KR102274877B1 - Novel cell penetrating peptides and use thereof - Google Patents
Novel cell penetrating peptides and use thereof Download PDFInfo
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- KR102274877B1 KR102274877B1 KR1020200183618A KR20200183618A KR102274877B1 KR 102274877 B1 KR102274877 B1 KR 102274877B1 KR 1020200183618 A KR1020200183618 A KR 1020200183618A KR 20200183618 A KR20200183618 A KR 20200183618A KR 102274877 B1 KR102274877 B1 KR 102274877B1
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Images
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- 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
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/069—Vascular Endothelial cells
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- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
Abstract
Description
본 발명은 생물학적 활성 물질을 세포 내로 전달하기 위한 세포 내 전달 기술에 관한 것으로서, 보다 구체적으로 세포 투과능이 우수한 신규한 세포 투과성 펩타이드 및 이의 용도에 관한 것이다. The present invention relates to an intracellular delivery technology for delivering a biologically active substance into a cell, and more particularly, to a novel cell-penetrating peptide having excellent cell-penetrating ability and use thereof.
현재까지 다양한 저분자 화합물, 단백질, 펩타이드, RNA, DNA 등 고분자 물질의 세포내 전달 및 이의 응용을 위한 연구들이 진행되어 왔으며, 특히 SOD, Catalase, SOCS와 같은 효소(enzyme), dnPI3K, ZAP70 돌연변이와 같은 세포 내 신호전달 단백질의 저해 형태의 단백질, Foxp3, RORgt와 같은 전사인자 단백질 혹은 전사인자의 DNA binding domain 부분과 같이 다양한 단백질 전달을 통해 세포 내 기능을 조절하고자 하는 시도가 되어져 왔다. 또한 사이클로스포린A(cyclosporinA) 와 같은 저분자 화합물 약물을 세포 내 및 조직 내로 전달하여 장기 및 세포 이식 시 발생하는 거부반응을 조절하고자 하는 시도, 건선과 같은 자가면역을 조절하고자 하는 시도가 진행되어 왔다.Until now, studies have been conducted for intracellular delivery of various low-molecular compounds, high molecular substances such as proteins, peptides, RNA, and DNA, and their applications. In particular, enzymes such as SOD, Catalase, SOCS, dnPI3K, ZAP70 mutants, etc. Attempts have been made to regulate intracellular functions through the transduction of various proteins, such as inhibitory forms of intracellular signaling proteins, transcription factor proteins such as Foxp3 and RORgt, or DNA binding domains of transcription factors. In addition, attempts to control rejection that occur during organ and cell transplantation by delivering low molecular weight compound drugs such as cyclosporin A into cells and tissues, and attempts to control autoimmunity such as psoriasis have been in progress.
그러나 일반적으로 친수성이거나 분자량이 크고 거대한 물질들은 세포막이라는 장벽에 의해 세포 안으로 들어갈 수 없다. 세포막은 펩타이드나 단백질, 핵산과 같은 거대 분자가 세포 내로 들어오지 못하게 막고, 세포막 수용체에 의한 엔도사이토시스(endocytosis)라는 생리적 기작을 통해 세포 내로 들어오더라도 세포의 용해소체 분획(lysosomal compartment)과 융합되어 결국 분해되므로, 상기 거대 분자들을 이용한 질병의 치료 및 예방에 있어서 많은 제약이 따른다. 또한, 항암제의 경우 세포 내로 약물을 전달하기 위해서는 다약제 저항성(multidrug resistance)과 같은 장애물을 극복해야만 한다. 이에 약물 분해를 막기 위해 다양한 거대 분자 및 약물이 함유된 전달체를 엔도사이토시스 과정을 통하지 않고, 직접 세포 내로 전달하는 많은 방법이 제시되었다. 이러한 방법들에는 마이크로 주입(microinjection), 전기천공법(electroporation) 등이 있는데, 이는 세포막에 손상을 줄 가능성이 있다. 또 다른 방법들로 세포 투과성 물질을 이용하는 방법 등이 있다. 하지만 이러한 방법을 통하여 세포 내로 약물들을 전달하였다 하더라도 약효를 발휘하기 위해서는 특정한 기관으로 이동되어야 하는 문제가 있다.However, in general, hydrophilic or large molecular weight substances cannot enter the cell by the barrier called the cell membrane. The cell membrane prevents macromolecules such as peptides, proteins, and nucleic acids from entering the cell, and even if it enters the cell through a physiological mechanism called endocytosis by cell membrane receptors, it is fused with the lysosomal compartment of the cell. As it is eventually degraded, there are many restrictions in the treatment and prevention of diseases using the macromolecules. In addition, in the case of anticancer drugs, in order to deliver the drug into cells, obstacles such as multidrug resistance must be overcome. Accordingly, in order to prevent drug degradation, many methods have been proposed to directly deliver various macromolecules and drug-containing carriers into cells without going through the endocytosis process. These methods include microinjection, electroporation, etc., which have the potential to damage cell membranes. Another method includes a method using a cell-permeable material. However, even if drugs are delivered into cells through this method, there is a problem in that they must be moved to a specific organ in order to exert the drug effect.
따라서 상기와 같은 한계점들을 극복하고 안정성 및 물질 전달 효율을 높일 수 있는 다양한 약물전달체들이 많이 연구 개발되어 왔으며, 대표적으로 리포좀(liposome)과 마이셀(micelle)이 있다. 리포좀은 인공적으로 만든 인지질 전달체로 친유성 및 친수성 약물 모두 봉입할 수 있으며, 생체적합성 물질이므로 독성이 없고 약물을 외부환경으로부터 보호한다. 하지만 흡수가 지연되고 분포의 제한을 받으며 대사율이 낮아지며, 간이나 비장의 세포에 포획되어 혈액으로부터 신속하게 제거되는 단점이 있다. 마이셀은 약물의 용해도 및 생체이용률을 높여줄 수 있는 특징이 있으나, 물질의 세포 내로의 이동에 관한 효과와 기초 의학적 및 임상적 적용가능성에 대해서는 아직도 많은 연구가 필요하다. 이러한 한계점 때문에, 생체물질을 생체 내로 효과적으로 전달할 수 있고 세포독성이 없으며, 특히 엔도사이토시스를 통하여 들어가지 않는 새로운 제제들이 필요하다.Therefore, various drug delivery systems that can overcome the above limitations and increase stability and mass delivery efficiency have been researched and developed, and representative examples include liposomes and micelles. Liposomes are artificially made phospholipid carriers that can encapsulate both lipophilic and hydrophilic drugs, and since they are biocompatible materials, they are nontoxic and protect drugs from external environments. However, absorption is delayed, distribution is limited, metabolic rate is low, and it is captured by cells of the liver or spleen and is rapidly removed from the blood. Although micelles have characteristics that can increase the solubility and bioavailability of drugs, many studies are still needed on the effects of substances on the movement of substances into cells and their basic medical and clinical applicability. Because of these limitations, there is a need for new agents that can effectively deliver biomaterials into the body, have no cytotoxicity, and do not enter through endocytosis in particular.
이러한 측면에서, 세포 투과성 펩타이드(cell penetrating peptide)가 새로운 대안으로써 주목받아왔다. 세포 투과성 펩타이드는 일종의 신호 펩타이드(Signal Peptide) 로서 단백질, DNA, RNA 등과 같은 고분자 물질을 세포 내로 전달하고자 하는 목적으로 사용되는 일종의 특정 아미노산 서열의 조합인 펩타이드이다. 예컨대, HIV 바이러스에서 유래된 TAT 단백질에 존재하는 11개 아미노산 서열이 베타-갈락토시다아제(β-galactosidase)(120 kDa) 단백질의 세포 내 및 조직 내 전달이 가능하다는 것을 보여준 1990년대 이후부터 본격적으로 관련 연구가 진행되었다. 초파리의 단백질에서 유래한 안테나페디아(Antennapedia(Penetratin)), HSV-1 바이러스로부터 유래된 VP22(Elliott, G. et al., Cell, 88:223, 1997), 시미안 바이러스 40 거대 항원 T(Simian Virus 40 large antigen T)로부터 유래한 Pep-1이 대표적인 1세대 세포 투과성 펩타이드로 여겨지며 TAT과 함께 많이 사용되어져왔다. 또한, 폴리 아르기닌(poly Arginine), 폴리 라이신(poly Lysine)과 같이 단순히 아르기닌 및 라이신과 같은 양이온성 아미노산이 반복적으로 여러 개 연결된 펩타이드도 세포 투과능이 뛰어난 것으로 보고되었으며, 다양한 물질 전달에 응용되고 있다. 그러나 대부분 이러한 세포 투과성 펩타이드는 면역원성, 독성의 가능성을 내포하고 있고, 인간세포에 전달되는 효능이 떨어지는 것으로 여겨진다. 따라서 독성이 유발하지 않고 생체 내에서의 안전성을 가지며 효과적으로 물질을 전달할 수 있는 세포 투과성 펩타이드의 개발이 필요하다. In this regard, cell penetrating peptides have been attracting attention as a new alternative. A cell-penetrating peptide is a kind of signal peptide and is a peptide that is a combination of a specific amino acid sequence used for the purpose of delivering high molecular substances such as proteins, DNA, RNA, etc. into cells. For example, since the 1990s, when the 11 amino acid sequence present in the TAT protein derived from the HIV virus showed that the intracellular and intracellular delivery of the beta-galactosidase (120 kDa) protein was possible, full-scale since the 1990s. related research was conducted. Antennapedia (Penetratin) derived from Drosophila protein, VP22 derived from HSV-1 virus (Elliott, G. et al., Cell, 88:223, 1997), Simian
본 발명자들은 상기와 같은 우수한 효과를 갖는 신규한 세포 투과성 펩타이드를 개발하기 위해 연구 노력한 결과, 본 발명에 따른 다양한 아미노산 서열로 이루어진 세포 투과성 펩타이드를 설계 및 합성하고 이의 우수한 세포 투과성을 확인하였는 바, 이로써 본 발명을 완성하였다. As a result of research efforts to develop novel cell-permeable peptides having the above-described excellent effects, the present inventors designed and synthesized cell-permeable peptides composed of various amino acid sequences according to the present invention and confirmed their excellent cell-permeability, thereby The present invention was completed.
이에, 본 발명은 신규한 세포 투과성 펩타이드를 제공하는 것을 목적으로 한다. Accordingly, an object of the present invention is to provide a novel cell-penetrating peptide.
또한, 본 발명은 상기 세포 투과성 펩타이드 및 생물학적 활성 물질을 포함하는 복합체를 제공하는 것을 다른 목적으로 한다. Another object of the present invention is to provide a complex comprising the cell-penetrating peptide and a biologically active substance.
또한, 본 발명은 상기 복합체를 포함하는 물질 전달용 조성물, 및 상기 조성물을 세포에 처리하는 단계를 포함하는 물질 전달방법을 제공하는 것을 또 다른 목적으로 한다. In addition, another object of the present invention is to provide a mass transfer composition comprising the complex, and a mass transfer method comprising the step of treating cells with the composition.
또한, 본 발명은 상기 세포 투과성 펩타이드를 암호화하는 폴리뉴클레오티드를 제공하는 것을 또 다른 목적으로 한다. Another object of the present invention is to provide a polynucleotide encoding the cell-penetrating peptide.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.
상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은 하기의 [일반식 I]로 표시되는 아미노산 서열로 이루어진, 세포 투과성 펩타이드를 제공한다. In order to achieve the object of the present invention as described above, the present invention provides a cell-penetrating peptide consisting of an amino acid sequence represented by the following [General Formula I].
[일반식 I][General Formula I]
X1-[Xa-L-Xb]-X2-V-X3 X 1 -[X a -LX b ]-X 2 -VX 3
X1은 GHHE, AHHE, GHAE, SHHE, AHAE, GHNE, GHHD, GHKE, GHGE, GHRE, GHVE, GHQE 또는 GHHG이고; X 1 is GHHE, AHHE, GHAE, SHHE, AHAE, GHNE, GHHD, GHKE, GHGE, GHRE, GHVE, GHQE or GHHG;
Xa는 Arg(R), Ala(A), Ile(I), Val(V), Leu(L), Gly(G), Asn(N), Lys(K) 또는 Gln(Q)이고;X a is Arg(R), Ala(A), Ile(I), Val(V), Leu(L), Gly(G), Asn(N), Lys(K) or Gln(Q);
Xb 는 Lys(K), Arg(R), His(H), Asn(N) 또는 Gln(Q)이고;X b is Lys(K), Arg(R), His(H), Asn(N) or Gin(Q);
X2는 SDEWS, ADEWS, CDEWS, LDEWS, QDEWS, VDEWS, NDEWS, TDEWS, RDEWS, SDSWS, SDEYS, GDEWS, SSEWS, SDFWS 또는 SDEFS이고; X 2 is SDEWS, ADEWS, CDEWS, LDEWS, QDEWS, VDEWS, NDEWS, TDEWS, RDEWS, SDSWS, SDEYS, GDEWS, SSEWS, SDFWS or SDEFS;
X3은 TSG, NSA, NSV, QSG, KSG, RSG, ISG, SSG, TRG, TNG, TKG, TQG 또는 NSG 이다.X 3 is TSG, NSA, NSV, QSG, KSG, RSG, ISG, SSG, TRG, TNG, TKG, TQG or NSG.
본 발명의 일구현예로, 상기 세포 투과성 펩타이드는 서열번호 1 내지 서열번호 62로 이루어진 군에서 선택되는 어느 하나의 아미노산 서열로 이루어지는 것일 수 있다. In one embodiment of the present invention, the cell-penetrating peptide may be composed of any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 62.
본 발명의 다른 구현예로, 상기 세포는 뇌혈관장벽 내피세포(brain endothelial cell), 암세포, 혈액세포(blood cell), 림프구(lymphocyte), 면역세포, 줄기세포, 유도만능줄기세포(induced pluripotent stem cell; iPSC), 신경줄기세포(neural stem cell; NSC), T 세포, B 세포, 자연살해세포(natural Killer cell; NK cell), 대식세포(macrophage), 미세아교세포(microglia), 신경세포(neuron), 신경아교세포(glial cells), 성상세포(astrocyte) 및 근육세포(muscle cell)로 이루어진 군에서 선택되는 것일 수 있다. In another embodiment of the present invention, the cell is a brain blood barrier endothelial cell (brain endothelial cell), cancer cell, blood cell (blood cell), lymphocyte (lymphocyte), immune cell, stem cell, induced pluripotent stem cell (induced pluripotent stem) cell; iPSC), neural stem cell (NSC), T cell, B cell, natural killer cell (NK cell), macrophage, microglia, neuron ), glial cells, astrocytes, and muscle cells may be selected from the group consisting of.
또한, 본 발명은 상기 세포 투과성 펩타이드 및 생물학적 활성 물질을 포함하는, 복합체를 제공한다. In addition, the present invention provides a complex comprising the cell-penetrating peptide and a biologically active substance.
본 발명의 일구현예로, 생물학적 활성 물질은 화합물(chemical compound), 단백질, 당단백질, 펩타이드, 항체(antibody), 효소(enzyme), 핵산분해효소(nuclease), 호르몬, 사이토카인(cytokine), 전사인자(transcription factor), 독소, 핵산, 탄수화물, 지질, 당지질, 천연물(natural product), 반합성 물질 (semi-synthetic drug), 약물(drug), 마이크로입자, 나노입자, 리포좀, 바이러스, 양자점(quantum dots) 및 형광색소(fluorochrome)로 이루어진 군으로부터 선택되는 하나 이상인 것일 수 있다. In one embodiment of the present invention, the biologically active material is a compound (chemical compound), protein, glycoprotein, peptide, antibody (antibody), enzyme (enzyme), nuclease (nuclease), hormone, cytokine (cytokine), transcription factors, toxins, nucleic acids, carbohydrates, lipids, glycolipids, natural products, semi-synthetic drugs, drugs, microparticles, nanoparticles, liposomes, viruses, quantum dots dots) and may be at least one selected from the group consisting of fluorochromes.
본 발명의 다른 구현예로, 상기 핵산분해효소는 CAS9(CRISPR associated protein 9), CAS12, CAS13, CAS14, CAS variants, Cfp1(CxxC-finger protein-1), ZEN(Zinc-finger nucleases) 및 TALEN(Transcription activator-like effector nuclease)으로 이루어진 군에서 선택되는 것일 수 있다. In another embodiment of the present invention, the nuclease is CAS9 (CRISPR associated protein 9), CAS12, CAS13, CAS14, CAS variants, Cfp1 (CxxC-finger protein-1), ZEN (Zinc-finger nucleases) and TALEN ( Transcription activator-like effector nuclease) may be selected from the group consisting of.
본 발명의 또 다른 구현예로, 상기 핵산은 DNA, RNA, ASO(Antisense oligonucleotide), 마이크로RNA(microRNA; miRNA), 작은 간섭 RNA(small interfering RNA; siRNA), 앱타머(aptamer), LNA(locked nucleic acid), PNA(peptide nucleic acid) 및 모폴리노(morpholino)로 이루어진 군으로부터 선택되는 것일 수 있다.In another embodiment of the present invention, the nucleic acid is DNA, RNA, ASO (Antisense oligonucleotide), microRNA (microRNA; miRNA), small interfering RNA (siRNA), aptamer (aptamer), LNA (locked) nucleic acid), peptide nucleic acid (PNA), and morpholino may be selected from the group consisting of.
또한, 본 발명은 상기 복합체를 포함하는 물질 전달용 조성물을 제공한다. In addition, the present invention provides a composition for mass delivery comprising the complex.
또한, 본 발명은 상기 조성물을 세포에 처리하는 단계를 포함하는, 물질 전달방법을 제공한다. In addition, the present invention provides a material delivery method comprising the step of treating the cell with the composition.
또한, 본 발명은 상기 펩타이드를 암호화하는, 폴리뉴클레오티드를 제공한다. In addition, the present invention provides a polynucleotide encoding the peptide.
본 발명에서는 신규한 세포 투과성 펩타이드를 합성하였고, 이의 우수한 세포 투과능을 확인하였다. 따라서 본 발명에 따른 세포 투과성 펩타이드는 생물학적 활성을 갖는 물질을 세포, 조직 등 생체 내로 효과적으로 전달할 수 있어 기초연구 분야, 다양한 질병의 진단 및 치료 분야 등에서 유용하게 활용될 수 있을 것으로 기대된다.In the present invention, a novel cell-penetrating peptide was synthesized, and its excellent cell-penetrating ability was confirmed. Therefore, the cell-penetrating peptide according to the present invention is expected to be usefully utilized in the field of basic research, diagnosis and treatment of various diseases, etc. because it can effectively deliver a biologically active substance into a living body such as cells and tissues.
도 1은 16개 아미노산으로 이루어진 펩타이드에서 N-말단으로부터 1~4번 위치의 영역에서 아미노산을 치환시킨 펩타이드 후보군에 대한 in vitro 세포 투과성 분석 결과를 나타낸 것으로, 도 1a 및 도 1b는 우수한 세포 투과성이 확인된 펩타이드군에 대한 결과이고, 도 1c는 세포 투과성이 나타나지 않은 펩타이드군에 대한 결과이다.
도 2는 16개 아미노산으로 이루어진 펩타이드에서 N-말단으로부터 5~7번 위치의 영역에서 아미노산을 치환시킨 펩타이드 후보군에 대한 in vitro 세포 투과성 분석 결과를 나타낸 것으로, 도 2a는 우수한 세포 투과성이 확인된 펩타이드군에 대한 결과이고, 도 2b는 세포 투과성이 나타나지 않은 펩타이드군에 대한 결과이다.
도 3은 16개 아미노산으로 이루어진 펩타이드에서 N-말단으로부터 8~12번 위치의 영역에서 아미노산을 치환시킨 펩타이드 후보군에 대한 in vitro 세포 투과성 분석 결과를 나타낸 것으로, 도 3a 및 도 3b는 우수한 세포 투과성이 확인된 펩타이드군에 대한 결과이고, 도 3c는 세포 투과성이 나타나지 않은 펩타이드에 대한 결과이다.
도 4는 16개 아미노산으로 이루어진 펩타이드에서 N-말단으로부터 13~16번 위치의 영역에서 아미노산을 치환시킨 펩타이드 후보군에 대한 in vitro 세포 투과성 분석 결과를 나타낸 것으로, 도 4a는 우수한 세포 투과성이 확인된 펩타이드군에 대한 결과이고, 도 4b 및 도 4c는 세포 투과성이 나타나지 않은 펩타이드군에 대한 결과이다.
도 5는 16개 아미노산으로 이루어진 펩타이드의 전체 영역에서 하나 이상의 아미노산을 동시에 치환시킨 펩타이드 후보군에 대한 in vitro 세포 투과성 분석 결과를 나타낸 것으로, 도 5a 및 도 5b는 우수한 세포 투과성이 확인된 펩타이드군에 대한 결과이고, 도 5c는 세포 투과성이 나타나지 않은 펩타이드에 대한 결과이다. 1 shows the results of in vitro cell permeability analysis of a peptide candidate group in which amino acids are substituted in the region of
FIG. 2 shows the results of in vitro cell permeability analysis of a peptide candidate group in which amino acids are substituted in the region of
3 shows the results of an in vitro cell permeability analysis of a peptide candidate group in which amino acids are substituted in the region of
4 shows the results of an in vitro cell permeability analysis of a peptide candidate group in which amino acids are substituted in the region of
5 shows the results of an in vitro cell permeability analysis for a peptide candidate group in which one or more amino acids are simultaneously substituted in the entire region of a peptide consisting of 16 amino acids, and FIGS. 5A and 5B show the peptide group with excellent cell permeability. Results, Figure 5c is a result for the peptide did not show cell permeability.
본 발명은 기초연구 분야, 다양한 질병의 진단 및 치료 분야 등에서 유용하게 활용될 수 있는 세포 투과성 펩타이드에 관한 것으로서, 한정되지 않은 수의 디자인으로 확장 가능한 기본 플랫폼 펩타이드 구조에 관한 것이다.The present invention relates to a cell-permeable peptide that can be usefully utilized in the field of basic research, diagnosis and treatment of various diseases, and the like, and relates to a basic platform peptide structure that can be expanded to an unlimited number of designs.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 하기의 [일반식 I]로 표시되는 아미노산 서열로 이루어진, 세포 투과성 펩타이드를 제공한다. The present invention provides a cell-penetrating peptide consisting of an amino acid sequence represented by the following [General Formula I].
[일반식 I][General Formula I]
X1-[Xa-L-Xb]-X2-V-X3 X 1 -[X a -LX b ]-X 2 -VX 3
X1은 GHHE, AHHE, GHAE, SHHE, AHAE, GHNE, GHHD, GHKE, GHGE, GHRE, GHVE, GHQE 또는 GHHG이고; X 1 is GHHE, AHHE, GHAE, SHHE, AHAE, GHNE, GHHD, GHKE, GHGE, GHRE, GHVE, GHQE or GHHG;
Xa는 Arg(R), Ala(A), Ile(I), Val(V), Leu(L), Gly(G), Asn(N), Lys(K) 또는 Gln(Q)이고;X a is Arg(R), Ala(A), Ile(I), Val(V), Leu(L), Gly(G), Asn(N), Lys(K) or Gln(Q);
Xb 는 Lys(K), Arg(R), His(H), Asn(N) 또는 Gln(Q)이고;X b is Lys(K), Arg(R), His(H), Asn(N) or Gin(Q);
X2는 SDEWS, ADEWS, CDEWS, LDEWS, QDEWS, VDEWS, NDEWS, TDEWS, RDEWS, SDSWS, SDEYS, GDEWS, SSEWS, SDFWS 또는 SDEFS이고; X 2 is SDEWS, ADEWS, CDEWS, LDEWS, QDEWS, VDEWS, NDEWS, TDEWS, RDEWS, SDSWS, SDEYS, GDEWS, SSEWS, SDFWS or SDEFS;
X3은 TSG, NSA, NSV, QSG, KSG, RSG, ISG, SSG, TRG, TNG, TKG, TQG 또는 NSG 이다.X 3 is TSG, NSA, NSV, QSG, KSG, RSG, ISG, SSG, TRG, TNG, TKG, TQG or NSG.
본 발명에서 사용된 아미노산 서열은 IUPAC-IUB 명명법에 따라 다음과 같이 약어로 기재하였다.The amino acid sequence used in the present invention is abbreviated as follows according to the IUPAC-IUB nomenclature.
본 발명에서 사용되는 용어, “세포 투과성”이란, 펩타이드가 세포(막)를 투과하여 세포 내부로 침투할 수 있는 능력 또는 성질을 의미한다.As used herein, the term “cell permeability” refers to the ability or property of a peptide to penetrate a cell (membrane) and penetrate into the cell.
본 발명에서, “펩타이드(peptide)”란 아미노산의 중합체로서, 보통 소수의 아미노산이 연결된 형태를 펩타이드라 하며, 많은 아미노산이 연결되면 단백질이라 부른다. 이러한 펩타이드 및 단백질 구조에서 아미노산 간의 연결은 아마이드(amide) 결합 또는 펩타이드 결합으로 이루어져 있다. 펩타이드 결합이란 카르복실기(-COOH)와 아미노기(-NH2) 사이에 물(H2O)이 빠져나가고 -CO-NH- 형태를 이루는 결합이다. In the present invention, a “peptide” is a polymer of amino acids, usually a form in which a few amino acids are linked is called a peptide, and when many amino acids are linked, it is called a protein. In these peptide and protein structures, the linkage between amino acids consists of an amide bond or a peptide bond. A peptide bond is a bond between a carboxyl group (-COOH) and an amino group (-NH 2 ) through which water (H 2 O) escapes and forms -CO-NH-.
본 발명에 따른 상기 [일반식 I]의 신규 펩타이드는 대표적으로 서열번호 1 내지 서열번호 62로 이루어진 군에서 선택되는 어느 하나의 아미노산 서열일 수 있으나, 이로 제한되는 것은 아니다. 이때, 상기 세포 투과성 펩타이드는 상기 서열번호 1 내지 62로 표시되는 아미노산 서열과 각각 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 가장 바람직하게는 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 이상의 서열 상동성을 가지는 아미노산 서열을 포함할 수도 있다.The novel peptide of [General Formula I] according to the present invention may be any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 62, but is not limited thereto. In this case, the cell-penetrating peptide is 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 91%, 92%, 93 of the amino acid sequence shown in SEQ ID NOs: 1 to 62, respectively. %, 94%, 95%, 96%, 97%, 98%, 99% or more of an amino acid sequence having sequence homology.
본 발명에 있어서, 상기 [일반식 I]로 나타내어지는 세포 투과성 펩타이드의 N-말단 및C-말단에는 상기 세포 투과성 펩타이드로서의 효과를 증대시킬 수 있는 다양한 아미노산을 추가로 부가하거나 결실시킬 수도 있다. In the present invention, various amino acids capable of enhancing the effect as the cell-permeable peptide may be additionally added or deleted to the N-terminus and C-terminus of the cell-permeable peptide represented by the [General Formula I].
본 발명에 있어서, 상기 세포 투과성 펩타이드가 투과 가능한 세포의 종류로는, 이에 제한되는 것은 아니나, 뇌혈관장벽 내피세포(brain endothelial cell), 암세포, 혈액세포(blood cell), 림프구(lymphocyte), 면역세포, 줄기세포, 유도만능줄기세포(induced pluripotent stem cell; iPSC), 신경줄기세포(neural stem cell; NSC), T 세포, B 세포, 자연살해세포(natural Killer cell; NK cell), 대식세포(macrophage), 미세아교세포(microglia), 신경세포(neuron), 신경아교세포(glial cells), 성상세포(astrocyte) 및 근육세포(muscle cell)로 이루어진 군에서 선택되는 어느 하나일 수 있다. In the present invention, the types of cells permeable to the cell-penetrating peptide include, but are not limited to, brain endothelial cells, cancer cells, blood cells, lymphocytes, and immune cells. Cells, stem cells, induced pluripotent stem cells (iPSCs), neural stem cells (NSCs), T cells, B cells, natural killer cells (NK cells), macrophages (macrophage) ), microglia, nerve cells (neuron), glial cells (glial cells), astrocytes (astrocyte) and may be any one selected from the group consisting of muscle cells (muscle cell).
본 발명의 펩타이드는 당업자에게 알려진 통상의 펩타이드 합성 방법 혹은 제조 방법을 통하여 각 펩타이드의 순도가 90% 이상이 되도록 제작할 수 있으며, 예컨대 직접 합성하거나 펩타이드 제조회사에 제조를 의뢰한 후 구입하여 사용할 수 있다. 상기 펩타이드는 당업자에게 알려진 통상의 펩타이드 합성 방법 혹은 제조 방법을 통하여 D-form이나 L-form, 서열 중 일부만 D-form이나 L-form으로 구성된 펩타이드, 또는 이들의 라세미체 형태로 모두 제작하여 사용될 수 있다. 또한, 펩타이드의 안정성을 높이기 위해 그 외의 당업계에 공지된 통상적인 변형이 가능하다. 본 발명에서는 바람직하게 고체상 펩타이드 합성(Solid state peptide synthesis) 방법을 이용하여 펩타이드를 합성하였으나, 전술한 바와 같이 펩타이드 합성 방법 및 조건이 이에 제한되는 것은 아니다.The peptide of the present invention can be manufactured so that the purity of each peptide is 90% or more through a conventional peptide synthesis method or manufacturing method known to those skilled in the art, for example, it can be synthesized directly or purchased after requesting production from a peptide manufacturer. . The peptide can be used by producing both D-form or L-form, a peptide composed of only a part of the D-form or L-form sequence, or a racemate form thereof through a conventional peptide synthesis method or manufacturing method known to those skilled in the art. can In addition, in order to increase the stability of the peptide, other conventional modifications known in the art are possible. In the present invention, the peptide was preferably synthesized using a solid state peptide synthesis method, but as described above, the peptide synthesis method and conditions are not limited thereto.
또한, 본 발명은 상기 펩타이드를 암호화하는, 폴리뉴클레오티드를 제공한다. In addition, the present invention provides a polynucleotide encoding the peptide.
본 발명에 있어서, 폴리뉴클레오티드는 RNA 또는 DNA의 형태일 수 있는데, 상기 DNA는 cDNA 및 합성 DNA를 포함한다. DNA는 단일 가닥이거나 이중 가닥일 수 있다. 만약 단일 가닥이라면, 이는 코딩 가닥 또는 비-코딩(안티센스) 가닥일 수 있고, 상기 코딩 서열은, 유전적 코드의 축퇴성(degeneracy) 또는 중복성(redundancy)의 결과로서, 동일한 폴리펩티드를 인코딩한다.In the present invention, the polynucleotide may be in the form of RNA or DNA, and the DNA includes cDNA and synthetic DNA. DNA can be single-stranded or double-stranded. If single-stranded, it may be the coding strand or the non-coding (antisense) strand, wherein the coding sequence encodes the same polypeptide, as a result of the degeneracy or redundancy of the genetic code.
본 발명의 폴리뉴클레오티드는 또한 상기에 기술된 폴리뉴클레오티드의 변이체를 포함할 수 있으며, 상기 폴리뉴클레오티드의 변이체는 폴리뉴클레오티드의 자연적으로 발생하는 대립(allelic) 변이체 또는 폴리뉴클레오티드의 비-자연적으로 발생하는 변이체일 수 있다. 대립 변이체는, 인코딩(암호화)되는 폴리뉴클레오티드의 기능을 실질적으로 변경하지 않는, 하나 이상의 뉴클레오티드들의 치환, 결실, 또는 부가를 가질 수 있는 폴리염기서열의 교대(alternate) 형태이다. 단일 아미노산이 하나 이상의 뉴클레오티드 코돈에 의해 인코딩될 수 있고 상기 폴리뉴클레오티드가 동일한 펩타이드를 암호화하는 교대 폴리뉴클레오티드를 제조하도록 용이하게 변형될 수 있음이 당업계에 잘 알려져 있다.The polynucleotides of the present invention may also include variants of the polynucleotides described above, wherein the variants of the polynucleotide are either naturally occurring allelic variants of the polynucleotide or non-naturally occurring variants of the polynucleotide. can be Allelic variants are alternate forms of a polynucleotide sequence that may have substitutions, deletions, or additions of one or more nucleotides that do not substantially alter the function of the polynucleotide being encoded (encoded). It is well known in the art that a single amino acid can be encoded by more than one nucleotide codon and that the polynucleotide can be readily modified to produce alternating polynucleotides encoding the same peptide.
본 발명자들은, 이전 선행 연구를 통해 세포 투과능이 우수한 세포 투과성 펩타이드를 발굴하였으며, 우수한 세포 투과능을 갖는 펩타이드를 추가적으로 발굴하기 위하여, 상기 발굴된 펩타이드의 서열 및 구조를 기반으로 하나 이상의 아미노산을 치환시켜 다양한 조합의 펩타이드 후보군을 설계 및 합성하였고, 이의 세포 투과능을 분석함으로써 본 발명에 따른 우수한 세포 투과능을 갖는 다양한 세포 투과성 펩타이드들을 발굴하였다. The present inventors have discovered cell-penetrating peptides having excellent cell-penetrating ability through previous prior studies, and in order to additionally discover peptides having excellent cell-penetrating ability, one or more amino acids are substituted based on the sequence and structure of the discovered peptides Various combinations of candidate peptides were designed and synthesized, and various cell-penetrating peptides having excellent cell-penetrating ability according to the present invention were discovered by analyzing their cell-penetrating ability.
보다 구체적으로, 본 발명의 실시예에서는 16개 아미노산을 4개 구역(N-말단으로부터 1~4번, 5~7번, 8~12번 및 13~16번 위치 영역)으로 나누고, 구역별로 또는 2개 이상의 구역을 동시에 하나 이상의 아미노산을 다양한 조합으로 치환시킨 세포 투과성 펩타이드 후보군들을 설계 및 합성하였으며, 상기 후보군들에 대하여 in vitro 세포 투과성 분석을 실시하여 최종적으로 총 62종의 세포 투과성 펩타이드를 발굴하였다(실시예 1 및 2 참조).More specifically, in the embodiment of the present invention, 16 amino acids are divided into 4 regions (
상기 실시예의 결과로부터, 본 발명에 따른 펩타이드는 우수한 세포 투과성을 가지며, 이를 기반으로 상기 펩타이드와 결합한 임의의 물질을 세포 내로 효과적으로 유입시킬 수 있는 전달체로서 활용 가능함을 알 수 있었다.From the results of the above examples, it was found that the peptide according to the present invention has excellent cell permeability, and based on this, it can be used as a carrier that can effectively introduce any substance bound to the peptide into the cell.
이에, 본 발명의 다른 양태로서, 본 발명은 상기 세포 투과성 펩타이드 및 생물학적 활성 물질을 포함하는 복합체를 제공한다.Accordingly, as another aspect of the present invention, the present invention provides a complex comprising the cell-penetrating peptide and a biologically active substance.
본 발명에 있어서, 상기 복합체는 펩타이드와 물질이 단순히 혼합(mixing)된 것, 펩타이드와 물질이 혼합되어 형성된 것, 또는 이들이 화학적 결합에 의해 연결되거나 컨쥬게이션되어 생성된 것을 모두 포함한다. 또한, 복합체는 물리적 결합, 화학적 결합, 공유 결합, 비공유 결합, 자기조립화로 연결되거나, 매개체를 이용하여 통합된 또는 융합된 형태로 연결될 수 있다.In the present invention, the complex includes all of a compound formed by simply mixing a peptide and a substance, a compound formed by mixing a peptide and a substance, or a compound formed by connecting or conjugating them by a chemical bond. In addition, the complex may be linked by a physical bond, a chemical bond, a covalent bond, a non-covalent bond, self-assembly, or may be linked in an integrated or fused form using a mediator.
또한, 상기 복합체는 상기 펩타이드와 생물학적 활성 물질이 서로 융합(fusion)된 상태로 발현되어 이룬 복합체가 될 수 있다. 예를 들어, 하나의 벡터 내에 상기 펩타이드와 생물학적 활성 물질을 발현하는 유전자를 삽입한 후, 상기 벡터로 생물을 형질전환시켜 벡터에 삽입된 유전자를 발현하도록 하면, 상기 펩타이드와 생물학적 활성 물질이 융합 단백질(fusion protein)로서 발현될 수 있다. 융합 단백질로 발현될 때, 상기 펩타이드와 생물학적 활성 물질 간에 임의의 링커가 포함되도록 할 수 있다. In addition, the complex may be a complex formed by expressing the peptide and the biologically active material in a fusion state. For example, when a gene expressing the peptide and a biologically active substance is inserted into one vector, and then an organism is transformed with the vector to express the gene inserted into the vector, the peptide and the biologically active substance are fusion proteins (fusion protein) can be expressed. When expressed as a fusion protein, any linker may be included between the peptide and the biologically active material.
또한, 본 발명에 따른 복합체에서, 상기 세포 투과성 펩타이드는 생물학적 활성 물질을 세포 내로 효율적으로 전달하기 위해서, 단일 또는 복수개가 결합된 형태를 모두 포함할 수 있으며, 전달하고자 하는 생물학적 활성물질에 따라 세포 투과성 펩타이드의 결합 개수는 당업자에게 용이하게 선택 또는 조절될 수 있다. In addition, in the complex according to the present invention, the cell-penetrating peptide may include both single or multiple combined forms in order to efficiently deliver a biologically active substance into a cell, and the cell-penetrating peptide may be cell-permeable depending on the biologically active substance to be delivered. The number of peptide bonds can be easily selected or adjusted by those skilled in the art.
본 발명에서, 세포 투과성 펩타이드에 결합되어 복합체를 형성할 수 있는 생물학적 활성 물질은 바람직하게는 '생물학적 또는 약제학적 활성을 갖는 물질'을 의미하며, 이는 세포 내(세포질 또는 핵 내)로 투과되어 생리활성조절에 관여하거나 약리효과를 발현할 수 있는 것 또는 운반되어 작용해야 하는 세포 내, 조직 내, 세포간질, 혈액 등 다양한 생체 내 부위에서도 생물학적 활성을 갖는 물질을 의미한다. 예컨대, 이에 제한되지는 않으나, 화합물(chemical compound), 단백질, 당단백질, 펩타이드 항체(antibody), 효소(enzyme), 핵산분해효소(nuclease), 호르몬, 사이토카인(cytokine), 전사인자(transcription factor), 독소, 핵산, 탄수화물, 지질, 당지질, 천연물(natural product), 반합성 물질(semi-synthetic drug), 약물(drug), 마이크로입자, 나노입자, 리포좀, 바이러스, 양자점(quantum dots), 및 형광색소(fluorochrome)로 이루어진 군으로부터 선택되는 하나 이상인 것일 수 있다. In the present invention, a biologically active substance capable of forming a complex by binding to a cell-penetrating peptide preferably means 'a substance having biological or pharmaceutical activity', which penetrates into the cell (in the cytoplasm or nucleus) and is physiologically It refers to a substance that is involved in the regulation of activity or can express a pharmacological effect, or has biological activity in various parts of the body, such as intracellular, tissue, intercellular, and blood, which must be transported and acted upon. For example, but not limited to, a chemical compound, a protein, a glycoprotein, a peptide antibody, an enzyme, a nuclease, a hormone, a cytokine, a transcription factor ), toxins, nucleic acids, carbohydrates, lipids, glycolipids, natural products, semi-synthetic drugs, drugs, microparticles, nanoparticles, liposomes, viruses, quantum dots, and fluorescence It may be one or more selected from the group consisting of dyes (fluorochromes).
상기 핵산분해효소는 CAS9(CRISPR associated protein 9), CAS12, CAS13, CAS14, CAS variants, Cfp1(CxxC-finger protein-1), ZEN(Zinc-finger nucleases) 및 TALEN(Transcription activator-like effector nuclease)으로 이루어진 군에서 선택되는 것일 수 있으나, 이에 제한되는 것은 아니다. The nucleases are CAS9 (CRISPR associated protein 9), CAS12, CAS13, CAS14, CAS variants, CxxC-finger protein-1 (Cfp1), Zinc-finger nucleases (ZEN) and transcription activator-like effector nuclease (TALEN). It may be selected from the group consisting of, but is not limited thereto.
상기 핵산은 DNA, RNA, ASO(Antisense oligonucleotide), 마이크로RNA(microRNA; miRNA), 작은 간섭 RNA(small interfering RNA; siRNA), 앱타머(aptamer), LNA(locked nucleic acid), PNA(peptide nucleic acid), 및 모폴리노(morpholino)로 이루어진 군으로부터 선택될 수 있고, 추가적으로 decoy DNA, plasmid, shRNA, 안티센스 RNA, 올리고리보뉴클레오티드, 또는 전달 RNA(transfer RNA) 등을 포함할 수 있으나, 이에 제한되지는 않는다. The nucleic acid is DNA, RNA, antisense oligonucleotide (ASO), microRNA (miRNA), small interfering RNA (siRNA), aptamer, LNA (locked nucleic acid), PNA (peptide nucleic acid) ), and may be selected from the group consisting of morpholino, and may additionally include decoy DNA, plasmid, shRNA, antisense RNA, oligoribonucleotide, or transfer RNA, but is not limited thereto. does not
상기 약물(drug)은 화합물 약물(chemical drug), 바이오 약물(bio drug), 핵산 약물(nucleic acid drug), 펩타이드 약물(peptide drug), 단백질 약물(protein drug), 천연물 약물(natural product drug), 호르몬(hormone), 조영제(contrast agent) 및 항체(antibody)로 이루어진 군으로부터 선택될 수 있으나, 이에 제한되는 것은 아니다. The drug (drug) is a compound drug (chemical drug), bio drug (bio drug), nucleic acid drug (nucleic acid drug), peptide drug (peptide drug), protein drug (protein drug), natural product drug (natural product drug), It may be selected from the group consisting of a hormone, a contrast agent, and an antibody, but is not limited thereto.
상기 “바이오 약물”은 (오리지널) 생물학적 치료제(biologics) 및 바이오제네릭(biogenerics), 바이오베터(biobetters), 바이오서피어리어(biosuperiors) 등 다양한 바이오 의약품을 의미한다. 상기 바이오 약물은 생물학적 기원으로부터 제조, 분비 또는 반합성된 임의의 약물을 의미하며, 백신, 혈액 제제, 항원, 세포 제제, 유전자 치료제, 줄기세포 등을 모두 포함하며 이에 제한되지는 않는다.The “bio drug” refers to various biologics such as (original) biologics, biogenerics, biobetters, and biosuperiors. The bio-drug means any drug manufactured, secreted or semi-synthesized from a biological origin, and includes, but is not limited to, vaccines, blood products, antigens, cell products, gene therapy products, stem cells, and the like.
상기 나노입자는 산화철, 금, 탄소나노튜브, 및 자기 비드로 이루어진 군으로부터 선택되는 것일 수 있으나, 이에 제한되지는 않는다.The nanoparticles may be selected from the group consisting of iron oxide, gold, carbon nanotubes, and magnetic beads, but is not limited thereto.
본 발명의 또 다른 양태로서, 본 발명은 상기 복합체를 유효성분으로 포함하는 물질 전달용 조성물을 제공한다. As another aspect of the present invention, the present invention provides a composition for mass transfer comprising the complex as an active ingredient.
상기 물질 전달용 조성물은 생물학적 활성 물질을 생체 조직 또는 혈중으로 전달시키거나 세포 투과를 촉진시키기 위해 사용될 수 있다. 상기 조성물은 생체 조직을 구성하는 세포 또는 세포 간 연접을 통하여 전달될 수 있으나 전달 방식에는 제한이 없다.The composition for mass delivery may be used to deliver a biologically active material to a living tissue or blood or to promote cell permeation. The composition may be delivered through cells constituting a living tissue or cell-to-cell junctions, but there is no limitation in the delivery method.
상기 생체 조직은 하나 이상의 상피조직, 근육조직, 신경조직, 결합조직을 의미하며 각 장기는 하나 이상의 조직으로 이루어질 수 있으므로, 점막, 피부, 뇌, 폐, 간, 신장, 비장, 폐장, 심장, 위장, 대장, 소화관, 방광, 요관, 요도, 난소, 정소, 생식기, 근육, 혈액, 혈관, 림프관, 림프절, 흉선, 췌장, 부신, 갑상선, 부갑상선, 후두, 편도, 기관지, 폐포의 다양한 생체 장기가 포함될 수 있으나, 이에 제한되지는 않는다.The biological tissue refers to one or more epithelial tissue, muscle tissue, nervous tissue, and connective tissue, and each organ may consist of one or more tissues, so mucosa, skin, brain, lung, liver, kidney, spleen, lung, heart, stomach , large intestine, digestive tract, bladder, ureter, urethra, ovary, testis, genitalia, muscle, blood, blood vessels, lymphatic vessels, lymph nodes, thymus, pancreas, adrenal gland, thyroid gland, parathyroid gland, larynx, tonsils, bronchi, alveoli However, it is not limited thereto.
상기 복합체를 특정 세포, 조직 또는 장기로 전달하고자 하는 경우, 상기 생물학적 활성을 갖는 물질은 특정 세포, 조직 또는 장기에서 특이적으로 발현하는 수용체와 선택적으로 결합할 수 있는 리간드의 세포외 부분 단백질, 또는 이들 수용체 또는 리간드와 특이적으로 결합할 수 있는 단일클론항체(mAb) 및 변형된 형태와 결합하여 복합체를 형성할 수 있다. 상기 펩타이드와 생물학적 활성을 갖는 물질의 결합은 뉴클레오티드 수준에서 발현 벡터를 이용한 클로닝 기법에 의한 간접적인 연결에 의하거나 펩타이드와 생물학적 활성을 갖는 물질의 화학적 또는 물리적 공유 결합 또는 비공유 결합에 의한 직접적인 연결에 의할 수 있다.When the complex is to be delivered to a specific cell, tissue or organ, the material having the biological activity is an extracellular partial protein of a ligand capable of selectively binding to a receptor specifically expressed in a specific cell, tissue or organ, or A complex can be formed by binding to monoclonal antibodies (mAbs) capable of specifically binding these receptors or ligands and modified forms. The binding between the peptide and the biologically active substance is either by indirect linking by cloning technique using an expression vector at the nucleotide level, or by direct linking by chemical or physical covalent or non-covalent binding between the peptide and the biologically active substance. can do.
본 발명에 있어서, 상기 복합체를 포함하는 조성물이 약제학적 조성물로 이용되는 경우, 상기 조성물은 조성물 총 중량에 대하여 상기 유효성분을 0.0001 내지 50 중량%로 포함할 수 있다. In the present invention, when the composition including the complex is used as a pharmaceutical composition, the composition may include the active ingredient in an amount of 0.0001 to 50% by weight based on the total weight of the composition.
본 발명의 조성물은 상기 유효성분에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다.The composition of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the active ingredients.
본 발명의 조성물은 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 제조할 수 있다. 약제학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올, 리포좀 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있으며, 표적 기관에 특이적으로 작용할 수 있도록 표적 기관 특이적 항체 또는 기타 리간드를 상기 담체와 결합시켜 사용할 수 있다. 더 나아가 당해 기술분야의 적정한 방법으로 또는 레밍턴의 문헌에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The composition of the present invention can be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration. The pharmaceutically acceptable carrier may be used in a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and one or more of these components, and, if necessary, an antioxidant , buffers, bacteriostatic agents, and other conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to form injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, and may act specifically on target organs. A target organ-specific antibody or other ligand may be used in combination with the carrier. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's literature.
상기 복합체를 유효성분으로 포함하는 조성물은 정맥내(intravenous), 복막내(intraperitoneal), 근육내(intramuscular), 척수강내(intrathecal), 뇌실질내(intracerebroventricular), 피하내(subcutaneous), 피내(intradermal), 비내(nasal), 점막내(mucosal), 흡입(inhalation) 및 경구(oral) 등의 경로로 주입함으로써 생체 내로 전달될 수 있다. 투여량은 대상의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다.The composition comprising the complex as an active ingredient is intravenous (intravenous), intraperitoneal (intraperitoneal), intramuscular (intramuscular), intrathecal (intrathecal), intracerebroventricular (intracerebroventricular), subcutaneous (subcutaneous), intradermal , intranasal (nasal), intramucosal (mucosal), inhalation (inhalation), it can be delivered into the living body by injecting orally (oral). The dosage range varies according to the subject's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease.
본 발명의 또 다른 양태로서, 본 발명은 상기 물질 전달용 조성물을 세포에 처리하는 단계를 포함하는 세포 내로의 물질 전달 방법을 제공한다. As another aspect of the present invention, the present invention provides a method for transferring a mass into a cell, comprising the step of treating the cell with the composition for mass transfer.
본 발명에 따른 물질 전달기능을 가지는 세포 투과성 펩타이드는 매우 작은 펩타이드이므로 혹시 발생할 수 있는 활성물질에 대한 생물학적 간섭을 최소화할 수 있다. Since the cell-penetrating peptide having a mass transfer function according to the present invention is a very small peptide, it is possible to minimize any biological interference with the active material that may occur.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.
[실시예][Example]
실시예 1. 세포 투과성 펩타이드 후보군 설계 및 합성Example 1. Cell-permeable peptide candidate group design and synthesis
1-1. 아미노산 치환을 통한 세포 투과성 펩타이드 후보군 설계1-1. Cell-penetrating peptide candidate group design through amino acid substitution
본 발명자들은 이전 선행 연구결과 우수한 세포 투과능을 갖는 16개 아미노산으로 이루어진 펩타이드를 발굴하였다(N-GHHERLKSDEWSVTSG-C). 나아가 우수한 세포 투과능을 갖는 펩타이드를 추가적으로 발굴하기 위하여, 상기 발굴된 펩타이드의 서열 및 구조를 분석하여 안정성, 세포 투과성 등을 고려해 16개 아미노산을 4개 구역(N-말단으로부터 1~4번, 5~7번, 8~12번 및 13~16번 위치 영역)으로 나누고, 구역별로 또는 2개 이상의 구역에서 하나 이상의 아미노산을 치환시킨 다양한 조합의 세포 투과성 펩타이드 후보군들을 설계 및 합성하였다. The present inventors discovered a peptide consisting of 16 amino acids having excellent cell permeability as a result of previous studies (N-GHHERLKSDEWSVTSG-C). Furthermore, in order to additionally discover peptides having excellent cell-penetrating ability, the sequence and structure of the discovered peptides were analyzed and 16 amino acids were divided into 4 regions (N-
1-2. 펩타이드 후보군의 합성 및 분리 정제1-2. Synthesis, separation and purification of peptide candidates
본 발명자들은 상기 실시예 1-1에 기재된 각 펩타이드를 합성하기 위하여 고체상 펩타이드 합성(Solid state peptide synthesis; SPPS) 방법을 이용하였다. 상기 방법은 레진의 N-말단에 N-말단이 F-moc로 보호되어 있는 아미노산의 C-말단을 하나씩 결합하는 유기합성법이다. 모든 반응의 용매는 N,N-다이메틸포름아마이드(N,N-dimethylformamide; DMF)를 이용하였으며, 아미노산 커플링 조건은 2M 농도의 아미노산 용액을 0.5M DIC(N,N′-Diisopropylcarbodiimide; DIC) 1ml, 1M Ethyl cyanohydroxyiminoacetate(Oxyma) 0.5ml와 혼합하여 마이크로웨이브 합성기(Microwave synthesizer)에서 반응시켜 진행하였다. 또한 각 아미노산 서열마다 반응시간, 온도 또는 마이크로웨이브의 전압을 달리하여 아미노산을 제조하였다. 다음 아미노산을 합성하기 위해서는 이전 아미노산의 F-moc을 제거해야 하기 때문에 이를 위해 80% DMF와 20% 피페리딘(piperidine) 용액을 사용해 F-moc 보호기를 80℃에서 2분 동안 2회 디프로텍팅(deprotecting) 시켰다. 모든 커플링 과정 및 디프로텍팅 과정 사이에는 DMF와 염화메틸렌(dichloromethane; DCM)을 이용해 교대로 3회씩 세척하는 과정을 수행하였다.The present inventors used a solid state peptide synthesis (SPPS) method to synthesize each of the peptides described in Example 1-1. The method is an organic synthesis method in which the C-terminus of an amino acid whose N-terminus is protected with F-moc is joined to the N-terminus of the resin one by one. The solvent for all reactions was N,N -dimethylformamide ( N,N- dimethylformamide; DMF), and the amino acid coupling condition was 0.5M DIC ( N,N ′-Diisopropylcarbodiimide; DIC) in an amino acid solution of 2M concentration. 1ml, 0.5ml of 1M Ethyl cyanohydroxyiminoacetate (Oxyma) was mixed and reacted in a microwave synthesizer. In addition, amino acids were prepared by varying the reaction time, temperature, or microwave voltage for each amino acid sequence. In order to synthesize the next amino acid, the F-moc of the previous amino acid must be removed. For this, deprotect the F-moc protecting group twice at 80 ° C for 2 minutes using 80% DMF and 20% piperidine solution ( deprotecting). Between all the coupling processes and the deprotecting process, a process of washing three times with DMF and methylene chloride (dichloromethane; DCM) was alternately performed.
상기 방법을 통해 합성된 펩타이드에 대하여, 추후 세포 투과성 여부를 관찰하고 정량화하기 위해 펩타이드의 N-말단에 카르복실기 (-COOH)를 포함하는 형광물질을 화학적 결합방법으로 연결할 수 있다. 이때 사용할 수 있는 상기 형광물질로는 5-FAM(5-carboxylfluorecein), FITC(Fluorescein-5-isothiocyanate), 시아닌 3 카르복시산(Cyanine 3 carboxylic acid), 시아닌 5 카르복시산(Cyanine5 carboxylic acid), 시아닌 7 카르복시산(Cyanine 7 carboxylic acid) 등이 있으며, 상기 형광물질 중 하나 이상을 사용할 수 있다.For the peptide synthesized through the above method, a fluorescent substance including a carboxyl group (-COOH) at the N-terminus of the peptide may be linked by a chemical bonding method in order to later observe and quantify cell permeability. In this case, the fluorescent material that can be used is 5-FAM (5-carboxylfluorecein), FITC (Fluorescein-5-isothiocyanate),
이를 위해, 본 발명자들은 먼저 고체상의 레진에 합성된 펩타이드의 마지막 아미노산을 합성한 뒤 5-FAM : DIC : Oxyma : 레진을 2 : 2.5 : 4 : 1 비율로 잘 혼합시킨 후 상기 레진에서의 반응을 상온에서 2시간 동안 진행하되, 교반기(magnetic stirrer)를 이용하였다. 다음으로, 상기 합성과정 중 레진 색이 노란색에서 진한 노랑 또는 주황빛으로 변하면 DMF와 염화메틸렌을 교대로 3회씩 세척하는 과정을 실시하였다. 이어서 고체상 레진으로부터 펩타이드/5-FAM을 분리하기 위해 트리플루오로아세트산(Trifluoroacetic acid; TFA) : 트리이소프로필실란(Triisopropylsilane; TIS) : 증류수가 95 : 2.5 : 2.5로 혼합된 클리비지 용액에서 합성이 완료된 레진을 교반기를 이용해 2시간 동안 반응을 진행한 후 석면필터로 레진을 걸러 내었다. 걸러진 용액에서 용액은 질소 기체 하에서 증발시키고 침전물이 생기면 차갑게 보관한 다이에틸 에테르(diethyl ether)로 침전시켰다. 침전된 펩타이드/5-FAM은 진공상태에서 건조시킨 후 증류수로 녹여 동결건조 하였다. To this end, the present inventors first synthesized the last amino acid of the peptide synthesized in a solid resin, and then mixed well with 5-FAM: DIC: Oxyma: resin in a 2: 2.5: 4: 1 ratio, and then reacted in the resin. The process was carried out at room temperature for 2 hours, but a magnetic stirrer was used. Next, when the color of the resin changed from yellow to dark yellow or orange during the synthesis process, a process of washing DMF and methylene chloride three times was alternately performed. Then, in order to separate the peptide/5-FAM from the solid resin, the synthesis was completed in a cleavage solution in which trifluoroacetic acid (TFA): triisopropylsilane (TIS): distilled water was mixed in a ratio of 95: 2.5: 2.5. After the resin was reacted for 2 hours using a stirrer, the resin was filtered out with an asbestos filter. From the filtered solution, the solution was evaporated under nitrogen gas, and when a precipitate was formed, it was precipitated with diethyl ether stored cold. The precipitated peptide/5-FAM was dried under vacuum and then dissolved in distilled water and freeze-dried.
동결건조된 펩타이드는 증류수나 아세토니트릴(Acetonitrile; ACN) 등으로 용해시키고 역상 고성능 액체 크로마토그래피(Reverse phase High-performance liquid chromatography)를 사용하여 분리 및 정제하였다. 이 때 상기 HPLC의 이동상 용매로는 Solvent A(증류수 99.9%, TFA 0.1%)와 Solvent B(증류수 9.9%, 아세토니트릴 90%, TFA 0.1%)를 사용하였다. HPLC 이동상은 Solvent A 90%와 Solvent B 10%로 시작하여 Solvent B를 1%/min gradient로 증가시키며 분리를 진행하였다. 이후 분리된 펩타이드 용액은 동결건조하여 용매를 제거하고 이후 원하는 용매에 녹여 실험을 진행하였다. The lyophilized peptide was dissolved in distilled water or acetonitrile (ACN), and separated and purified using reverse phase high-performance liquid chromatography. At this time, solvent A (distilled water 99.9%, TFA 0.1%) and solvent B (distilled water 9.9%, acetonitrile 90%, TFA 0.1%) were used as mobile phase solvents of the HPLC. The HPLC mobile phase started with Solvent A 90% and
실시예 2. Example 2. In vitroin vitro 세포 투과성 분석 Cell permeability assay
2-1. 실험 방법2-1. Experimental method
본 발명자들은 상기 실시예 1에서 합성한 펩타이드 후보군들의 세포 투과성을 평가하기 위하여, in vitro에서 뇌혈관장벽세포에 대한 투과성 분석을 실시하였다.In order to evaluate the cell permeability of the peptide candidates synthesized in Example 1, the present inventors performed an in vitro permeability analysis on the blood-brain barrier cells.
구체적으로, 인간 뇌혈관장벽세포인 hCMEC/D3 세포를 96웰 플레이트에 분주하고, 세포가 플레이트 면적의 80~90%에 도달할 때까지 상피세포 성장 배지인 EGM(endothelial cell growth medium)에서 37℃, CO2 조건으로 밤새 배양하였다. 이어서 상기 실시예 1-2에서 제조한 FAM이 연결된 후보군 펩타이드들 및 음성대조군인 FAM 단독을 4uM로 성장인자가 보충된 상기 배지에 희석하여 각 웰당 100μL씩 처리할 양을 제조하였다. 이어서 세포를 배양한 플레이트에서 배양액을 흡입(suction)하여 제거하고, 상기 펩타이드를 희석하여 미리 제조한 용액을 처리한 후 2시간 동안 37℃, CO2 조건으로 배양하였다. 다음으로, 각 웰에서 펩타이드를 처리한 용액을 모두 흡입기로 제거하고, EGM 100μL를 첨가하여 5~6회 tapping한 후, 제거하는 과정을 2회 반복하였다. 이후 Hoechst 33342를 EGM에 1:5000으로 희석하여 각 웰에 100μL씩 첨가하고, 30분 동안 37℃, CO2 조건으로 배양한 후 2회 씻어내었다. 다음으로 Cytation 5장비로 DAPI를 기준으로 하여 초점을 맞춘 후 DAPI를 중심으로 30μM 크기로 세포의 구획을 정하고 이 크기의 세포만을 선택하여 FAM 형광을 이미지 리딩(image reading)한 후, 영상 처리하여 Mean-FAM value를 구하고 FAM 실험군을 음성대조군으로 보정한 그래프를 만들어 뇌혈관장벽세포 투과도를 계산하였다. 통계는 ONE-WAY ANOVA 분석으로 진행했고 multiple comparison(다중 비교)은 FAM 값을 대조군으로 하여 구하고 Bonferroni 방법(95% 신뢰도)으로 보정하였다(mean±SEM, *p<0.1, **p<0.01, ***p<0.001, ****p<0.0001).Specifically, hCMEC/D3 cells, which are human blood-brain barrier cells, are seeded in a 96-well plate, and the epithelial cell growth medium is EGM (endothelial cell growth medium) until the cells reach 80-90% of the plate area at 37°C. , and incubated overnight under CO 2 conditions. Then, the FAM-linked candidate peptides prepared in Example 1-2 and the negative control FAM alone were diluted in the medium supplemented with the growth factor at 4uM to prepare an amount to be treated by 100 μL per well. Then, the culture solution was removed from the cell culture plate by suction, and the peptide was diluted and treated with a solution prepared in advance, and then cultured at 37° C. and CO 2 conditions for 2 hours. Next, all the peptide-treated solutions from each well were removed with an inhaler, 100 μL of EGM was added, tapping 5-6 times, and the removal process was repeated twice. After that, Hoechst 33342 was diluted 1:5000 in EGM, 100 μL was added to each well, and incubated for 30 minutes at 37° C., CO 2 conditions, and washed twice. Next, after focusing based on DAPI with
2-2. 제1 영역 치환에 따른 세포 투과성 검증2-2. Cell permeability verification according to the first region substitution
상기 실시예 1-1에서 설계한 대로, 이전 선행 연구결과를 통해 발굴한 16개 아미노산으로 구성된 펩타이드에서 N-말단으로부터 1~4번 위치의 아미노산 영역이 치환된 다양한 펩타이드 후보를 설계하였고, 구체적인 서열정보를 하기 표 1에 나타내었다.As designed in Example 1-1, various peptide candidates in which the amino acid region at
상기 펩타이드 군에서의 세포 투과성을 확인한 결과, 도 1a 및 도 1b에 나타낸 바와 같이 상기 표 1의 #1 내지 #11, 및 #49에 해당하는 서열의 펩타이드가 우수한 뇌혈관장벽세포로의 유입 효과를 나타내는 것을 확인하였다. 이에 반해, 도 1c에서 볼 수 있는 바와 같이 상기 표 1의 #63 내지 #71에 해당하는 서열의 펩타이드의 경우 음성대조군인 FAM과 비교하여 형광 강도에 거의 차이가 없거나 더 낮은 것을 통해 유의한 세포 투과성이 없는 것을 확인할 수 있었다. As a result of confirming cell permeability in the peptide group, As shown in FIGS. 1A and 1B , it was confirmed that the peptides having sequences corresponding to #1 to #11 and #49 in Table 1 showed an excellent effect on influx into the blood-brain barrier cells. In contrast, as can be seen in FIG. 1c , in the case of the peptides of the sequence corresponding to #63 to #71 in Table 1, there is little or no difference in fluorescence intensity compared to the negative control FAM, or significant cell permeability through lower I was able to confirm that this was not there.
상기 결과로부터, N-말단으로부터 1~4번 위치의 아미노산 영역이 AHHE, GHAE, SHHE, AHAE, GHNE, GHHD, GHKE, GHGE, GHRE, GHVE, GHQE 또는 GHHG으로 치환되어 구성되는 경우에는 우수한 세포 투과성을 유지하나, 이를 벗어나는 경우에는 세포 투과성이 유지되지 않음을 알 수 있었다.From the above results, when the amino acid region at
2-3. 제2 영역 치환에 따른 세포 투과성 검증2-3. Cell permeability verification according to the second region substitution
다음으로, 이전 선행 연구결과를 통해 발굴한 16개 아미노산으로 구성된 펩타이드에서 N-말단으로부터 5~7번 위치의 아미노산 영역이 치환된 다양한 펩타이드 후보를 설계하였고, 구체적인 서열정보를 하기 표 2에 나타내었다.Next, various peptide candidates were designed in which the amino acid region at
상기 펩타이드 군에서의 세포 투과성을 확인한 결과, 도 2a에 나타낸 바와 같이 상기 표 2의 #12 내지 #23에 해당하는 서열의 펩타이드가 정도의 차이는 있으나 우수한 뇌혈관장벽세포로의 유입 효과를 나타내는 것을 확인하였다. 이에 반해, 도 2b에서 볼 수 있는 바와 같이 상기 표 2의 #72 및 #73에 해당하는 서열의 펩타이드의 경우 음성대조군인 FAM과 비교하여 형광 강도에 유의한 차이가 없는 것을 확인할 수 있었다. As a result of confirming cell permeability in the peptide group, As shown in Fig. 2a, it was confirmed that the peptides of the
상기 결과로부터, N-말단으로부터 5~7번 위치의 아미노산 영역에서 5번 위치의 아미노산이 R, I, V, L, N, K, Q, G 또는 A로 구성되거나, 7번 위치의 아미노산이 K, R, H, N 또는 Q로 구성되는 경우에는 우수한 세포 투과성이 유지되는 반면, 이를 벗어나는 경우에는 세포 투과성이 유지되지 않는 것을 알 수 있었다. From the above results, in the amino acid region at positions 5-7 from the N-terminus, the amino acid at
2-4. 제3 영역 치환에 따른 세포 투과성 검증2-4. Cell permeability verification according to the third region substitution
다음으로, 이전 선행 연구결과를 통해 발굴한 16개 아미노산으로 구성된 펩타이드에서 N-말단으로부터 8~12번 위치의 아미노산 영역이 치환된 다양한 펩타이드 후보를 설계하였고, 구체적인 서열정보를 하기 표 3에 나타내었다.Next, various peptide candidates were designed in which the amino acid region at
상기 펩타이드 군에서의 세포 투과성을 확인한 결과, 도 3a 및 도 3b에 나타낸 바와 같이 상기 표 3의 #24 내지 #37에 해당하는 서열의 펩타이드가 정도의 차이는 있으나 우수한 뇌혈관장벽세포로의 유입 효과를 나타내는 것을 확인하였다. 이에 반해, 도 3c에서 볼 수 있는 바와 같이 상기 표 3의 #74 내지 #76에 해당하는 서열의 펩타이드의 경우 음성대조군인 FAM과 비교하여 형광 강도가 현저히 낮은 것을 통해 세포 투과성이 없는 것을 알 수 있었다. As a result of confirming cell permeability in the peptide group, As shown in FIGS. 3A and 3B , it was confirmed that the peptides of the
상기 결과로부터, N-말단으로부터 8~12번 위치의 아미노산 영역이 ADEWS, CDEWS, LDEWS, QDEWS, VDEWS, NDEWS, TDEWS, RDEWS, SDSWS, SDEYS, GDEWS, SSEWS, SDFWS 또는 SDEFS로 치환되어 구성되는 경우에는 우수한 세포 투과성이 유지되는 반면, 이를 벗어나는 경우에는 세포 투과성이 유지되지 않는 것을 알 수 있었다.From the above results, when the amino acid region at positions 8-12 from the N-terminus is substituted with ADEWS, CDEWS, LDEWS, QDEWS, VDEWS, NDEWS, TDEWS, RDEWS, SDSWS, SDEYS, GDEWS, SSEWS, SDFWS or SDEFS. On the other hand, it was found that excellent cell permeability is maintained, but when it is outside the cell permeability is not maintained.
2-5. 제4 영역 치환에 따른 세포 투과성 검증2-5. Cell permeability verification according to the fourth region substitution
다음으로, 이전 선행 연구결과를 통해 발굴한 16개 아미노산으로 구성된 펩타이드에서 N-말단으로부터 13~16번 위치의 아미노산 영역이 치환된 다양한 펩타이드 후보를 설계하였고, 구체적인 서열정보를 하기 표 4에 나타내었다.Next, various peptide candidates were designed in which the amino acid region at
상기 펩타이드 군에서의 세포 투과성을 확인한 결과, 도 4a에 나타낸 바와 같이 상기 표 4의 #38 내지 #47에 해당하는 서열의 펩타이드가 정도의 차이는 있으나 우수한 뇌혈관장벽세포로의 유입 효과를 나타내는 것을 확인하였다. 이에 반해, 도 4b 및 도 4c에서 볼 수 있는 바와 같이 상기 표 4의 #77 내지 #80에 해당하는 서열의 펩타이드의 경우 음성대조군인 FAM과 비교하여 형광 강도가 현저히 낮거나 유사한 것을 확인하였다. 특히, 도 4b에서와 같이 N-말단으로부터 13번 위치에 있는 Val(V)이 단독으로 다른 아미노산으로 치환된 경우 세포 투과성이 유지되지 않는 것을 확인하였다. As a result of confirming cell permeability in the peptide group, As shown in Fig. 4a, it was confirmed that the peptides of the
상기 결과로부터, N-말단으로부터 13~16번 위치의 아미노산 영역만 단독으로 치환되는 경우, 13번 위치의 아미노산은 Val(V)으로써 다른 아미노산으로 치환되지 않고, 14~16번 위치의 아미노산이 QSG, KSG, RSG, ISG, SSG, TRG, TNG, TKG, TQG 또는 NSG로 치환되어 구성되는 경우에는 우수한 세포 투과성이 유지되는 반면, 이를 벗어나는 경우에는 세포 투과성이 유지되지 않는 것을 알 수 있었다.From the above results, when only the amino acid region at
2-6. 2개 이상의 영역에서 아미노산 치환에 따른 세포 투과성 검증2-6. Verification of cell permeability according to amino acid substitution in two or more regions
다음으로, 이전 선행 연구결과를 통해 발굴한 16개 아미노산으로 구성된 펩타이드에서 N-말단으로부터 상기 제1 영역 내지 제4 영역에서 2개 이상의 영역의 아미노산이 치환된 다양한 펩타이드 후보를 설계하였고, 구체적인 서열정보를 하기 표 5에 나타내었다.Next, various peptide candidates were designed in which amino acids of two or more regions in the first to fourth regions were substituted from the N-terminus in the peptide composed of 16 amino acids discovered through the results of previous prior research, and detailed sequence information is shown in Table 5 below.
상기 펩타이드 군에서의 세포 투과성을 확인한 결과, 도 5a 및 도 5b에 나타낸 바와 같이 상기 표 5의 #48 내지 #62에 해당하는 서열의 펩타이드가 정도의 차이는 있으나 우수한 뇌혈관장벽세포로의 유입 효과를 나타내는 것을 확인하였다. 이에 반해, 도 5c에서 볼 수 있는 바와 같이 상기 표 5의 #81 및 #82에 해당하는 서열의 펩타이드의 경우 음성대조군인 FAM과 비교하여 형광 강도가 현저히 낮은 것을 통해 세포 투과성이 없는 것을 확인하였다. As a result of confirming cell permeability in the peptide group, As shown in FIGS. 5A and 5B , it was confirmed that the peptides having sequences corresponding to #48 to #62 in Table 5 showed an excellent effect on influx into the blood-brain barrier cells, although there was a difference in degree. In contrast, as can be seen in FIG. 5c , in the case of the peptides having sequences corresponding to #81 and #82 in Table 5, the fluorescence intensity was significantly lower than that of the negative control FAM, indicating that there was no cell permeability.
상기 결과로부터, 상기 #48, #50 내지 #62의 서열에 해당하는 아미노산 서열과 같이, 제1 영역 내지 제4 영역 중 2개 이상의 영역의 아미노산을 치환하되, 제1 영역, 제2 영역 및 제3 영역은 상기 실시예 2-2, 2-3 및 2-4에 기재된 범위로, 제4 영역의 경우 상기 실시예 2-5에 기재된 범위 외에 NSA 또는 NSV으로 치환되어 구성되는 경우까지 우수한 세포 투과성이 유지되는 반면, 이를 벗어나는 경우에는 세포 투과성이 유지되지 않는 것을 알 수 있었다.From the above results, as in the amino acid sequence corresponding to the sequences of #48, #50 to #62, amino acids of two or more regions among the first to fourth regions are substituted, but the first region, the second region and the
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.
<110> IMNEWRUN BIOSCIENCES, INC. RESEARCH & BUSINESS FOUNDATION SUNGKYUNKWAN UNIVERSITY <120> Novel cell penetrating peptides and use thereof <130> PD20-452 <160> 82 <170> KoPatentIn 3.0 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #1 <400> 1 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #2 <400> 2 Gly His Asn Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #3 <400> 3 Gly His His Asp Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #4 <400> 4 Gly His Lys Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #5 <400> 5 Gly His Gly Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #6 <400> 6 Gly His Arg Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #7 <400> 7 Gly His Val Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #8 <400> 8 Gly His Gln Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #9 <400> 9 Gly His His Gly Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 10 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #10 <400> 10 Ala His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 11 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #11 <400> 11 Gly His Ala Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 12 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #12 <400> 12 Gly His His Glu Ile Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 13 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #13 <400> 13 Gly His His Glu Val Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 14 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #14 <400> 14 Gly His His Glu Leu Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 15 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #15 <400> 15 Gly His His Glu Asn Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 16 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #16 <400> 16 Gly His His Glu Lys Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 17 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #17 <400> 17 Gly His His Glu Gln Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 18 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #18 <400> 18 Gly His His Glu Arg Leu Arg Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 19 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #19 <400> 19 Gly His His Glu Arg Leu His Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 20 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #20 <400> 20 Gly His His Glu Gly Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 21 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #21 <400> 21 Gly His His Glu Arg Leu Asn Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 22 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #22 <400> 22 Gly His His Glu Arg Leu Gln Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 23 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #23 <400> 23 Gly His His Glu Ala Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 24 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #24 <400> 24 Gly His His Glu Arg Leu Lys Leu Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 25 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #25 <400> 25 Gly His His Glu Arg Leu Lys Asn Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 26 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #26 <400> 26 Gly His His Glu Arg Leu Lys Gln Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 27 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #27 <400> 27 Gly His His Glu Arg Leu Lys Thr Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 28 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #28 <400> 28 Gly His His Glu Arg Leu Lys Val Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 29 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #29 <400> 29 Gly His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 30 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #30 <400> 30 Gly His His Glu Arg Leu Lys Ser Asp Ser Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 31 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #31 <400> 31 Gly His His Glu Arg Leu Lys Ser Asp Glu Tyr Ser Val Thr Ser Gly 1 5 10 15 <210> 32 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #32 <400> 32 Gly His His Glu Arg Leu Lys Gly Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 33 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #33 <400> 33 Gly His His Glu Arg Leu Lys Ser Ser Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 34 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #34 <400> 34 Gly His His Glu Arg Leu Lys Ser Asp Phe Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 35 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #35 <400> 35 Gly His His Glu Arg Leu Lys Ser Asp Glu Phe Ser Val Thr Ser Gly 1 5 10 15 <210> 36 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #36 <400> 36 Gly His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 37 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #37 <400> 37 Gly His His Glu Arg Leu Lys Arg Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 38 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #38 <400> 38 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Asn Ser Gly 1 5 10 15 <210> 39 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #39 <400> 39 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Gln Ser Gly 1 5 10 15 <210> 40 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #40 <400> 40 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Lys Ser Gly 1 5 10 15 <210> 41 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #41 <400> 41 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Arg Ser Gly 1 5 10 15 <210> 42 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #42 <400> 42 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Ile Ser Gly 1 5 10 15 <210> 43 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #43 <400> 43 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Ser Ser Gly 1 5 10 15 <210> 44 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #44 <400> 44 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Arg Gly 1 5 10 15 <210> 45 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #45 <400> 45 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Asn Gly 1 5 10 15 <210> 46 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #46 <400> 46 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Lys Gly 1 5 10 15 <210> 47 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #47 <400> 47 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Gln Gly 1 5 10 15 <210> 48 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #48 <400> 48 Ala His His Glu Ala Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 49 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #49 <400> 49 Ala His Ala Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 50 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #50 <400> 50 Gly His Ala Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 51 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #51 <400> 51 Ala His Ala Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 52 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #52 <400> 52 Ala His Ala Glu Ala Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 53 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #53 <400> 53 Gly His Ala Glu Ala Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 54 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #54 <400> 54 Gly His His Glu Ala Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 55 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #55 <400> 55 Gly His Ala Glu Ala Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 56 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #56 <400> 56 Ser His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 57 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #57 <400> 57 Gly His His Gly Gly Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 58 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #58 <400> 58 Ala His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 59 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #59 <400> 59 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Asn Ser Ala 1 5 10 15 <210> 60 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #60 <400> 60 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Asn Ser Val 1 5 10 15 <210> 61 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #61 <400> 61 Ser His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Val 1 5 10 15 <210> 62 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #62 <400> 62 Ser His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Ala 1 5 10 15 <210> 63 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #63 <400> 63 Gly His Ile Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 64 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #64 <400> 64 Leu His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 65 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #65 <400> 65 Val His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 66 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #66 <400> 66 Lys His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 67 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #67 <400> 67 Ile His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 68 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #68 <400> 68 Arg His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 69 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #69 <400> 69 Gly His Leu Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 70 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #70 <400> 70 Gly His Gly Gly Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 71 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #71 <400> 71 Gly His His Tyr Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 72 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #72 <400> 72 Gly His His Glu Gly Gly Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 73 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #73 <400> 73 Gly His His Glu Arg Leu Gly Gly Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 74 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #74 <400> 74 Gly His His Glu Arg Leu Lys Ser Phe Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 75 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #75 <400> 75 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Tyr Val Thr Ser Gly 1 5 10 15 <210> 76 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #76 <400> 76 Gly His His Glu Arg Leu Lys Ser Asp Glu Ala Ser Val Thr Ser Gly 1 5 10 15 <210> 77 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #77 <400> 77 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Ile Thr Ser Gly 1 5 10 15 <210> 78 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #78 <400> 78 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Phe Thr Ser Gly 1 5 10 15 <210> 79 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #79 <400> 79 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Leu Thr Ser Gly 1 5 10 15 <210> 80 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #80 <400> 80 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Glu 1 5 10 15 <210> 81 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #81 <400> 81 Ser His Leu Glu Arg Thr Lys Ser Asp Glu Trp Ser Ile Ile Ser Glu 1 5 10 15 <210> 82 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #82 <400> 82 Ser His Leu Glu Arg Thr Lys Cys Asp Glu Trp Ser Ile Ile Ser Glu 1 5 10 15 <110> IMNEWRUN BIOSCIENCES, INC. RESEARCH & BUSINESS FOUNDATION SUNGKYUNKWAN UNIVERSITY <120> Novel cell penetrating peptides and use thereof <130> PD20-452 <160> 82 <170> KoPatentIn 3.0 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #1 <400> 1 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #2 <400> 2 Gly His Asn Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #3 <400> 3 Gly His His Asp Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #4 <400> 4 Gly His Lys Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #5 <400> 5 Gly His Gly Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #6 <400> 6 Gly His Arg Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #7 <400> 7 Gly His Val Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #8 <400> 8 Gly His Gln Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #9 <400> 9 Gly His His Gly Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 10 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #10 <400> 10 Ala His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 11 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #11 <400> 11 Gly His Ala Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 12 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #12 <400> 12 Gly His His Glu Ile Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 13 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #13 <400> 13 Gly His His Glu Val Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 14 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #14 <400> 14 Gly His His Glu Leu Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 15 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #15 <400> 15 Gly His His Glu Asn Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 16 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #16 <400> 16 Gly His His Glu Lys Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 17 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #17 <400> 17 Gly His His Glu Gln Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 18 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #18 <400> 18 Gly His His Glu Arg Leu Arg Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 19 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #19 <400> 19 Gly His His Glu Arg Leu His Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 20 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #20 <400> 20 Gly His His Glu Gly Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 21 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #21 <400> 21 Gly His His Glu Arg Leu Asn Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 22 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #22 <400> 22 Gly His His Glu Arg Leu Gln Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 23 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #23 <400> 23 Gly His His Glu Ala Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 24 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #24 <400> 24 Gly His His Glu Arg Leu Lys Leu Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 25 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #25 <400> 25 Gly His His Glu Arg Leu Lys Asn Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 26 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #26 <400> 26 Gly His His Glu Arg Leu Lys Gln Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 27 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #27 <400> 27 Gly His His Glu Arg Leu Lys Thr Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 28 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #28 <400> 28 Gly His His Glu Arg Leu Lys Val Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 29 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #29 <400> 29 Gly His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 30 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #30 <400> 30 Gly His His Glu Arg Leu Lys Ser Asp Ser Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 31 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #31 <400> 31 Gly His His Glu Arg Leu Lys Ser Asp Glu Tyr Ser Val Thr Ser Gly 1 5 10 15 <210> 32 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #32 <400> 32 Gly His His Glu Arg Leu Lys Gly Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 33 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #33 <400> 33 Gly His His Glu Arg Leu Lys Ser Ser Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 34 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #34 <400> 34 Gly His His Glu Arg Leu Lys Ser Asp Phe Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 35 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #35 <400> 35 Gly His His Glu Arg Leu Lys Ser Asp Glu Phe Ser Val Thr Ser Gly 1 5 10 15 <210> 36 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #36 <400> 36 Gly His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 37 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #37 <400> 37 Gly His His Glu Arg Leu Lys Arg Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 38 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #38 <400> 38 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Asn Ser Gly 1 5 10 15 <210> 39 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #39 <400> 39 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Gln Ser Gly 1 5 10 15 <210> 40 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #40 <400> 40 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Lys Ser Gly 1 5 10 15 <210> 41 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #41 <400> 41 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Arg Ser Gly 1 5 10 15 <210> 42 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #42 <400> 42 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Ile Ser Gly 1 5 10 15 <210> 43 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #43 <400> 43 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Ser Ser Gly 1 5 10 15 <210> 44 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #44 <400> 44 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Arg Gly 1 5 10 15 <210> 45 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #45 <400> 45 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Asn Gly 1 5 10 15 <210> 46 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #46 <400> 46 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Lys Gly 1 5 10 15 <210> 47 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #47 <400> 47 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Gln Gly 1 5 10 15 <210> 48 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #48 <400> 48 Ala His His Glu Ala Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 49 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #49 <400> 49 Ala His Ala Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 50 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #50 <400> 50 Gly His Ala Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 51 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #51 <400> 51 Ala His Ala Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 52 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #52 <400> 52 Ala His Ala Glu Ala Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 53 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #53 <400> 53 Gly His Ala Glu Ala Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 54 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #54 <400> 54 Gly His His Glu Ala Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 55 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #55 <400> 55 Gly His Ala Glu Ala Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 56 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #56 <400> 56 Ser His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 57 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #57 <400> 57 Gly His His Gly Gly Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 58 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #58 <400> 58 Ala His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 59 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #59 <400> 59 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Asn Ser Ala 1 5 10 15 <210> 60 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #60 <400> 60 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Asn Ser Val 1 5 10 15 <210> 61 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #61 <400> 61 Ser His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Val 1 5 10 15 <210> 62 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #62 <400> 62 Ser His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Ala 1 5 10 15 <210> 63 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #63 <400> 63 Gly His Ile Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 64 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #64 <400> 64 Leu His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 65 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #65 <400> 65 Val His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 66 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #66 <400> 66 Lys His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 67 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #67 <400> 67 Ile His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 68 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #68 <400> 68 Arg His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 69 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #69 <400> 69 Gly His Leu Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 70 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #70 <400> 70 Gly His Gly Gly Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 71 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #71 <400> 71 Gly His His Tyr Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 72 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #72 <400> 72 Gly His His Glu Gly Gly Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 73 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #73 <400> 73 Gly His His Glu Arg Leu Gly Gly Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 74 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #74 <400> 74 Gly His His Glu Arg Leu Lys Ser Phe Glu Trp Ser Val Thr Ser Gly 1 5 10 15 <210> 75 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #75 <400> 75 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Tyr Val Thr Ser Gly 1 5 10 15 <210> 76 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #76 <400> 76 Gly His His Glu Arg Leu Lys Ser Asp Glu Ala Ser Val Thr Ser Gly 1 5 10 15 <210> 77 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #77 <400> 77 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Ile Thr Ser Gly 1 5 10 15 <210> 78 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #78 <400> 78 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Phe Thr Ser Gly 1 5 10 15 <210> 79 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #79 <400> 79 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Leu Thr Ser Gly 1 5 10 15 <210> 80 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #80 <400> 80 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Glu 1 5 10 15 <210> 81 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #81 <400> 81 Ser His Leu Glu Arg Thr Lys Ser Asp Glu Trp Ser Ile Ile Ser Glu 1 5 10 15 <210> 82 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> #82 <400> 82 Ser His Leu Glu Arg Thr Lys Cys Asp Glu Trp Ser Ile Ile Ser Glu 1 5 10 15
Claims (11)
[일반식 I]
X1-[Xa-L-Xb]-X2-V-X3
X1은 GHHE, AHHE, GHAE, SHHE, AHAE, GHNE, GHHD, GHKE, GHGE, GHRE, GHVE, GHQE 또는 GHHG이고;
Xa는 Arg(R), Ala(A), Ile(I), Val(V), Leu(L), Gly(G), Asn(N), Lys(K) 또는 Gln(Q)이고;
Xb 는 Lys(K), Arg(R), His(H), Asn(N) 또는 Gln(Q)이고;
X2는 SDEWS, ADEWS, CDEWS, LDEWS, QDEWS, VDEWS, NDEWS, TDEWS, RDEWS, SDSWS, SDEYS, GDEWS, SSEWS, SDFWS 또는 SDEFS이고;
X3은 TSG, NSA, NSV, QSG, KSG, RSG, ISG, SSG, TRG, TNG, TKG, TQG 또는 NSG 이고,
상기 세포 투과성 펩타이드는 서열번호 1 내지 서열번호 62로 이루어진 군에서 선택되는 어느 하나의 아미노산 서열로 이루어지는 것을 특징으로 하는, 세포 투과성 펩타이드.
A cell-penetrating peptide consisting of an amino acid represented by the following [General Formula I]:
[General Formula I]
X 1 -[X a -LX b ]-X 2 -VX 3
X 1 is GHHE, AHHE, GHAE, SHHE, AHAE, GHNE, GHHD, GHKE, GHGE, GHRE, GHVE, GHQE or GHHG;
X a is Arg(R), Ala(A), Ile(I), Val(V), Leu(L), Gly(G), Asn(N), Lys(K) or Gln(Q);
X b is Lys(K), Arg(R), His(H), Asn(N) or Gin(Q);
X 2 is SDEWS, ADEWS, CDEWS, LDEWS, QDEWS, VDEWS, NDEWS, TDEWS, RDEWS, SDSWS, SDEYS, GDEWS, SSEWS, SDFWS or SDEFS;
X 3 is TSG, NSA, NSV, QSG, KSG, RSG, ISG, SSG, TRG, TNG, TKG, TQG or NSG,
The cell-permeable peptide is characterized in that it consists of any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 62, cell-permeable peptide.
상기 세포는 뇌혈관장벽 내피세포(brain endothelial cell), 암세포, 혈액세포(blood cell), 림프구(lymphocyte), 면역세포, 줄기세포, 유도만능줄기세포(induced pluripotent stem cell; iPSC), 신경줄기세포(neural stem cell; NSC), T 세포, B 세포, 자연살해세포(natural Killer cell; NK cell), 대식세포(macrophage), 미세아교세포(microglia), 신경세포(neuron), 신경아교세포(glial cells), 성상세포(astrocyte) 및 근육세포(muscle cell)로 이루어진 군에서 선택되는 것임을 특징으로 하는, 세포 투과성 펩타이드.
According to claim 1,
The cells include brain endothelial cells, cancer cells, blood cells, lymphocytes, immune cells, stem cells, induced pluripotent stem cells (iPSCs), neural stem cells ( neural stem cells (NSC), T cells, B cells, natural killer cells (NK cells), macrophages, microglia, neurons, glial cells , Astrocytes (astrocyte) and muscle cells (muscle cell), characterized in that selected from the group consisting of, cell-penetrating peptide.
A complex comprising the cell penetrating peptide of claim 1 or 3 and a biologically active substance.
생물학적 활성 물질은 화합물(chemical compound), 단백질, 당단백질, 펩타이드, 항체(antibody), 효소(enzyme), 핵산분해효소(nuclease), 호르몬, 사이토카인(cytokine), 전사인자(transcription factor), 독소, 핵산, 탄수화물, 지질, 당지질, 천연물(natural product), 반합성 물질(semi-synthetic drug), 약물(drug), 마이크로입자, 나노입자, 리포좀, 바이러스, 양자점(quantum dots), 및 형광색소(fluorochrome)로 이루어진 군으로부터 선택되는 하나 이상인 것을 특징으로 하는, 복합체.
5. The method of claim 4,
Biologically active substances include chemical compounds, proteins, glycoproteins, peptides, antibodies, enzymes, nucleases, hormones, cytokines, transcription factors, toxins , nucleic acids, carbohydrates, lipids, glycolipids, natural products, semi-synthetic drugs, drugs, microparticles, nanoparticles, liposomes, viruses, quantum dots, and fluorochromes ), characterized in that at least one selected from the group consisting of, the complex.
상기 핵산분해효소는 CAS9(CRISPR associated protein 9), CAS12, CAS13, CAS14, CAS variants, Cfp1(CxxC-finger protein-1), ZEN(Zinc-finger nucleases) 및 TALEN(Transcription activator-like effector nuclease)으로 이루어진 군에서 선택되는 것을 특징으로 하는, 복합체.
6. The method of claim 5,
The nucleases are CAS9 (CRISPR associated protein 9), CAS12, CAS13, CAS14, CAS variants, CxxC-finger protein-1 (Cfp1), Zinc-finger nucleases (ZEN) and transcription activator-like effector nuclease (TALEN). Characterized in that selected from the group consisting of, the complex.
상기 핵산은 DNA, RNA, ASO(Antisense oligonucleotide), 마이크로RNA(microRNA; miRNA), 작은 간섭 RNA(small interfering RNA; siRNA), 앱타머(aptamer), LNA(locked nucleic acid), PNA(peptide nucleic acid), 및 모폴리노(morpholino)로 이루어진 군으로부터 선택되는 것을 특징으로 하는, 복합체.
6. The method of claim 5,
The nucleic acid is DNA, RNA, antisense oligonucleotide (ASO), microRNA (miRNA), small interfering RNA (siRNA), aptamer, LNA (locked nucleic acid), PNA (peptide nucleic acid) ), and morpholino (morpholino), characterized in that selected from the group consisting of, the complex.
A composition for mass transfer, comprising the complex of claim 4.
A material delivery method comprising the step of treating cells of a mammal other than a human with the composition of claim 8.
상기 세포는 뇌혈관장벽 내피세포(brain endothelial cell), 암세포, 혈액세포(blood cell), 림프구(lymphocyte), 면역세포, 줄기세포, 유도만능줄기세포(induced pluripotent stem cell; iPSC), 신경줄기세포(neural stem cell; NSC), T 세포, B 세포, 자연살해세포(natural Killer cell; NK cell), 대식세포(macrophage), 미세아교세포(microglia), 신경세포(neuron), 신경아교세포(glial cells), 성상세포(astrocyte) 및 근육세포(muscle cell)로 이루어진 군에서 선택되는 것임을 특징으로 하는, 물질 전달방법.
10. The method of claim 9,
The cells include brain endothelial cells, cancer cells, blood cells, lymphocytes, immune cells, stem cells, induced pluripotent stem cells (iPSCs), neural stem cells ( neural stem cells (NSC), T cells, B cells, natural killer cells (NK cells), macrophages, microglia, neurons, glial cells , Astrocytes (astrocyte) and muscle cells (muscle cell), characterized in that selected from the group consisting of, the mass delivery method.
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| KR101647804B1 (en) * | 2015-06-24 | 2016-08-11 | 한국과학기술연구원 | Novel Cell Penetrating Peptides and Uses Thereof |
| KR20170015852A (en) * | 2015-07-31 | 2017-02-09 | 이화여자대학교 산학협력단 | Novel cell penetrating peptide |
| KR20200104524A (en) * | 2019-02-27 | 2020-09-04 | 주식회사 아임뉴런바이오사이언스 | Novel cell penetrating peptides and use thereof |
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| KR20150145132A (en) * | 2014-06-18 | 2015-12-29 | 한국과학기술연구원 | Novel Cell Penetrating Peptides and Uses Thereof |
| KR101647804B1 (en) * | 2015-06-24 | 2016-08-11 | 한국과학기술연구원 | Novel Cell Penetrating Peptides and Uses Thereof |
| KR20170015852A (en) * | 2015-07-31 | 2017-02-09 | 이화여자대학교 산학협력단 | Novel cell penetrating peptide |
| KR20200104524A (en) * | 2019-02-27 | 2020-09-04 | 주식회사 아임뉴런바이오사이언스 | Novel cell penetrating peptides and use thereof |
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