WO2024117321A1 - Peptide de pénétration de membrane cellulaire et véhicule intracellulaire le comprenant - Google Patents

Peptide de pénétration de membrane cellulaire et véhicule intracellulaire le comprenant Download PDF

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Publication number
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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English (en)
Korean (ko)
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백이용
성보경
구혜정
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㈜에빅스젠
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Priority to PCT/KR2022/019332 priority Critical patent/WO2024117321A1/fr
Publication of WO2024117321A1 publication Critical patent/WO2024117321A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • 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

La présente invention concerne un peptide de pénétration de membrane cellulaire et un véhicule intracellulaire le comprenant. Le véhicule intracellulaire selon la présente invention a l'avantage de pouvoir administrer efficacement une substance dans une cellule même à une faible concentration, par comparaison avec un peptide de pénétration cellulaire dérivé d'un virus.
PCT/KR2022/019332 2022-12-01 2022-12-01 Peptide de pénétration de membrane cellulaire et véhicule intracellulaire le comprenant WO2024117321A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20070083218A (ko) * 2006-02-20 2007-08-23 이화여자대학교 산학협력단 막투과 단백질 도메인 기능을 갖는 펩타이드
WO2014046478A1 (fr) * 2012-09-19 2014-03-27 주식회사 카엘젬백스 Peptide de pénétration cellulaire, conjugué le comprenant, et composition comprenant le conjugué
KR20180070496A (ko) * 2016-12-16 2018-06-26 (주) 에빅스젠 세포막 투과 펩티드 및 이를 포함하는 세포내 전달체
KR20230016804A (ko) * 2021-07-27 2023-02-03 (주) 에빅스젠 세포막 투과 펩티드 및 이를 포함하는 세포내 전달체

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20070083218A (ko) * 2006-02-20 2007-08-23 이화여자대학교 산학협력단 막투과 단백질 도메인 기능을 갖는 펩타이드
WO2014046478A1 (fr) * 2012-09-19 2014-03-27 주식회사 카엘젬백스 Peptide de pénétration cellulaire, conjugué le comprenant, et composition comprenant le conjugué
KR20180070496A (ko) * 2016-12-16 2018-06-26 (주) 에빅스젠 세포막 투과 펩티드 및 이를 포함하는 세포내 전달체
KR20230016804A (ko) * 2021-07-27 2023-02-03 (주) 에빅스젠 세포막 투과 펩티드 및 이를 포함하는 세포내 전달체

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 11 October 2005 (2005-10-11), ANONYMOUS: "HIV SAVINE 1 [synthetic construct]", XP093175574, retrieved from NCBI Database accession no. AAX19842.1 *
XIAOPENG QI, ET. AL.: "Cell-Penetrating Peptides Derived from Viral Capsid Proteins", MOLECULAR PLANT-MICROBE INTERACTIONS, VOL. 24, N.1, 1 January 2011 (2011-01-01), pages 25 - 36, XP055669148, Retrieved from the Internet <URL:https://apsjournals.apsnet.org/doi/pdf/10.1094/MPMI-07-10-0147> [retrieved on 20200217], DOI: 10.1094/MPMI-07-10-0147 *

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