5 펩티드 및 FITC-Tat 펩티드를 HeLa 세포에 처리한 후 각 펩티드의 세포막 투과 정도를 확인하였다.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.
구체적으로, HeLa 세포는 10% FBS(fetal bovine serum) 및 100 U/㎖ 페니실린/스트렙토마이신이 추가된 DMEM 배지에서 배양하였다. HeLa 세포는 37℃ 5% CO2의 가습 항온기에서 배양하였다. 배양한 HeLa 세포를 글라스가 들어 있는 12웰 플레이트에 1x105 세포/웰(well)의 밀도로 분주하여 24시간 동안 배양하였다. 이후, FITC-ACP 펩티드 (3μM, 3h), FITC-ACP4 펩티드(30μM, 3h /60μM, 3h), FITC-ACP5 펩티드(60μM, 2h /120μM, 2h) 및 FITC-Tat 펩티드 (60μM, 3h) 각각 세포에 처리한 후 세포를 PBS로 3회 세척하였다. 세척한 세포를 3.7% 포름알데하이드로 20분 동안 고정시키고, 0.2% 트리톤 X-100(Triton X-100)이 포함된 PBS를 처리하여 세포막 투과성을 증가시켰다. 다음으로 세포에 3% BS를 처리하여 1시간 동안 블로킹(blocking)시키고, tubulin 항체(Santa Cruz Biotechnology, sc-5286)와 상온에서 2시간 반응시킨 후 PBS로 3회 세척하였다. 세척 후 Cy3 2차 항체를 처리하여 상온에서 1시간 동안 반응시키고, PBS로 2회 세척한 후 DAPI(4', 6-diamidino-2-phenylindol)로 10분 동안 염색하였다. 세포가 부착된 글라스를 떼어내어 슬라이드 글라스 위에 올려놓고, 공초점 레이저 주사현미경(confocal laser scanning microscopy; LSM 700, Zeiss, 독일)으로 관찰하였다. 실험에 사용된 ACP 펩티드, ACP 4 펩티드 및 ACP5 펩티드의 서열은 아래와 같다.Specifically, HeLa cells were cultured in DMEM medium supplemented with 10% FBS (fetal bovine serum) and 100 U/ml penicillin/streptomycin. HeLa cells were cultured in a humidified thermostat at 37°C and 5% CO2. Cultured HeLa cells were distributed at a density of 1x10 5 cells/well in a 12-well plate containing glass and cultured for 24 hours. Then, 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), respectively. After treatment, the cells were washed three times with PBS. Washed cells were fixed with 3.7% formaldehyde for 20 minutes and treated with PBS containing 0.2% Triton X-100 to increase cell membrane permeability. Next, the cells were treated with 3% BS and blocked for 1 hour, reacted with a tubulin antibody (Santa Cruz Biotechnology, sc-5286) at room temperature for 2 hours, and then washed three times with PBS. After washing, the cells were treated with Cy3 secondary antibody and reacted at room temperature for 1 hour, washed twice with PBS, and stained with DAPI (4', 6-diamidino-2-phenylindol) for 10 minutes. The glass with attached cells was removed, placed on a glass slide, and observed using a confocal laser scanning microscope (LSM 700, Zeiss, Germany). The sequences of ACP peptide, ACP 4 peptide, and ACP5 peptide used in the experiment are as follows.
이름name 서열번호sequence number 아미노산 서열amino acid sequence
ACP 펩티드ACP peptide 서열번호 1SEQ ID NO: 1 MQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQANMQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQAN
ACP4 펩티드ACP4 peptide 서열번호 2SEQ ID NO: 2 ARAPRKKGARAPRKKGARAPRKKGARAPRKKG
ACP5 펩티드ACP5 peptide 서열번호 3SEQ ID NO: 3 ARAPRKKGARAPRKKG
그 결과, 도 1에 나타난 바와 같이 FITC-ACP4를 30 mM, 60 mM 처리한 경우 세포 내에서 형광을 관찰하였고, FITC-ACP5를 120 mM 처리한 경우 세포 내에서 형광을 관찰할 수 있었다. 반면 FITC-Tat를 60 mM 처리한 경우 형광 신호가 약하게 관찰되어 세포 내에 거의 침투하지 못한 것을 알 수 있었다.As a result, as shown in Figure 1, fluorescence was observed within cells when FITC-ACP4 was treated at 30mM and 60mM, and fluorescence was observed within cells when FITC-ACP5 was treated at 120mM. On the other hand, when treated with 60 mM FITC-Tat, the fluorescence signal was observed weakly, indicating that it hardly penetrated into the cells.
이를 통해 본 발명의 세포투과 펩티드는 낮은 농도에서도 세포막 투과능이 우수하여 세포 내 물질 전달에 유용하게 활용될 수 있음을 알 수 있다. This shows that the cell-penetrating peptide of the present invention has excellent cell membrane penetrating ability even at low concentrations and can be usefully used for intracellular material transfer.
실험예 2: 본 발명의 세포 투과 펩티드의 조직 투과 활성 비교Experimental Example 2: Comparison of tissue penetrating activity of cell penetrating peptides of the present invention
FITC-ACP 펩티드, FITC-ACP4 펩티드, FITC-ACP5 펩티드 및 FITC-Tat 펩티드를 피부 조직에 처리한 후 각 펩티드의 조직 투과 정도를 확인하였다.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.
6웰 플레이트에 거즈(gauze)를 깔고, RPMI 배지를 3 ㎖씩 넣어 주었다. 무모 마우스(㈜오리엔트 바이오: 대한민국)로부터 분리한 0.5 ㎝ x 0.5 ㎝ 크기의 피부 조직을 거즈 위에 살며시 올려 놓고, 마우스 피부 조직 위에 와트만 페이퍼(Whatman paper)를 올려 놓았다. 이후 FITC-ACP, FITC-ACP4, FITC-ACP5 및 FITC-Tat를 와트만 페이퍼에 처리하고 (각각, ACP-FITC, ACP4-FITC, ACP5-FITC 및 Tat-FITC로 표시), 24시간 동안 반응시켰다. 마우스 피부 조직을 4% 파라포름알데히드로 1시간 동안 고정하고, 피부 조직이 가라앉을 때까지 30% 수크로스(sucrose)에 담가 두었다. OCT 컴파운드(OCT compound: Leica, 독일)로 피부 조직을 냉동하고, 냉동 절편기로 피부 조직 절편 슬라이드를 제작하였다. DAPI로 10분 동안 염색한 후 공초점 레이저 주사현미경으로 관찰하였다.Gauze was spread on a 6-well plate, and 3 ml of RPMI medium was added at a time. Skin tissue measuring 0.5 cm Afterwards, FITC-ACP, FITC-ACP4, FITC-ACP5, and FITC-Tat were treated on Whatman paper (indicated as ACP-FITC, ACP4-FITC, ACP5-FITC, and Tat-FITC, respectively) and reacted for 24 hours. . Mouse skin tissue was fixed with 4% paraformaldehyde for 1 hour and soaked in 30% sucrose until the skin tissue settled. Skin tissue was frozen with OCT compound (Leica, Germany), and skin tissue section slides were produced using a cryosection machine. After staining with DAPI for 10 minutes, it was observed with a confocal laser scanning microscope.
그 결과, 도 2에서 확인되는 바와 같이 FITC-ACP4, FITC-ACP5를 처리한 경우 피부 조직의 표피층 및 진피층에서까지 형광 신호가 강하게 나타나 피부 조직에 깊숙이 침투한 것을 확인할 수 있었다. 반면 FITC-Tat를 처리한 경우 형광 신호가 약하게 관찰되어 피부 조직에 거의 침투하지 못한 것을 알 수 있었다. As a result, as seen in Figure 2, when FITC-ACP4 and FITC-ACP5 were treated, a strong fluorescence signal appeared in the epidermal and dermal layers of the skin tissue, confirming that it had penetrated deep into the skin tissue. On the other hand, when treated with FITC-Tat, the fluorescence signal was observed weakly, showing that it hardly penetrated the skin tissue.
이를 통해 본 발명의 세포투과 펩티드는 낮은 농도에서도 세포막 투과능이 우수하여 세포 내 물질 전달에 유용하게 활용될 수 있음을 알 수 있다. This shows that the cell-penetrating peptide of the present invention has excellent cell membrane penetrating ability even at low concentrations and can be usefully used for intracellular material transfer.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form 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 in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (7)

  1. 서열번호 3으로 표시되는 아미노산 서열이 1 내지 7번 반복되는 폴리펩티드를 포함하는 세포 투과용 조성물.A cell-penetrating composition comprising a polypeptide in which the amino acid sequence shown in SEQ ID NO: 3 is repeated 1 to 7 times.
  2. 제1항에 있어서, 상기 폴리펩티드는 서열번호 3으로 표시되는 아미노산 서열이 하나 또는 두 번 반복되는 것인 세포 투과용 조성물.The cell-penetrating composition according to claim 1, wherein the polypeptide has the amino acid sequence represented by SEQ ID NO: 3 repeated once or twice.
  3. 서열번호 3으로 표시되는 아미노산 서열이 한 번 내지 7번 반복되는 폴리펩티드를 포함하고, Contains a polypeptide in which the amino acid sequence represented by SEQ ID NO: 3 is repeated one to seven times,
    상기 폴리펩티드의 N-말단 또는 C-말단에 세포 내 운반 대상의 카고(cargo)가 결합되어 있는 세포내 전달체.An intracellular delivery vehicle in which the cargo of the intracellular transport target is bound to the N-terminus or C-terminus of the polypeptide.
  4. 제3항에 있어서, 상기 폴리펩티드는 서열번호 3으로 표시되는 아미노산 서열이 한 번 또는 두 번 반복되는 것인 세포내 전달체.The intracellular delivery system according to claim 3, wherein the polypeptide is one in which the amino acid sequence represented by SEQ ID NO: 3 is repeated once or twice.
  5. 제3항에 있어서, 상기 세포 내 운반 대상의 카고는 화학물질, 폴리펩티드, 핵산, 탄수화물 또는 지질인 것인 세포내 전달체.The intracellular delivery vehicle according to claim 3, wherein the cargo of the intracellular transport target is a chemical substance, polypeptide, nucleic acid, carbohydrate, or lipid.
  6. 제3항의 세포내 전달체를 유효성분으로 포함하는 세포 또는 조직 내 카고 전달용 조성물.A composition for cargo delivery within cells or tissues, comprising the intracellular delivery vehicle of claim 3 as an active ingredient.
  7. 제6항에 있어서, 상기 세포 내 운반 대상의 카고는 화학물질, 폴리펩티드, 핵산, 탄수화물 또는 지질인 것인 조성물.The composition according to claim 6, wherein the cargo to be transported within the cell is a chemical substance, polypeptide, nucleic acid, carbohydrate, or lipid.
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