KR101669203B1 - Novel Cell Penetrating Peptides and Uses Thereof - Google Patents
Novel Cell Penetrating Peptides and Uses Thereof Download PDFInfo
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- KR101669203B1 KR101669203B1 KR1020140074573A KR20140074573A KR101669203B1 KR 101669203 B1 KR101669203 B1 KR 101669203B1 KR 1020140074573 A KR1020140074573 A KR 1020140074573A KR 20140074573 A KR20140074573 A KR 20140074573A KR 101669203 B1 KR101669203 B1 KR 101669203B1
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Abstract
본 발명은 신규한 세포투과성 펩타이드 및 이의 용도에 관한 것이다. 본 발명의 펩타이드는 인간 아넥신 단백질-유래된 아미노산 서열로, 우수한 세포투과성을 가질 뿐 아니라, 세포독성을 나타내지 않고 혈청에서 느린 분해율을 보인다. 또한, 본 발명의 펩타이드에 결합된 목적 카르고(예컨대, 베타-갈락토시다제)는 안정적이고 효과적으로 세포 내(세포질)로 운반된다. 따라서, 본 발명의 펩타이드를 포함하는 약물전달체, 그리고 이를 이용하는 목적 카르고의 전달방법 및 조성물은 질병 또는 질환의 치료, 특정 세포의 검출, 질병(예컨대, 암)의 진단, 특정 세포의 위치 추적 및 인 비보 이미징 등에 이용될 수 있다.The present invention relates to novel cell permeable peptides and uses thereof. The peptide of the present invention is a human anexin protein-derived amino acid sequence, which not only has excellent cell permeability but also shows a slow degradation rate in serum without cytotoxicity. In addition, the objective cargo (e.g., beta-galactosidase) bound to the peptide of the present invention is stably and effectively transported intracellularly (cytoplasmic). Accordingly, the drug delivery system comprising the peptide of the present invention, and the method and composition for delivering the target cargo using the peptide of the present invention are useful for the treatment of diseases or diseases, detection of specific cells, diagnosis of diseases (e.g. cancer) In-vivo imaging and the like.
Description
본 발명은 신규 세포투과성 펩타이드 및 이의 용도에 관한 것이다.
The present invention relates to novel cell permeable peptides and uses thereof.
질병의 발생 및 진행에 중요한 역할을 하는 주요단백질의 기능이 밝혀지면서 세포 내 질환 관련 단백질을 조절할 수 있는 물질로 저분자 화합물이 주요 약물로 이용되고 있으나, 넓은 면적의 단백질 간 상호작용 부위는 저분자 약물이 결합하여 표적단백질의 활성을 조절하기에는 한계가 있음이 알려져 있었다. 이를 극복하기 위하여, 단백질과 단백질간의 상호작용을 조절하기에 적합한 펩타이드나 단백질 기반의 약물 개발이 시도되고 있다. 세포 내 존재하는 표적단백질의 활성 조절을 목적으로 하는 단백질 약물의 성공적인 개발을 위해서는 단백질의 세포 내 전달 및 수송이 필수적으로 요구되는데, 일반적으로 단백질의 세포 내 수송은 어려운 것으로 알려져 있었다. 이러한 단점을 보완하기 위해 단백질을 세포 내 투과를 도와주는 물질로써, 단백질 전달 도메인(protein transduction domain, PTD), 즉 세포투과 펩타이드(cell penetrating peptide, CPP)라고 불리는 전달체를 이용하고 있다. 세포 내로 전달이 불가능했던 신약 후보물질들을 PTD 또는 CPP와 결합시켜 세포 내로 전달하는 새로운 약물 전달체(drug delivery system, DDS)가 각광 받음으로써 새로운 치료제 개발에 다양한 파급효과를 나타내고 있다. 지금까지 가장 잘 알려진 CPP는 HIV 바이러스 전사인자에서 유래된 TAT-CPP로, 인 비트로(in vitro)와 인 비보(in vivo) 실험에서 탁월한 효과를 가지고 있고, 이와 비슷한 성질을 지닌 합성 CPP나 바이러스에서 유래된 CPP들의 효능도 좋다고 알려져 있었다. 하지만, 상술한 CPP, 특히 아미노산 서열이 긴 CPP의 경우 세포내 발현을 통해 단백질을 얻는 방법으로는 돌연변이가 생성될 가능성이 있고 대량생산이 힘들어 임상적으로 사용되기에는 제한이 있다. 따라서, 임상에서 사용되는 CPP는 극히 드물고 바이러스에서 유래된 CPP의 경우 면역반응에 영향을 미치는 문제점을 지니고 있어, 바이러스에서 유래되지 않으면서 전달효율이 높은 전달체 발굴이 시급하다.
As the function of major proteins plays an important role in the development and progression of diseases, low molecular compounds are used as main drugs to control intracellular disease-related proteins. However, It has been known that there is a limitation in controlling the activity of the target protein by binding. To overcome this, attempts have been made to develop peptides and protein-based drugs suitable for controlling the interaction between proteins and proteins. In order to successfully develop a protein drug for the purpose of regulating the activity of a target protein present in a cell, intracellular delivery and transport of the protein are indispensably required. In general, it has been known that intracellular transport of the protein is difficult. To overcome this disadvantage, a protein transduction domain (PTD), a cell penetrating peptide (CPP), is used as a substance that facilitates intracellular permeation of proteins. A new drug delivery system (DDS), which binds PTD or CPP to cells and delivers new drug candidate substances that could not be delivered into cells, has been spotlighted. CPP is the best known so far as a TAT-CPP derived from HIV virus transcription factors in vitro (in vitro) and in vivo (in vivo ) It has been known that CPPs derived from synthetic CPPs or viruses with similar properties and with similar properties are also effective. However, in the case of the above-mentioned CPP, especially CPP having a long amino acid sequence, there is a possibility that a protein is obtained through intracellular expression, and mutation is generated, and mass production is difficult. Therefore, the CPP used in clinical practice is extremely rare, and CPP derived from viruses have problems affecting the immune response, so it is urgent to find a carrier having high delivery efficiency without being derived from virus.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
본 발명자들은 목적 카르고(cargo of interest)를 세포 내로 전달할 수 있는 신규한 분자/물질 및 이를 이용한 약물전달체(drug delivery system)를 개발하고자 노력하였다. 그 결과, 본 발명자들은 12종류의 인간 아넥신 단백질로부터 신규한 세포투과성 펩타이드들을 동정하였고, 상기 펩타이드들은 세포독성을 나타내지 않을 뿐 아니라 혈청 내에서 오랜 기간 동안 분해되지 않고 존재하여 목적 운반 카르고(특히, 약물)의 세포 내 전달에 효율적으로 적용(예컨대, 본 발명의 펩타이드와 융합된 베타-갈락토시다제)될 수 있다는 것을 확인함으로써 본 발명을 완성하였다.The present inventors have sought to develop a novel molecule / substance capable of delivering a cargo of interest into cells and a drug delivery system using the same. As a result, the present inventors identified novel cell permeable peptides from 12 kinds of human annexin proteins, and found that the peptides do not exhibit cytotoxicity but also exist in the serum for a long time without being degraded, , Drug) of the present invention (for example, beta-galactosidase fused with the peptide of the present invention).
따라서, 본 발명의 목적은 세포투과성 펩타이드를 제공하는데 있다.Accordingly, an object of the present invention is to provide a cell permeable peptide.
본 발명의 다른 목적은 상술한 펩타이드를 인코딩하는 뉴클레오타이드 서열을 제공하는 데 있다.Another object of the present invention is to provide a nucleotide sequence encoding the above-mentioned peptide.
본 발명의 다른 목적은 상술한 뉴클레오타이드 서열을 포함하는 재조합 벡터를 제공하는 데 있다.Another object of the present invention is to provide a recombinant vector comprising the above-described nucleotide sequence.
본 발명의 또 다른 목적은 약물전달체를 제공하는 데 있다.Still another object of the present invention is to provide a drug delivery system.
본 발명의 또 다른 목적은 목적 카르고의 세포 내 전달 방법을 제공하는 데 있다.It is another object of the present invention to provide a method for intracellular delivery of a target cargo.
본 발명의 또 다른 목적은 목적 카르고 운반용 조성물을 제공하는 데 있다.It is another object of the present invention to provide a composition for cargo transportation.
본 발명의 다른 목적은 목적 카르고의 트랜스펙션 키트를 제공하는 데 있다.
Another object of the present invention is to provide a transfection kit of a target cargo.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 서열번호 1 내지 서열번호 6의 아미노산 서열로 구성된 군으로부터 선택된 아미노산 서열로 이루어진 세포투과성 펩타이드(cell penetrating peptide, CPP)를 제공한다.According to one aspect of the present invention, there is provided a cell penetrating peptide (CPP) comprising an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 6.
본 발명의 다른 양태에 따르면, 본 발명은 (a) 상술한 세포투과성 펩타이드; (b) 상기 펩타이드에 결합된 목적 카르고(cargo of interest)를 포함하는 약물전달체(drug delivery system)를 제공한다.According to another aspect of the present invention, the present invention provides a method for producing a cell-permeable peptide comprising: (a) the above-described cell permeable peptide; (b) a cargo of interest bound to the peptide.
본 발명의 또 다른 양태에 따르면, 본 발명은 상술한 세포투과성 펩타이드를 인코딩하는 뉴클레오타이드 서열을 제공한다.According to another aspect of the present invention, the present invention provides a nucleotide sequence encoding the aforementioned cell permeable peptide.
본 발명의 또 다른 양태에 따르면, 본 발명은 상술한 뉴클레오타이드 서열을 포함하는 재조합 발현벡터를 제공한다.
According to another aspect of the present invention, the present invention provides a recombinant expression vector comprising the above-described nucleotide sequence.
본 발명자들은 목적 카르고를 세포 내로 전달할 수 있는 신규한 분자/물질 및 이를 이용한 약물전달체를 개발하고자 노력하였다. 그 결과, 본 발명자들은 12종류의 인간 아넥신 단백질로부터 신규한 세포투과성 펩타이드들을 동정하였고, 상기 펩타이드들은 세포독성을 나타내지 않을 뿐 아니라 혈청 내에서 오랜 기간 동안 분해되지 않고 존재하여 목적 운반 카르고(특히, 약물)의 세포 내 전달에 효율적으로 적용(예컨대, 본 발명의 펩타이드와 융합된 베타-갈락토시다제)될 수 있다는 것을 확인하였다.The present inventors have sought to develop a novel molecule / substance capable of delivering a target cargo into cells and a drug delivery system using the same. As a result, the present inventors identified novel cell permeable peptides from 12 kinds of human annexin proteins, and found that the peptides do not exhibit cytotoxicity but also exist in the serum for a long time without being degraded, , Drug) into the intracellular delivery of beta-galactosidase (e. G., Beta-galactosidase fused with the peptide of the invention).
세포투과성 펩타이드가 세포 내로 전달될 때에는 세포 표면의 프로테오글리칸(proteoglycans)과 교류하여 정전기적 결합을 하는 것이 첫 번째 단계인데, 대부분의 세포투과성 펩타이드의 경우, 세포 표면의 HSPG(heparin sulfate proteoglycans)와 상호작용을 통해 들어간다는 점에서 본 발명자들은 세포막과 결합/상호작용하는 단백질들을 조사하여, 세포투과기능을 가지는 아미노산 서열을 가질 가능성이 높은 아넥신 단백질들로부터 본 발명의 펩타이드들을 최종 선별하였다.When the cell permeable peptide is delivered into the cell, the first step is to exchange the proteoglycans with the proteoglycans on the cell surface to perform the electrostatic binding. For most of the cell permeable peptides, interaction with HSPG (heparin sulfate proteoglycans) The present inventors investigated proteins binding to / interacting with the cell membrane and finally selected the peptides of the present invention from annexin proteins likely to have an amino acid sequence having cell penetration function.
아넥신(annexin)은 칼슘 이온에 의해 세포막의 인지질에 결합함으로서 세포막을 통해 이온 농도를 조절하고 이온의 세포막 내외로의 수송에 관여하고, 세포 구조에 관여하는 단백질인 F-액틴(actin)과 결합하면서 세포 구조의 역학을 조절한다. 아넥신은 생물체에 따라 5가지 주요 그룹(A 내지 E 그룹)으로 분류된다(즉, 인간, 곤충, 곰팡이, 식물 및 원생생물). 또한, 아넥신은 세포막과 결합할 뿐 아니라 엔도사이토시스(endocytosis) 및 엑소사이토시스(exocytosis)를 통해 세포 안팎을 드나드는 기능이 있다는 것을 2005년에 Gerke V 등(Nature Review Mol Cell Biol ., 6: 449-461, 2005)이 보고하였다.Annexin binds to the phospholipid of the cell membrane by calcium ion, which regulates the ion concentration through the cell membrane and is involved in the transport of the ion into and out of the cell membrane. It binds to F-actin, a protein involved in cell structure While controlling the dynamics of the cell structure. Anexin is classified into five major groups (A to E groups) (ie, humans, insects, fungi, plants and protists), depending on the organism. In addition, it has been reported by Gerke V et al ( Nature , 2005) that anexin binds to cell membranes and has the ability to move in and out of cells through endocytosis and exocytosis Review Mol Cell Biol . , 6: 449-461, 2005).
본 발명의 펩타이드는 아넥신 단백질의 N-말단에 위치된 아미노산 서열을 포함하며, 구체적으로는 서열번호 1 내지 서열번호 12의 아미노산 서열을 포함하고, 보다 구체적으로는 서열번호 3 내지 서열번호 6, 서열번호 11 및 서열번호 13의 아미노산 서열을 포함하고, 보다 더 구체적으로는 서열번호 3 및 서열번호 5의 아미노산 서열을 포함한다(참고: 표 1).The peptide of the present invention includes an amino acid sequence located at the N-terminus of the annexin protein, specifically, an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 12, more specifically, SEQ ID NO: 3 to SEQ ID NO: SEQ ID NO: 11 and SEQ ID NO: 13, and more specifically, the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 5 (see Table 1).
본 발명의 어떤 구현예에서, 상기 서열번호 1 내지 서열번호 12의 아미노산 서열은 각각 아넥신 A1 내지 아넥신 A11, 그리고 아넥신 A13으로부터 유래된 아미노산 서열이고, 보다 구체적으로는 인간 아넥신 단백질들로부터 유래된 아미노산 서열이다.In some embodiments of the present invention, the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 12 are amino acid sequences derived from annexin A1 to annexin A11 and annexin A13, respectively, and more specifically to human annexin proteins Derived amino acid sequence.
본 명세서에서 사용되는 용어 “펩타이드”는 펩타이드 결합에 의해 아미노산 잔기들이 서로 결합되어 형성된 선형의 분자를 의미한다. 본 발명의 펩타이드는 당업계에 공지된 화학적 합성 방법, 특히 고상 합성 기술(solid-phase synthesis techniques)에 따라 제조될 수 있다(Merrifield, J. Amer. Chem . Soc . 85:2149-54(1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chem. Co.: Rockford, 111(1984)).As used herein, the term " peptide " refers to a linear molecule formed by peptide bonds and amino acid residues joined together. The peptides of the present invention can be prepared according to chemical synthesis methods known in the art, particularly solid-phase synthesis techniques (Merrifield, J. Amer. Chem . Soc . 85: 2149-54 (1963) ; Stewart, et al., Solid Phase Peptide Synthesis , 2nd. ed., Pierce Chem. Co .: Rockford, 111 (1984)).
본 발명의 펩타이드는 그 자체로서 천연의 아넥신보다 안정성이 훨씬 우수하지만, 아미노산의 변형에 의해 안정성이 더욱 향상될 수 있다.The peptide of the present invention is much more stable than natural annexin itself, but the stability can be further improved by modification of the amino acid.
본 발명의 어떤 구현예에서, 상기 펩타이드의 N-말단 또는 C-말단은 아세틸기, 플루오레닐 메톡시 카르보닐기, 포르밀기, 팔미토일기, 미리스틸기, 스테아릴기, 폴리에틸렌글리콜(PEG) 및 아미노산으로 구성된 군으로부터 선택되는 보호기가 추가적으로 결합되어 있다. 또한, 본 발명의 펩타이드의 N-말단은 히드록시기(-OH) 또는 아미노기(-NH2)로 변형되어 안정성을 증가시킬 수 있다. In some embodiments of the present invention, the N-terminal or C-terminal of the peptide is an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, a polyethylene glycol (PEG) An amino acid, and a protecting group selected from the group consisting of amino acids. In addition, the N-terminal of the peptide of the present invention may be modified into a hydroxyl group (-OH) or an amino group (-NH 2 ) to increase the stability.
상술한 보호기는 생체 내의 단백질 절단효소의 공격으로부터 본 발명의 펩타이드를 보호하는 작용을 한다. 또한, 상기 변형된 펩타이드는 보호기로 인해 우수한 열안정성을 나타내며, 또한 산과 알칼리 등의 물리화학적 인자에 대한 안정성을 우수하다. 따라서, 본 발명의 펩타이드는 장기 보존성이 뛰어나게 변형될 수 있으므로, 의약품, 의약외품, 화장품 및 구강용품과 같은 장기간 저장이 요구되는 제품에 유리하게 적용될 수 있다. 상술한 아미노산의 변형은 본 발명의 펩타이드 안정성을 크게 개선하는 작용을 하고, 본 명세서에서 언급되는 용어 “안정성”은 인 비보 안정성 뿐만 아니라, 저장 안정성(예컨대, 상온 저장 안정성)도 의미한다.The above-mentioned protecting group acts to protect the peptide of the present invention from attack of a protein cleaving enzyme in vivo. In addition, the modified peptide exhibits excellent thermal stability due to a protecting group, and is excellent in stability against physicochemical factors such as acid and alkali. Therefore, the peptide of the present invention can be advantageously applied to products requiring long-term storage such as medicines, quasi-drugs, cosmetics, and oral care products because they can be highly modified for long-term preservation. The above-mentioned modification of the amino acid acts to greatly improve the peptide stability of the present invention, and the term "stability" as referred to herein also means storage stability (for example, room temperature storage stability) as well as in vivo stability.
일반적으로, 단백질 운반 도메인(PTD)은 라이신/아르기닌 등 기본 아미노산 잔기들을 주로 포함하고 있어서 이와 융합된 단백질들을 세포막을 투과하여 세포내로 침투시키는 역할을 한다. 상기 단백질 운반 도메인(PTD)은 HIV-1 Tat 단백질, 드로소필라 안테나페디아의 호메오도메인(페네트라틴), HSV VP22 전사조절단백질, vFGF에서 유도된 MTS 펩타이드, 트랜스포탄 또는 Pep-1 펩타이드에서 유래된 서열 등을 포함하나, 이에 한정되는 것은 아니다. 이와 같이, 바이러스 또는 양이온성 펩타이드로부터 동정/합성된 다양한 세포투과성 펩타이드들(예를 들어, TAT48 -60, 페네트라틴(pAntp)43 -58, 폴리아르기닌, Pep-1, 트랜스포르탄, 등)은 생물학적 활성 물질, 약물 운반 벡터들의 이동을 매개하기 위해 세포 내재화(internalization)가 가능한 활성을 가진다. 하지만, 종래의 세포투과성 펩타이드를 이용한 약물들에 대한 임상 결과는 아직까지 성공적이지 못 하다. 예를 들어, 폴리아르기닌과 사이클로스포린의 컨쥬게이트인 PsorBan?의 건선 치료제로서의 임상은 임상 II기에서 중단되었다. 또한, 최초의 세포투과성 펩타이드인 TATp를 이용한 임상 Irl가 현재 진행중이다(ISS P-002; Istituto Superiore di Sanita and Novartis). 이 외에도, 암 치료용 PTD(protein transduction domain) 컨스트럭트(TAT-DRBD(double stranded RNA binding domain))에 대한 연구가 Traversa, Inc.에 의해 수행되고 있다. 상술한 연구들은 종결되거나 현재 진행형이지만 아직까지 임상적으로 승인되지 않은 상태인데, 이러한 측면에서 세포투과성 펩타이드 연구에서 중요한 점은 세포독성 및 혈청에서의 분해율을 꼽을 수 있다. 특히, 혈청에서의 펩타이드 분해율은 목적 카르고를 세포에 전달하기 위해 매우 중요하다.Generally, the protein transport domain (PTD) mainly contains basic amino acid residues such as lysine / arginine, and functions to penetrate the cell membrane and permeate into the cell. The protein transport domain (PTD) can be selected from the group consisting of HIV-1 Tat protein, the homeom domain of Drosophila Anapadia (Phenetradine), the HSV VP22 transcriptional regulatory protein, the vFGF-derived MTS peptide, the transposon or the Pep- Derived sequences, and the like. In this way, the virus or cationic peptides variety of cell-permeable peptides identified / synthesized from (e.g., TAT 48 -60, page net Latin (pAntp) 43 -58, polyarginine, Pep-1, trans-formyl carbon, etc. ) Has an activity capable of internalizing cells for mediating the movement of biologically active substances, drug delivery vectors. However, the clinical results of drugs using conventional cell permeable peptides are not yet successful. For example, poly-arginine and the conjugate of cyclosporin PsorBan? The clinical trial for psoriasis was discontinued at Phase II. In addition, clinical Irl using the first cell permeable peptide, TATp, is currently under development (ISS P-002; Istituto Superiore di Sanita and Novartis). In addition, a study on the PTD (TAT-DRBD (double stranded RNA binding domain)) for cancer therapy is being conducted by Traversa, Inc. The above studies have been terminated or are currently in progress, but have not yet been clinically approved. In this respect, cytotoxicity and serum degradation rates are important in cell permeable peptide studies. In particular, the degradation rate of peptides in serum is very important for delivering the target cargo to cells.
본 발명의 펩타이드는 세포질에 존재할 뿐 아니라 낮은 세포독성과 혈청에서의 느린 분해율을 나타냈기 때문에(참고: 실험결과 2-3 및 2-4), 약물전달체로서의 개발 가능성이 매우 높다. 따라서, 본 발명은 (a) 상술한 세포투과성 펩타이드; 및 (b) 상기 펩타이드에 결합된 목적 카르고(cargo of interest)를 포함하는 약물전달체를 제공한다. Since the peptides of the present invention not only exist in the cytoplasm but also exhibit low cytotoxicity and a slow degradation rate in serum (Reference: Experimental Results 2-3 and 2-4), the possibility of development as a drug delivery system is very high. Accordingly, the present invention relates to (a) a cell permeable peptide as described above; And (b) a cargo of interest bound to the peptide.
본 발명의 어떤 구현예에서, 본 발명의 펩타이드는 세포질에 존재한다.In some embodiments of the invention, the peptides of the invention are present in the cytoplasm.
본 발명의 어떤 구현예에서, 본 발명의 펩타이드는 세포독성을 나타내지 않는다. 보다 구체적으로는, 본 발명의 펩타이드를 100 μM의 농도로 처리하는 경우에 세포 내에서 약 50-60%의 세포독성을 나타내지만, 10 μM의 농도로 처리하는 경우 20% 이하의 세포독성을 나타내며, 1 μM 이하의 농도로 처리하는 경우에는 세포독성을 나타내지 않는다(참고: 도 4).In some embodiments of the invention, the peptides of the invention do not exhibit cytotoxicity. More specifically, when the peptide of the present invention is treated at a concentration of 100 μM, it exhibits about 50-60% cytotoxicity in the cell, but when treated at a concentration of 10 μM it exhibits cytotoxicity of 20% or less , And does not show cytotoxicity when treated at a concentration of 1 μM or less (see FIG. 4).
본 발명의 어떤 구현예에서, 본 발명의 펩타이드는 혈청 투여 시 24시간까지 최소 60%까지 분해되지 않는다(참고: 도 5).In some embodiments of the invention, the peptides of the present invention are not degraded by at least 60% until 24 hours upon serum administration (see Figure 5).
통상적으로, 세포투과성 펩타이드는 목적 카르고와 비공유결합 또는 공유결합을 통해 연결되어 구성될 수 있다. 특히, 세포투과성 펩타이드와 목적 카르고가 화학적 교차-연결(cross-linking)을 통해 공유결합 컨쥬게이트를 형성하는 것이 바람직하다(Zatsepin, TS, et al ., Curr . Pharm . Des ., 11: 3639-3654(2005)). 따라서, 본 발명의 펩타이드는 목적 카르고와 공유결합 또는 비공유결합적으로 연결되어 세포 또는 조직 내로 전달시키는 기능을 할 수 있다. 본 발명에 따르면, 본 발명의 펩타이드는 목적 카르고로서 베타-갈락토시다제와 공유결합적으로 연결된 약물전달체는 세포막을 투과하여 상기 베타-갈락토시다제를 안정적이고 높은 효율로 전달하였다(참고: 도 9). 이를 고려할 때, 본 발명의 펩타이드와 결합되어 운반될 수 있는 목적 카르고가 폴리펩타이드인 경우 그 크기는 120 kDa 이하이지만, 이보다 큰 폴리펩타이드도 운반할 수 있을 것으로 예상된다. 본 발명의 어떤 구현예에서, 본 발명의 펩타이드와 결합되어 운반될 수 있는 목적 카르고가 (폴리)펩타이드인 경우 그 크기는 120 kDa 이하이다. 본 명세서에서 사용되는 용어 “목적 카르고(cargo of interest)”는 공유결합 또는 비공유결합을 통해 본 발명의 펩타이드와 컨쥬게이트를 이루어 세포 내로 운반되고자 하는 물질을 의미하며, 예를 들어, 화학물질, 나노입자, 펩타이드, 폴리펩타이드, 안티센스 올리고뉴클레오타이드, siRNA, shRNA, miRNA 및 PNA(peptide-nucleic acid)를 포함하지만, 이에 한정되는 것은 아니다.Typically, a cell permeable peptide can be constructed by linking a non-covalent or covalent bond with a target cargo. In particular, it is preferred that the cell permeable peptide and the target cargo form a covalent bond conjugate through chemical cross-linking (Zatsepin, TS, et al . , Curr . Pharm . Des . , ≪ / RTI > 11: 3639-3654 (2005)). Therefore, the peptide of the present invention may function as a covalent bond or a non-covalent bond with the target cargo and deliver the same into cells or tissues. According to the present invention, the drug delivery system in which the peptide of the present invention is covalently linked to a beta-galactosidase as a target cargo transmits the beta-galactosidase through the cell membrane in a stable and highly efficient manner : Fig. 9). Considering this, it is expected that the target carbohydrate polypeptide which can be carried in combination with the peptide of the present invention is capable of carrying a polypeptide having a size of 120 kDa or less, but larger than that. In some embodiments of the invention, the size of the target cargo (poly) peptide that can be delivered in combination with the peptide of the invention is less than 120 kDa. The term " cargo of interest " as used herein refers to a substance that is conjugated with the peptide of the present invention through covalent or non-covalent bonds and is intended to be transported into cells, for example, Nanoparticles, peptides, polypeptides, antisense oligonucleotides, siRNA, shRNA, miRNA and peptide-nucleic acid (PNA).
또한, 목적 카르고가 폴리펩타이드인 경우, 본 발명의 약물전달체는 본 발명의 펩타이드와 목적 카르고 사이에 링커를 추가적으로 포함할 수 있다. 본 발명에서 이용되는 링커로는 당업계에 공지된 다양한 링커를 이용할 수 있다. 상기 링커는 본 발명 펩타이드의 활성, 즉 세포투과 활성을 최대화하기 위하여 특별하게 선택된 길이 및/또는 서열을 가질 수 있다. 구체적으로는, 상기 링커는 복수의 아미노산 잔기로 이루어진 링커이다. 아미노산 서열로 이루어진 링커는 Huston, et al., Methods in Enzymology, 203:46-88(1991), 및 Whitlow, et al., Protein Eng ., 6:989(1993))에 개시되어 있으며, 상기 문헌은 본 명세서에 참조로서 삽입된다. 본 발명에 적합한 링커는 친수성 아미노산, 특히 아르기닌 및 글루타민으로 구성되며, 그 길이는 5-15개의 아미노산일 수 있다. 본 발명의 어떤 구현예에서, 상기 링커는 복수의 아미노산 잔기로 이루어져 있으며, 보다 구체적으로는 10-12개의 아르기닌 및 글루타민 잔기로 이루어져 있다. 본 발명의 링커는 서열번호 13의 아미노산 서열로 예시되어 있다(참고: 표 3).In addition, when the target cargo is a polypeptide, the drug delivery system of the present invention may additionally include a linker between the peptide of the present invention and the target cargo. As the linker used in the present invention, various linkers known in the art can be used. The linker may have a length and / or sequence specifically selected to maximize the activity of the peptides of the invention, i. E., The cytotoxic activity. Specifically, the linker is a linker composed of a plurality of amino acid residues. Linkers consisting of amino acid sequences are described in Huston, et al., Methods in Enzymology , 203: 46-88 (1991), and Whitlow, et al., Protein Eng . , 6: 989 (1993)), which is incorporated herein by reference. Suitable linkers for the present invention are composed of hydrophilic amino acids, in particular arginine and glutamine, the length of which may be from 5 to 15 amino acids. In some embodiments of the invention, the linker is composed of a plurality of amino acid residues, more specifically 10-12 arginine and glutamine residues. The linker of the present invention is exemplified by the amino acid sequence of SEQ ID NO: 13 (see Table 3).
본 발명의 약물전달체는 다양한 목적으로 이용될 수 있다. 구체적으로는, 본 발명의 약물전달체는 화학물질, 핵산, 나노입자, 등의 물질의 운반에 이용될 수 있으며, 상기 핵산은 안티센스 올리고뉴클레오타이드, siRNA, shRNA, miRNA 및 PNA를 포함하지만, 이에 한정되는 것은 아니다. 또한, 본 발명의 약물전달체는 운반 대상의 카르고에 따라, 질병 또는 질환의 치료, 특정 세포의 검출, 질병(예컨대, 암)의 진단, 특정 세포의 위치 추적 및 인 비보 이미징 등에 이용될 수 있다. 본 명세서에서 사용되는 용어 “핵산 분자”는 DNA(gDNA 및 cDNA) 그리고 RNA 분자를 포괄적으로 포함하는 의미를 갖으며, 핵산 분자에서 기본 구성 단위인 뉴클레오타이드는 자연의 뉴클레오타이드 뿐만 아니라, 당 또는 염기 부위가 변형된 유사체 (analogue)도 포함할 수 있다(Scheit, Nucleotide Analogs, John Wiley, New York(1980); Uhlman 및 Peyman, Chemical Reviews, 90:543-584(1990)).The drug delivery system of the present invention can be used for various purposes. Specifically, the drug delivery system of the present invention can be used for transportation of substances such as chemicals, nucleic acids, nanoparticles, etc., and the nucleic acid includes antisense oligonucleotides, siRNA, shRNA, miRNA and PNA It is not. In addition, the drug delivery system of the present invention can be used for treatment of diseases or diseases, detection of specific cells, diagnosis of diseases (for example, cancer), tracking of specific cells and in vivo imaging according to cartoons to be delivered . As used herein, the term " nucleic acid molecule " is meant to encompass both DNA (gDNA and cDNA) and RNA molecules, and the nucleotide, which is the basic building block in the nucleic acid molecule, includes not only natural nucleotides, But may also include modified analogues (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)).
또한, 본 발명의 약물전달체를 구성하는 펩타이드 및 목적 카르고에 대해 다양한 표지 물질이 이용될 수 있으며, 예를 들어 형광물질[예컨대, 플루오레신, FITC(fluoresein Isothiocyanate), 로다민 6G(rhodamine 6G), 로다민 B(rhodamine B), TAMRA(6-carboxy-tetramethyl-rhodamine), Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI(4,6-diamidino-2-phenylindole) 및 Coumarin], 형광 단백질(fluorescence protein; GFP, RFP, CFP, YFP, BFP, 루시퍼라제 또는 이의 변이체), 방사능동위원소(예컨대, C14, I125, P32 및 S35), 화학물질(예컨대, 바이오틴), 발광물질, 화학발광물질(chemiluminescent) 및 FRET(fluorescence resonance energy transfer)을 포함한다. 본 발명의 약물전달체가 방사능동위원소를 포함하여 구성되는 경우(예컨대, 본 발명의 펩타이드 및 나노입자 구성), 단일 광자 방출 컴퓨터 단층촬영(SPECT, Single Photon Emission Computed Tomography) 또는 양전자 방출 단층촬영(PET, Positron Emission Tomography)에 적용되어 조직의 이미징에 이용될 수도 있다.In addition, various labels may be used for the peptide and the target cargo constituting the drug delivery system of the present invention. For example, fluorescent substances such as fluorescein, FITC (fluorescein isothiocyanate), rhodamine 6G Rhodamine B, TAMRA (6-carboxy-tetramethyl-rhodamine), Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI (4,6-diamidino- , a fluorescent protein (fluorescence protein; GFP, RFP, CFP, YFP, BFP, luciferase or variants thereof), a radioactive isotope (e.g., 14 C, 125 I, 32 P And S 35 ), chemicals (e.g., biotin), luminescent materials, chemiluminescent materials, and fluorescence resonance energy transfer (FRET). When the drug delivery system of the present invention comprises radioactive isotopes (for example, peptides and nanoparticles of the present invention), single photon emission computed tomography (SPECT) or positron emission tomography (PET) , Positron Emission Tomography) and may be used for tissue imaging.
본 발명의 어떤 구현예에서, 본 발명의 약물전달체는 상술한 펩타이드의 N-말단에 FITC가 결합되어 있다.In some embodiments of the present invention, the drug delivery vehicle of the present invention has FITC bound to the N-terminus of the above-mentioned peptide.
한편, 본 발명은 상술한 세포투과성 펩타이드(서열번호 1 내지 서열번호 6)를 인코딩하는 뉴클레오타이드 서열을 제공하며, 상기 뉴클레오타이드 서열은 상술한 펩타이드를 인코딩하는 뉴클레오타이드 서열이라면 제한되지 않고, 이들의 구체적인 예는 서열번호 1 내지 서열번호 6에 대해 각각 서열번호 7 내지 서열번호 12의 뉴클레오타이드 서열로 예시될 수 있다. 보다 구체적으로, 상기 서열번호 1 내지 서열번호 6은 각각 GenBank 접근번호 NP_005130.1, NP_001145.1, P09525, NP_001146.2, P50995 및 P27216로부터 유래되며, 이의 뉴클레오타이드 서열의 구체적인 예는 GenBank 접근번호 NM_005139.2, NM_001154.3, NM_001153.3, NM_001155.4, AJ278464.1 및 BC125158로부터 유래된 것일 수 있다.Meanwhile, the present invention provides a nucleotide sequence encoding the above-mentioned cell permeable peptide (SEQ ID NO: 1 to SEQ ID NO: 6), and the nucleotide sequence is not limited as long as it is a nucleotide sequence encoding the above-mentioned peptide, The nucleotide sequence of SEQ ID NO: 7 to SEQ ID NO: 12 for SEQ ID NO: 1 to SEQ ID NO: 6, respectively. More specifically, SEQ ID NO: 1 to SEQ ID NO: 6 are derived from GenBank Accession Nos. NP_005130.1, NP_001145.1, P09525, NP_001146.2, P50995 and P27216, respectively. Specific examples of nucleotide sequences thereof are given in GenBank Accession No. NM_005139. 2, NM_001154.3, NM_001153.3, NM_001155.4, AJ278464.1, and BC125158.
본 명세서에서 언급되는 용어 “뉴클레오타이드”는 단일가닥 또는 이중가닥 형태로 존재하는 디옥시리보뉴클레오타이드 또는 리보뉴클레오타이드이며, 다르게 특별하게 언급되어 있지 않은 한 자연의 뉴클레오타이드의 유사체를 포함한다(Scheit, Nucleotide Analogs, John Wiley, New York(1980); Uhlman 및 Peyman, Chemical Reviews, 90:543-584(1990)).As used herein, the term " nucleotide " is a deoxyribonucleotide or ribonucleotide present in single-stranded or double-stranded form and includes analogs of natural nucleotides unless otherwise specifically indicated (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)).
또한, 본 발명은 상술한 세포투과성 펩타이드를 인코딩하는 뉴클레오타이드 서열을 포함하는 재조합 발현벡터를 제공할 수 있다. 보다 상세하게는, 본 발명의 재조합 발현벡터는 (a) 프로모터; 및 (b) 상기 프로모터에 작동적으로 연결된(operatively linked) 서열번호 7 내지 서열번호 12로 구성된 군으로부터 선택된 뉴클레오타이드 서열을 포함한다. 이러한 벡터 시스템은 당업계에 공지된 다양한 방법을 통해 구축될 수 있으며, 이에 대한 구체적인 방법은 Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press(2001)에 개시되어 있으며, 이 문헌은 본 명세서에 참조로서 삽입된다. 본 명세서에서 사용되는 용어 “프로모터”는 인코딩 서열 또는 기능적 RNA의 발현을 조절하는 DNA 서열을 의미한다. 본 명세서에서 사용되는 용어 “작동적으로 결합된(operatively linked)”은 핵산 발현 조절 서열(예: 프로모터 서열, 시그널 서열, 또는 전사조절인자 결합 위치의 어레이)과 다른 핵산 서열 사이의 기능적인 결합을 의미하며, 이에 의해 상기 조절 서열은 상기 다른 핵산 서열의 전사 및/또는 해독(translation)을 조절하게 된다.
In addition, the present invention can provide a recombinant expression vector comprising a nucleotide sequence encoding the aforementioned cell permeable peptide. More specifically, the recombinant expression vector of the present invention comprises (a) a promoter; And (b) a nucleotide sequence selected from the group consisting of SEQ ID NO: 7 to SEQ ID NO: 12 operatively linked to the promoter. Such vector systems can be constructed through a variety of methods known in the art, and specific methods for this can be found in Sambrook et < RTI ID = 0.0 > al ., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference. The term " promoter " as used herein refers to a DNA sequence that regulates the expression of an encoding sequence or a functional RNA. As used herein, the term " operatively linked " refers to a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter sequences, signal sequences, or transcription factor binding sites) , Whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequences.
본 발명의 또 다른 양태에 따르면, 본 발명은 상술한 약물전달체를 세포에 접촉시키거나 또는 혈액 내로 주입시키는 단계를 포함하는 목적 카르고의 세포 내 전달방법을 제공한다.According to still another aspect of the present invention, there is provided an intracellular delivery method of a target cargo comprising the steps of contacting the above-mentioned drug carrier with a cell or injecting it into blood.
본 발명의 또 다른 양태에 따르면, 본 발명은 상술한 약물전달체를 포함하는 목적 카르고 운반용 조성물을 제공한다.According to still another aspect of the present invention, there is provided a composition for carrying a cargo comprising the above-mentioned drug carrier.
본 발명의 또 다른 양태에 따르면, 본 발명은 상술한 약물전달체를 포함하는 트랜스펙션 키트를 제공한다.According to another aspect of the present invention, there is provided a transfection kit comprising the above-mentioned drug delivery vehicle.
본 발명의 방법, 조성물 및 키트는 상술한 세포투과성 펩타이드 및 이의 약물전달체를 유효성분으로 포함하기 때문에, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.Since the methods, compositions and kits of the present invention include the above-described cell permeable peptide and its drug carrier as active ingredients, the description common to both of them is omitted in order to avoid the excessive complexity of the present specification.
본 발명의 조성물은 약제학적으로 허용되는 담체와 함께 투여될 수 있다. 약제학적으로 허용되는 담체는 제제 시에 통상적으로 이용되는 것으로서, 락토오스, 덱스트로오스, 수크로오스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로오스, 폴리비닐피롤리돈, 셀룰로오스, 물, 시럽, 메틸 셀룰로오스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences(19th ed., 1995)에 상세히 기재되어 있다.The compositions of the present invention may be administered with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are those conventionally used in pharmaceutical preparations and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Suitable pharmaceutically acceptable carriers and formulations include, but are not limited to, Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명의 조성물은 비경구 방식으로 투여되는 것이 바람직하다. 비경구 투여를 하는 경우, 정맥내 주입, 피내 주입, 병변내(intralesional) 주입, 근육내 주입, 복강내 주입 등으로 투여할 수 있다. 본 발명의 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 결정될 수 있으며 일반적으로 0.0001-100 mg/kg의 범위일 수 있다.The composition of the present invention is preferably administered parenterally. In the case of parenteral administration, it can be administered by intravenous injection, intradermal injection, intralesional injection, intramuscular injection, intraperitoneal injection, and the like. The appropriate dosage of the composition of the present invention may be variously determined by such factors as the formulation method, the manner of administration, the age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, It may generally range from 0.0001-100 mg / kg.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화 함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person skilled in the art to which the present invention belongs Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.
본 발명의 어떤 구현예에서, 본 발명의 방법 및 조성물은 포유동물, 보다 구체적으로는 인간, 마우스, 래트, 기니어 피그, 토끼, 원숭이, 돼지, 말, 소, 양, 영양, 개 및 고양이에 적용될 수 있지만, 이에 한정되는 것은 아니다.In some embodiments of the invention, the methods and compositions of the present invention are administered to a mammal, more particularly a human, a mouse, a rat, a guinea pig, a rabbit, a monkey, a pig, a horse, But is not limited thereto.
한편, 본 발명의 트랜스펙션 키트(transfection kit)는 외부의 DNA/RNA를 포유동물의 세포 내로 도입이 용이하도록 최적화된 시스템이다. 현재까지 주로 의 트랜스펙션 키트는 칼슘포스페이트법, 지질결합체를 이용하는 방법, 덱스트란 복합체를 이용하는 방법 등이 있으나 이들의 효율성은 1/106 내지 1/102 정도이고 세포의 종류에 의존성이 있는 한계가 있다. 이를 극복하기 위해 세포투과성 펩타이드/단백질을 이용한 트랜스펙션 키트를 이용할 수 있다.Meanwhile, the transfection kit of the present invention is a system optimized for facilitating the introduction of external DNA / RNA into mammalian cells. Until now, the main transfection kits include calcium phosphate method, lipid conjugate method, and dextran conjugate method, but their efficiency is about 1/10 6 to 1/10 2 and they are dependent on the kind of cells There is a limit. To overcome this, a transfection kit using a cell permeable peptide / protein can be used.
본 발명의 트랜스펙션 키트는 추가로 상기 펩타이드와 목적 카르고 간의 링커/결합인자를 추가로 포함할 수 있다. 상기 결합인자는 전사인자 또는 바이러스 단백질을 포함하는 특정 DNA/RNA 서열 또는 단백질 등의 목적 카르고와 결합할 수 있는 단백질 전체 또는 일부를 포함한다. 예를 들어, Gal4는 DNA 결합인자로, 진핵세포생물, 원핵세포 생물, 바이러스에서 널리 사용되는 전사인자이다. DNA/RNA 결합인자는 인 비보 및 인 비트로에서 PTD와 융합단백질을 제조할 수 있는 단백질 발현벡터를 적용하여 이용가능하다. 또한, DNA/RNA 결합인자와 PTD 간의 융합은 화학적 결합, 물리적 공유결합, 또는 비공유 결합에 의해서도 가능하다.The transfection kit of the present invention may further comprise a linker / binding factor between the peptide and the target cargo. The binding factor includes all or a portion of a protein capable of binding a target cargo such as a specific DNA / RNA sequence or protein including a transcription factor or a viral protein. For example, Gal4 is a DNA binding factor, a transcription factor widely used in eukaryotic, prokaryotic, and viruses. DNA / RNA binding factor may be used by applying in vivo, protein expression vectors capable of producing a fusion protein and the PTD in vitro. Fusion between DNA / RNA binding factors and PTDs can also be achieved by chemical bonding, physical covalent bonding, or noncovalent bonding.
본 발명의 세포투과성 펩타이드와 DNA/RNA과의 융합 컨스트럭트를 만들어 이를 세포 외부에 처리해 주면 효율성과 세포 종류에 의존적인 한계를 극복할 수 있게 된다. 본 발명의 펩타이드와 DNA/RNA 결합인자를 이용하여 인 비보 및 인 비트로에서 다양한 세포의 세포질로 DNA/RNA를 효율적으로 도입할 수 있게 된다. 예를 들어, 전달 방법은 근육내, 복강내, 정맥, 경구, 피하, 피내, 비강내, 흡입을 포함하는 다양한 방법을 통해서 가능할 수 있다. 따라서, 본 발명의 트렌스펙션 키트는 본 발명의 방법에 의해서 유전자 치료와 DNA/RNA 백신에 대한 새로운 방법을 제공할 수 있으며, 일시적 또는 영구적으로 발현이 가능하며 DNA/RNA 백신과 유전자치료와 같은 의학임상 적용에서 뿐만 아니라 기초 연구의 활용에서도 다양하게 사용될 수 있다.
By constructing a fusion construct of the cell permeable peptide of the present invention with DNA / RNA and treating it outside the cell, it is possible to overcome the limitations depending on the efficiency and the cell type. It is possible to efficiently introduce DNA / RNA into cytoplasm of various cells in In vivo and in vitro using the peptide of the present invention and a DNA / RNA binding factor. For example, the delivery method may be through a variety of methods including intramuscular, intraperitoneal, intravenous, oral, subcutaneous, intradermal, intranasal, and inhalation. Therefore, the transfection kit of the present invention can provide a novel method for gene therapy and DNA / RNA vaccine by the method of the present invention, and can be transiently or permanently expressed and used for DNA / RNA vaccines and gene therapy It can be used in clinical applications as well as in basic research applications.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 신규한 세포투과성 펩타이드 및 이의 용도에 관한 것이다.(a) The present invention relates to novel cell permeable peptides and uses thereof.
(b) 본 발명의 펩타이드는 인간 아넥신 단백질-유래된 아미노산 서열로, 우수한 세포투과성을 가질 뿐 아니라, 세포독성을 나타내지 않고 혈청에서 느린 분해율을 보인다.(b) The peptide of the present invention is a human anexin protein-derived amino acid sequence, which not only has excellent cell permeability but also shows a slow degradation rate in serum without cytotoxicity.
(c) 또한, 본 발명의 펩타이드에 결합된 목적 카르고(예컨대, 베타-갈락토시다제)는 안정적이고 효과적으로 세포 내(세포질)로 운반된다.(c) In addition, the objective cargo (for example, beta-galactosidase) bound to the peptide of the present invention is stably and effectively transported intracellularly (cytoplasmic).
(d) 따라서, 본 발명의 펩타이드를 포함하는 약물전달체, 그리고 이를 이용하는 목적 카르고의 전달방법 및 조성물은 질병 또는 질환의 치료, 특정 세포의 검출, 질병(예컨대, 암)의 진단, 특정 세포의 위치 추적 및 인 비보 이미징 등에 이용될 수 있다.
(d) Therefore, the drug delivery system comprising the peptide of the present invention, and the method and composition for delivering the target cargo using the peptide of the present invention are useful for the treatment of diseases or diseases, the detection of specific cells, the diagnosis of diseases Location tracking and in-beam imaging.
도 1은 처리 농도에 따른 12종류의 아넥신-유래 펩타이드의 세포투과능을 측정한 FACS 결과이다. X축, 녹색 형광(Green Fluorescence); 및 Y축, 세포수(count).
도 2는 본 발명의 펩타이드(AA3H 및 AA5H)와 TAT-CPP 펩타이드 간의 세포 투과 효율을 비교한 결과이다. X축, 녹색 형광; 및 Y축, 세포수.
도 3은 본 발명의 펩타이드(AA3H 및 AA5H)의 농도-의존적 세포 투과능을 보여주는 결과이다. X축, 녹색 형광; 및 Y축, 세포수.
도 4는 본 발명의 펩타이드(AA3H 및 AA5H)와 TAT-CPP 펩타이드의 세포독성을 비교한 결과이다.
도 5는 본 발명의 펩타이드 AA3H의 혈청 내 분해능을 시간에 따라 측정한 결과이다.
도 6은 엔도사이토시스 억제제를 통한 본 발명의 펩타이드 AA3H-CPP의 세포 투과 경로를 확인한 결과이다.
도 7은 세포내 소기관의 마커 단백질들을 이용한 본 발명의 펩타이드 AA3H-CPP의 세포 내 투과 위치를 확인한 결과이다.
도 8은 본 발명의 펩타이드 AA3H와 결합된 Gal의 세포 내 투과를 보여주는 결과이다. X축, 녹색 형광; 및 Y축, 세포수.
도 9는 본 발명의 펩타이드 AA3H-CPP를 이용한 β-갈락토시다제의 세포 내 전달을 보여주는 염색 결과이다.Fig. 1 shows FACS results of measuring the cell permeability of 12 kinds of annexin-derived peptides according to the treatment concentration. X-axis, Green Fluorescence; And the Y axis, the number of cells (count).
FIG. 2 shows the results of comparing the cell permeation efficiency between the peptides (AA3H and AA5H) of the present invention and the TAT-CPP peptide. X-axis, green fluorescence; And Y axis, cell number.
Figure 3 shows the results showing the concentration-dependent cellular permeability of the peptides of the invention (AA3H and AA5H). X-axis, green fluorescence; And Y axis, cell number.
Fig. 4 shows the results of comparing the cytotoxicity of the peptides (AA3H and AA5H) of the present invention with the TAT-CPP peptide.
FIG. 5 shows the results of measuring the resolution of the peptide AA3H of the present invention in serum.
Figure 6 shows the results of confirming the cell permeation pathway of the peptide AA3H-CPP of the present invention through an endocytosis inhibitor.
FIG. 7 shows the results of confirming intracellular permeation sites of the peptide AA3H-CPP of the present invention using intracellular organelle marker proteins.
Figure 8 shows the intracellular permeation of Gal bound to the peptide AA3H of the present invention. X-axis, green fluorescence; And Y axis, cell number.
FIG. 9 shows the result of staining showing intracellular delivery of? -Galactosidase using the peptide AA3H-CPP of the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
실시예Example
실험방법 및 실험결과Experimental methods and experimental results
본 발명자들은 사람에서 발견된 아넥신 단백질 중 12종류를 선택하여 각 서열의 특성을 분석하였고, 참고문헌들을 통해 구조적으로 번역 후 변형 위치들(예컨대, 미리스토일화(myristolylation)) 및 세포막 및 세포막 단백질들과의 결합에 중요한 N-말단으로부터 10개의 아미노산 서열을 각 종류의 아넥신으로부터 선택하여 이들과 세포투과 능력 간의 연관성을 조사하였다. 상기 12종류의 아넥신 단백질은 세포의 각 소기관에 다양하게 위치해 있음이 알려져 있는데, 이에 대해 소기관 위치 데이터베이스(Locate: subcellular localization database)에서 확인하여 표 1에서 재구성하였다(표 1).The present inventors selected twelve kinds of annexin proteins found in humans and analyzed the characteristics of each sequence, and structurally translated post-translationally modified positions (for example, myristolylation) and cell membrane and cell membrane proteins The 10 amino acid sequences from the N-terminal which are important for binding to the ssDNA were selected from each kind of annexin and the relationship between them and the cell permeability was examined. The 12 kinds of annexin proteins are known to be located in various organelles of cells. Locations (subcellular localization database) were confirmed and they were reconstituted in Table 1 (Table 1).
(서열번호 1)Annexin A3
(SEQ ID NO: 1)
(서열번호 3)Annexin A4
(SEQ ID NO: 3)
(서열번호 2)Annexin A5
(SEQ ID NO: 2)
(서열번호 4)Annexin A6
(SEQ ID NO: 4)
(서열번호 5)Annexin A11
(SEQ ID NO: 5)
(서열번호 6)Annexin A13
(SEQ ID NO: 6)
아넥신 서브패밀리에서의 서열.
Sequence in the annexin subfamily.
선택된 펩타이드 서열을 형광 물질로 표지한 뒤 이들이 세포 내로 잘 수송 되는 지의 여부를 여러 실험을 통해 확인하고자 하였다. 대조군으로는 이미 잘 알려진 TAT-CPP를 선정하여 비교하였다. 아넥신 12종류와 대조군 TAT-CPP의 서열과 등전점 및 크기에 대한 정보는 상기 표 1에 기재되어 있다.After labeling the selected peptide sequence with a fluorescent substance, it was tried to confirm whether or not these peptides were transported well into cells by various experiments. As a control group, well-known TAT-CPP was selected and compared. Information on the sequence, isoelectric point and size of 12 kinds of annexin and control TAT-CPP are shown in Table 1 above.
종래에는 바이러스나 양이온성 펩타이드, 또는 세포막 투과에 탁월한 서열을 선택하여 합성한 펩타이드인 반면에, 본 발명은 인간 내에 존재하는 단백질로부터 전달체 기능을 가진 서열을 동정하여 새로운 CPP를 개발하였다.
In the past, the peptide was synthesized by selecting a virus or a cationic peptide, or a sequence superior in cell membrane permeability. On the other hand, the present invention developed a novel CPP by identifying a sequence having a transporter function from a protein existing in a human.
2-1. 2-1. 아넥신Annexin 단백질로부터 From protein 유래된Derived 세포투과 아미노산 서열 합성 Cell-mediated amino acid sequence synthesis
고체상 펩타이드 합성 방법을 이용하여 각 아미노산 서열을 합성하였고, 링크-아미드 MBHA 레진(rink-amide methylbenzylhydrylamine resin; Novabiochem, #855003)을 사용하여 C-말단에서부터 각 아미노산을 연결하고 N-말단에 형광물질인 FITC 이소머 I(fluorescein isothiocyanate isomer I; Sigma, #F7250)를 붙였다. 마지막 단계에서 95% TFA(trifluoroacetic acid; Sigma, #302031)/2.5% 트리이소프로필실란(triisopropylsilane; Sigma, #233781)/2.5% H2O를 섞어 2시간 동안 상온에서 반응하고 디에틸 에테르(Sigma, #309966)로 침전시켰다. 합성된 펩타이드는 역상 HPLC C18 컬럼을 통해 추출하였고, 0.1% TFA가 들어있는 물과 아세토니트릴(Fisher scientific, #955-1)의 5-80% 농도에서 각 펩타이드를 획득하였다. 각 펩타이드의 분자 크기는 MALDI-TOF 질량분석기(mass spectroscopy)를 통해 측정하여 확인하였다.
Each amino acid sequence was synthesized using a solid phase peptide synthesis method, and each amino acid was linked from the C-terminus using a link-amide methylbenzylhydrylamine resin (Novabiochem, # 855003) FITC isomer I (fluorescein isothiocyanate isomer I; Sigma, # F7250). In the final stage 95% TFA (trifluoroacetic acid; Sigma , # 302031) /2.5% triisopropylsilane (triisopropylsilane; Sigma, # 233781) /2.5% H 2 O reaction mix at room temperature for 2 hours, diethyl ether (Sigma , # 309966). The synthesized peptides were extracted on a reversed phase HPLC C18 column and each peptide was obtained at 5-80% concentration of water containing 0.1% TFA and acetonitrile (Fisher scientific, # 955-1). Molecular size of each peptide was confirmed by mass spectroscopy (MALDI-TOF).
2-2. 2-2. 아넥신Annexin 단백질로부터 From protein 유래된Derived 세포 투과 Cell permeation 펩타이드의Of peptide 세포 내 수송 효과 Intracellular transport effect
상기 합성된 12종류의 아넥신-CPP 후보물질들의 세포 내 수송 능력을 확인하기 위해, 인간 자궁암 세포주(HeLa)에 0, 0.1, 1, 10 μM 농도로 4시간 동안 처리한 후 유세포 분석기(Guava easyCyte, milipore, USA)를 이용하여 관찰하였다(도 1). 도 1에 표기된 AA1 내지 AA11 및 AA13은 각각 아넥신 A1 내지 A11 및 AA13 인간 형태(Human form)를 나타내고 아넥신 AA1 내지 AA11 및 AA13으로 지시되었으며, 이 중 AA3H(Annexin A3 Human form)-CPP 또는 AA5H-CPP 후보물질이 1 μM일 때 TAT-CPP보다 더 좋은 수송 능력을 갖는다는 것을 확인하였다(도 2). 또한, 상기 후보물질이 세포 내에 농도-의존적으로 투과하는지 확인하기 위해 5가지 농도(0, 0.625, 1.25, 2.5, 5, 10 μM)를 인간 자궁암세포에 처리한 후, 유세포 분석기로 확인한 결과 농도-의존적으로 세포 내로 들어가는 것을 확인할 수 있었다(도 3).
In order to confirm the intracellular transport capacity of the synthesized 12 kinds of Annexin-CPP candidate substances, human cervical cancer cell line (HeLa) was treated at a concentration of 0, 0.1, 1, 10 μM for 4 hours and then analyzed using a flow cytometer (Guava easyCyte , Milipore, USA) (Fig. 1). FIG AA1 through AA11 and AA13 displayed on the first respectively annexin A1 to A11 and AA13 represents the human form (Human form) Ah has been indicated by annexin AA1 through AA11 and AA13, of which AA3H (A nnexin A 3 H uman form) - CPP or AA5H-CPP candidates at 1 [mu] M had better transport capacity than TAT-CPP (Fig. 2). Five concentrations (0, 0.625, 1.25, 2.5, 5, and 10 μM) of human cancer cells were treated with human cervical cancer cells to determine whether the candidate substances were permeated in cells in a concentration- Dependent manner into the cells (Fig. 3).
2-3. 인간 자궁암세포에서의 2-3. In human uterine cancer cells AA3HAA3H -- CPPCPP 및 And AA5HAA5H -- CPPCPP 의 세포독성 평가Of cytotoxicity
인간 자궁암세포에서 AA3H-CPP 및 AA5H-CPP의 농도-의존적으로 세포막 투과능을 보였고, 이러한 투과능이 세포에 영향을 미치는지 확인하기 위해 세포 카운팅 키트(CCK-8; Dojindo, Japan)를 이용하여 세포 독성을 측정하였다. 96-웰 플레이트에서 인간 자궁암세포를 80% 정도의 컨플루언스로 배양하고, 비교군인 TAT-CPP, AA3H-CPP 및 AA5H-CPP를 각각 0.01, 0.1, 1, 10 및 100 μM 농도로 처리하여 24시간 동안 반응시켰다. 각 CPP가 처리된 세포에 CCK-8 용액 10 μl를 넣고 2시간 동안 반응하고 450 nm 흡광도에서 측정하였다. 그 결과, AA3H-CPP, AA5H-CPP 및 TAT-CPP가 100 μM 농도일 때 세포독성을 나타냈는데, AA3H-CPP 및 AA5H-CPP는 각각 약 50% 및 약 60%의 세포독성이 보인 반면에, 비교군인 TAT-CPP는 약 70%의 독성을 보였다. 또한, 10 μM 농도로 처리된 경우에 TAT-CPP는 20% 이상의 독성을 나타내는 반면에, AA5H-CPP는 10 μM 농도에서 약 24%의 세포독성을 보였으나 100 μM 농도에서 상대적으로 덜한 세포독성을 나타냈으며, AA3H-CPP는 10 μM 및 100 μM 농도에서 전혀 세포독성을 나타내지 않았다(도 4).
Cell permeability was determined in the concentration-dependent manner of AA3H-CPP and AA5H-CPP in human uterine cancer cells. Cell permeability was determined by using cell counting kit (CCK-8; Dojindo, Japan) Were measured. Human uterine cancer cells were cultured in a 96-well plate at a confluence of about 80%, and the comparative groups TAT-CPP, AA3H-CPP and AA5H-CPP were treated at concentrations of 0.01, 0.1, 1, Lt; / RTI > 10 μl of CCK-8 solution was added to the cells treated with each CPP, reacted for 2 hours, and measured at 450 nm absorbance. As a result, AA3H-CPP, AA5H-CPP and TAT-CPP showed cytotoxicity at a concentration of 100 μM, while AA3H-CPP and AA5H-CPP showed about 50% and 60% The comparative group TAT-CPP showed about 70% toxicity. In addition, TAT-CPP showed more than 20% toxicity when treated at 10 μM concentration, whereas AA5H-CPP showed about 24% cytotoxicity at 10 μM concentration, but relatively less cytotoxicity at 100 μM concentration , And AA3H-CPP showed no cytotoxicity at 10 μM and 100 μM concentrations (FIG. 4).
2-4. 마우스 혈청 안에서의 2-4. In mouse serum AA3HAA3H -- CPPCPP 분해 평가 Decomposition evaluation
AA3H-CPP 및 AA5H-CPP 중 AA3H-CPP가 세포막 투과 효율이 더 좋은 것으로 나타났기 때문에, AA3H-CPP를 이후의 연구에 주로 이용하였다. CPP는 다양한 질병 모델에서 약물 전달 물질로 이용되고 있는데, 조직이나 동물에 물질을 전달하는 경우 혈청에 의해 분해되어 효율이 낮아지는 단점이 있다. 이에, AA3H-CPP의 혈청 내 분해능을 확인하기 위해 10% 마우스 혈청에 10 μM의 AA3H-CPP를 넣고 시간에 따라(0.25, 2, 4, 8, 10 및 24시간) 분해 정도를 HPLC로 확인하였다(도 5). 도 5A는 시간별 혈청 내에 존재하는 AA3H-CPP를 측정한 HPLC 결과이고 도 5B는 시간별 확인된 HPLC 값을 백분율로 나타낸 결과이다. AA3H-CPP는 마우스 혈청에서 2시간 동안 반응시켰을 때에는 약 20% 정도 분해되었으며, 8시간째에는 약 30% 정도 분해되었고, 24시간 동안 반응시켜도 약 40% 정도의 분해율을 나타냈다. 따라서, 마우스 AA3H-CPP를 혈청 내에서 24시간 동안 반응시켜도 약 60% 이상의 AA3H-CPP가 남아있다는 점에서, 본 발명의 AA3H-CPP는 혈청에서 쉽게 분해되지 않는다는 것을 확인할 수 있었다. 또한, 혈청과 AA3H-CPP의 반응은 1시간 이내에 약 20% 정도만 분해될 뿐이고 이후 2시간부터 24시간까지 분해율에 거의 변함이 없어 혈청 내 분해 속도가 매우 느리다는 것을 확인하였다.
Since AA3H-CPP among AA3H-CPP and AA5H-CPP showed better cell membrane permeation efficiency, AA3H-CPP was mainly used in later studies. CPP is used as a drug delivery agent in various disease models. When CPP is delivered to tissues or animals, the CPP is degraded by the serum and the efficiency is lowered. In order to confirm the resolution of AA3H-CPP in serum, 10 μM AA3H-CPP was added to 10% mouse serum and the degree of degradation was determined by HPLC over time (0.25, 2, 4, 8, 10 and 24 hours) (Fig. 5). FIG. 5A shows the HPLC results of AA3H-CPP in the serum according to time, and FIG. 5B shows the HPLC values as a percentage of the time. AA3H-CPP degraded about 20% when reacted with mouse serum for 2 hours, decomposed by about 30% at 8 hours, and about 40% when reacted for 24 hours. Therefore, it was confirmed that the AA3H-CPP of the present invention was not easily degraded in serum because about 60% or more of AA3H-CPP remained even when the mouse AA3H-CPP was reacted in the serum for 24 hours. In addition, the reaction between serum and AA3H-CPP was only degraded by about 20% within 1 hour, and then it showed almost no degradation rate from 2 hours to 24 hours, indicating that the degradation rate in serum was very slow.
2-5. 인간 자궁암세포에서 2-5. In human uterine cancer cells AA3HAA3H -- CPPCPP 의 세포 내 투과 경로 및 위치 확인Of intracellular permeation pathways and location of
CPP가 세포 내로 들어가는 기전은 주로 엔도사이토시스(Endocytosis)이지만, CPP의 성격 및 전달 물질들에 따라 세포 내 수송 경로가 달라지고 최근에는 직접적 전위(translocation)로도 이동한다는 보고가 있었다. 엔도사이토시스는 마크로피노사이토시스(macropinocytosis), 클래트린(clathrin)-매개된 경로, 카베올래/지질-라프트(lipid-raft)-매개된 경로, 또는 클래트린, 카베올래-비의존적 경로의 네 가지 경로로 나뉜다. 이들 중 어느 경로를 통해 AA3H-CPP가 들어가는지 알아보기 위해 인간 자궁암세포에서 각 경로에 특이적인 저해제(표 2)를 30분 동안 전 처리한 후, 세포 내 투과 변화를 관찰하였다.The mechanism by which CPP enters the cell is mainly endocytosis, but it has been reported that the intracellular transport path varies depending on the nature of CPP and the transporter, and recently, it also translocates to direct translocation. Endocytosis may be caused by macropinocytosis, clathrin-mediated pathway, caveolae / lipid-raft-mediated pathway, or clathrin, The path is divided into two. In order to investigate the pathway of AA3H-CPP through these pathways, pretreatment of each pathway-specific inhibitor (Table 2) in human cervical cancer cells was carried out for 30 minutes and then the intracellular permeation change was observed.
(카탈로그 넘버)(Catalog number)
마크로피노사이토시스
카베올래/지질-라프트-매개된 엔도사이토시스
엔도좀/리소좀의 파괴
액틴의 파괴Clathrin-mediated endocytosis
Macropinocytosis
Caveolae / lipid-raft-mediated endocytosis
Endosomes / destruction of lysosomes
Actin destruction
5-(N-에틸-니소프로필) 아밀로리드(EPIA)
메틸-β-사이클로덱스트린(MβCD),
니스타틴
클로로퀸(CQ)
사이토칼라신 D(CYD)Chlorpromazine (CPZ)
5- (N-ethyl-nisopropyl) amiloride (EPIA)
Methyl-beta-cyclodextrin (M? CD),
Nystatin
Chloroquine (CQ)
Cytochalasin D (CYD)
시그마(A3085)
시그마(C4555, N6261)
시그마(C6628)
시그마(C8273)Sigma (C8138)
Sigma (A3085)
Sigma (C4555, N6261)
Sigma (C6628)
Sigma (C8273)
엔도사이토시스 타입에 따라 특이적인 억제제들.
Specific inhibitors depending on endocytosis type.
유세포 분석기를 통해 확인한 결과, AA3H-CPP는 두 가지 경로를 통해 세포 내로 들어가는 것으로 보인다. 도 6에서 볼 수 있듯이, 주로 마크로피노사이토시스(macropinocytosis)를 통해 들어가고, 부분적으로는 카베올래/지질-라프트를 통해 세포 내로 투과되는 것이 관찰되었다. 유세포 분석 그래프에서 X-축은 FITC의 형광도를 나타내고 Y-축은 세포수를 나타내는데, AA3H-CPP의 N-말단에 FITC가 붙어있어 세포 내에 들어간 AA3H-CPP를 명확하게 확인할 수 있다. 또한, 세포막에 붙어있는 AA3H-CPP를 제거하기 위해 0.01%의 트립신으로 10분 동안 처리하여 세포막 투과의 비특이적 활성을 억제하였다.Through the flow cytometry analysis, AA3H-CPP appears to enter the cell through two pathways. As can be seen in FIG. 6, it was observed that it penetrated mainly through macropinocytosis, and partly through the caveolae / lipid-raft into the cells. In the flow cytometry graph, the X-axis represents the fluorescence of FITC and the Y-axis represents the number of cells. The AA3H-CPP is attached to the N-terminus of AA3H-CPP. In addition, in order to remove AA3H-CPP attached to the cell membrane, it was treated with 0.01% trypsin for 10 minutes to inhibit nonspecific activity of cell membrane permeation.
다음으로, AA3H-CPP가 세포 내 수송될 때 어느 기관에 위치하는지 알아보기 위해 각 기관의 마커(표 3)를 사용하여 공초점 현미경(confocal microscopy; LSM700, GER)으로 확인하였다. Next, confocal microscopy (LSM700, GER) was used to confirm the location of AA3H-CPP in the organs when intracellularly transported, using markers from each organ (Table 3).
(카탈로그 넘버)(Catalog number)
핵
골지
ER
액틴Lysosome
nucleus
Golgi
ER
Actin
호에스트(Hoechst)
셀라이트 골지-RFP 백맴 2.0
셀라이트 ER-RFP 백맴 2.0
셀라이트 액틴-RFP 백맴 2.0Litho tracker
Hoechst
Celite Golgi - RFP Backing 2.0
Cell Light ER-RFP Backing 2.0
Celite Actin-RFP Backing 2.0
인비트로젠(33342)
인비트로젠(C10593)
인비트로젠(C10591)
인비트로젠(C10502)Invitrogen (L12492)
Invitrogen (33342)
Invitrogen (C10593)
Invitrogen (C10591)
Invitrogen (C10502)
각 소기관의 마커들.
Markers of each organelle.
세포에 AA3H-CPP를 4시간 동안 처리한 후 상기 세포를 각 소기관의 마커로 염색하여 공초점 현미경으로 확인한 결과, 리소좀, 핵, 골지, ER 및 액틴에 위치하지 않았다. 상술한 결과를 통해, AA3H-CPP는 리소좀에 의해 분해되지 않고 핵 내에도 위치하지 않는다는 것을 확인할 수 있었다. 이 밖에도, 다른 소기관인 골지 및 ER에서도 관찰되지 않는다는 점에서 세포질에 존재할 가능성이 높을 것으로 예상할 수 있다(도 7).
Cells were treated with AA3H-CPP for 4 hours. The cells were stained with markers of each organelle and confocal microscopy confirmed that they were not located in lysosomes, nuclei, corpus, ER, and actin. From the above results, it can be confirmed that AA3H-CPP is not decomposed by the lysosome and is not located in the nucleus. In addition, it can be expected that it is highly likely to be present in the cytoplasm in that it is not observed in other organelles such as Golgi and ER (FIG. 7).
2-6. 2-6. AA3HAA3H -- CPPCPP 의 응용을 위한 For the application of 세포내Intracellular 베타- beta- 갈락토시다제Galactosidase (( galactosidase가alat 시드세제 ) 전달) relay
AA3H-CPP를 전달체로 사용하기 위해 세포 내 전달 물질 단백질로 베타-갈락토시다제를 선택하였다. 베타-갈락토시다제는 120 kDa 이상의 크기의 단백질이기 때문에, 베타-갈락토시다제를 세포 내로 전달 할 수 있다면 120 kDa 이하의 크기를 가지는 물질을 수송할 수 있는 가능성을 제시할 수 있어 CPP 적용에 많이 사용되어온 단백질이다. AA3H-CPP가 베타-갈락토시다제를 세포 내로 전달할 수 있는지 확인하기 위해, 베타-갈락토시다제 융합 펩타이드를 AA3H-CPP에 연결하여 합성하였다. 베타-갈락토시다제와 융합할 수 있는 서열은 Kristopher M, 등(Scientific reports . 1661(3): 1-7, 2013)이 발표한 논문을 참고하였다. 베타-갈락토시다제 융합 펩타이드 합성서열과 크기, 등전점을 하기 표 4에 서술되어 있다.In order to use AA3H-CPP as a carrier, beta-galactosidase was selected as an intracellular transporter protein. Since beta-galactosidase is a protein with a size of more than 120 kDa, if beta-galactosidase can be delivered into a cell, it may be possible to transport a substance with a size below 120 kDa. It is a protein that has been widely used for. To confirm that AA3H-CPP can deliver beta-galactosidase into cells, beta-galactosidase fusion peptide was synthesized by linking to AA3H-CPP. Sequences that can be fused with beta-galactosidase are described by Kristopher M, et al. ( Scientific reports . 1661 (3): 1-7, 2013). The synthesis sequence, size and isoelectric point of the beta-galactosidase fusion peptide are described in Table 4 below.
AA3H-galGal-linking
AA3H-gal
FITC-MASIWVGHRG-RRRQQQQQQRRRRRQQQQQQRRR
FITC-MASIWVGHRG-RRRQQQQQQRRR
13.2813.20
13.28
3434.062225.56
3434.06
베타-갈락토시다제와 AA3H-CPP 간의 융합을 위한 펩타이드 서열.
Peptide sequence for fusion between beta-galactosidase and AA3H-CPP.
베타-갈락토시다제 융합 아미노산 서열과 AA3H-CPP 서열 사이는 링커(6-(fmoc-아미노)카프로익산; Fluka, #04067)로 연결하였으며, 이를 AA3H-gal이라 명명하였다. AA3H-CPP의 N-말단에는 FITC 형광물질을 연결하여 베타-갈락토시다제를 융합시킨 후에도 세포막 투과를 하는지 유세포 분석기를 통해 확인하였다(도 8). 그 결과, 베타-갈락토시다제 융합 펩타이드를 연결하여도 세포 내로 잘 투과되는 것을 알 수 있었고, 한편으로는 베타-갈락토시다제 융합 펩타이드만 단독으로 인간 자궁암세포에 처리하여도 세포 내로 수송되는 현상을 볼 수 있었다. 상술한 융합 펩타이드 자체의 세포 내 수송이 베타-갈락토시다제를 융합시켰을 때에도 나타나는지 확인하기 위해, 베타-갈락토시다제를 세포 내로 전달했을 때 베타-갈락토시다제 발현된 세포만 염색하는 방법을 통해 확인하였다.The beta-galactosidase fusion amino acid sequence and the AA3H-CPP sequence were linked by a linker (6- (fmoc-amino) caproic acid; Fluka, # 04067) and named AA3H-gal. After the FITC fluorescent substance was ligated to the N-terminal of AA3H-CPP and the β-galactosidase was fused, permeability of the cell membrane was confirmed by flow cytometry (FIG. 8). As a result, it was found that even when the beta-galactosidase fusion peptide was ligated, the beta-galactosidase fusion peptide was well permeated into the cell. On the other hand, only the beta-galactosidase fusion peptide was transported into cells I could see the phenomenon. In order to confirm whether intracellular transport of the above-mentioned fusion peptide itself also occurs when beta-galactosidase is fused, a method of staining only beta-galactosidase-expressing cells when beta-galactosidase is delivered into cells Respectively.
AA3H-gal과 베타-갈락토시다제(Sigma, #GALS)를 융합하기 위해 융합 완충액(50 mM Tris-HCl pH7.4, 5 mM CaCl2, 1 mM DTT)에 트랜스글루타미나제(transgultaminase; Sigma, #T5398)를 넣고 상온에서 한 시간 반응하였고, 이를 인간 자궁암세포에 3시간 동안 처리하였다. 이후, PBS로 두 번 씻어내고 베타-갈락토시다제 리포터 유전자 염색 키트(Sigma, #GALS)를 이용하여 세포에 전달된 베타-갈락토시다제를 X-gal(5-브로모-4-클로로-3-인돌릴-베타-D-갈락토피라노시드) 기질을 처리하여 염색하고 현미경(nuance microscopy; Thermo Fisher Scientific)으로 관찰하였다. 세포 내로 베타-갈락토시다제가 전달되어 발현이 되면 X-gal과 반응하여 푸른색을 나타내며 그렇지 않을 때에는 색이 나타나지 않는다. 이 실험에 있어서, 대조군으로는 베타-갈락토시다제만 세포에 처리한 군과 Gal-linking에 베타-갈락토시다제를 융합한 군(이하, Gal-linking/베타-갈락토시다제)을 사용하였고, AA3H-gal과 베타-갈락토시다제를 융합한 물질(이하, AA3H-gal/베타-갈락토시다제)과의 전달 효율을 비교하였다(도 9). AA3H-gal/베타-갈락토시다제는 베타-갈락토시다제가 세포 내로 잘 전달되어 X-gal로 염색했을 때 짙은 푸른색을 나타내었다. 대조군인 Gal-linking/베타-갈락토시다제의 경우에도 베타-갈락토시다제가 세포 내로 전달되어 푸른색으로 염색이 되었다. 상술한 도 8의 유세포 분석에서 보여진 과와 마찬가지로, 베타-갈락토시다제와 Gal-linking 펩타이드를 융합하였을 때에도 베타-갈락토시다제가 세포 내로 전달되었다. 이는 Gal-linking 펩타이드에 양성 아르기닌 서열이 포함되어 있어 세포 내 투과에 영향을 미친 것으로 추정된다. 하지만, 대조군(베타-갈락토시다제 또는 Gal-linking/베타-갈락토시다제)과 비교 시 AA3H-CPP에 의한 베타-갈락토시다제 전달능이 훨씬 뛰어남을 확인할 수 있었다(도 9).(50 mM Tris-HCl pH 7.4, 5 mM CaCl 2 , 1 mM DTT) was added to fusions of AA3H-gal and beta-galactosidase (Sigma, # GALS) in a fusion buffer (transgultaminase; Sigma, # T5398) and incubated at room temperature for one hour. The cells were treated with human cervical cancer cells for 3 hours. Then, the cells were washed twice with PBS, and the beta-galactosidase transferred to the cells was stained with X-gal (5-bromo-4-chloro- -3-indolyl-beta-D-galactopyranoside) substrate was treated and stained and observed with a microscope (nuance microscopy; Thermo Fisher Scientific). When beta-galactosidase is transferred into the cell and expressed, it reacts with X-gal to display a blue color, otherwise it does not appear. In this experiment, beta-galactosidase-treated cells and Gal-linking-conjugated beta-galactosidase (hereinafter referred to as Gal-linking / beta-galactosidase) (AA3H-gal / beta-galactosidase) fused with AA3H-gal and beta-galactosidase (FIG. 9). AA3H-gal / beta-galactosidase showed a deep blue color when beta-galactosidase was well transferred into cells and stained with X-gal. In the case of Gal-linking / beta-galactosidase as a control, beta-galactosidase was also transferred into cells and stained blue. Similar to the flow cytometry analysis shown in FIG. 8, when beta-galactosidase and Gal-linking peptide were fused, beta-galactosidase was delivered into the cells. This is thought to have affected the intracellular permeability due to the presence of a positive arginine sequence in the Gal-linking peptide. However, it was confirmed that the beta-galactosidase transfer ability by AA3H-CPP was much superior to that of the control (beta-galactosidase or Gal-linking / beta-galactosidase) (FIG.
CPP를 이용한 약물전달의 난제 중 혈액 내에서 빠른 분해로 인해 목표 지점까지 전달하지 못한다는 단점이 있으며, 최근 전달물질로 각광을 받고 있는 siRNA의 경우 세포 내로 투과시킬 경우 핵 내에 응축되거나 엔도좀(endosome)에서 빠져나오지 못해 세포질에서 역할을 수행하지 못한다는 단점이 있다. 사람의 아넥신 단백질의 N-말단에서 유래된 AA3H-CPP는 혈청에 대한 저항성이 강하고 독성이 없어 종래의 전달체와 차별성을 가진다. 또한, AA3H-CPP는 혈청에 의해 분해능이 낮고 핵이나 리소좀 등에 위치하지 않는다는 장점을 지니고 있어 상기의 한계를 극복할 수 있을 것으로 전망한다.
In the case of siRNA, which has been spotlighted as a transporter in recent years, when it is permeated into a cell, it is condensed in the nucleus or condensed in the endosome ) Can not escape from the cytoplasm is not able to play a role in the disadvantage. AA3H-CPP derived from the N-terminus of human annexin protein is highly resistant to serum and is not toxic and has a differentiability from conventional carriers. In addition, AA3H-CPP has the advantage that it has low resolution by serum and is not located in nucleus or lysosome, so that it can overcome the above limitations.
이상으로 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 일 구현예일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
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<110> Korea Institute of Science and Technology <120> Nove Cell Penetrating Peptides and Uses Thereof <130> F07151 <160> 12 <170> KopatentIn 2.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA3H <400> 1 Met Ala Ser Ile Trp Val Gly His Arg Gly 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA5H <400> 2 Met Ala Gln Val Leu Arg Gly Thr Val Thr 1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA4H <400> 3 Met Ala Thr Lys Gly Gly Thr Val Lys Ala 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA6H <400> 4 Met Ala Lys Pro Ala Gln Gly Ala Lys Tyr 1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA11H <400> 5 Met Ser Tyr Pro Gly Tyr Pro Pro Pro Pro 1 5 10 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA13H <400> 6 Met Gly Asn Arg His Ala Lys Ala Ser Ser 1 5 10 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA3H_NT <400> 7 atggcatcta tctgggttgg acaccgagga 30 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA5H_NT <400> 8 atggcacagg ttctcagagg cactgtgact 30 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA4H_NT <400> 9 atggccatgg caaccaaagg aggtactgtc 30 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA6H_NT <400> 10 atggccaaac cagcacaggg tgccaagtac 30 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA11H_NT <400> 11 atgagctacc ctggctatcc cccgccccca 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA13H_NT <400> 12 atgggcaatc gtcatgctaa agcgagcagt 30 <110> Korea Institute of Science and Technology <120> Nove Cell Penetrating Peptides and Uses Thereof <130> F07151 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA3H <400> 1 Met Ala Ser Ile Trp Val Gly His Arg Gly 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA5H <400> 2 Met Ala Gln Val Leu Arg Gly Thr Val Thr 1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA4H <400> 3 Met Ala Thr Lys Gly 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA6H <400> 4 Met Ala Lys Pro Ala Gln Gly Ala Lys Tyr 1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA11H <400> 5 Met Ser Tyr Pro Gly Tyr Pro Pro Pro Pro 1 5 10 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> AA13H <400> 6 Met Gly Asn Arg His Ala Lys Ala Ser Ser 1 5 10 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA3H_NT <400> 7 atggcatcta tctgggttgg acaccgagga 30 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA5H_NT <400> 8 atggcacagg ttctcagagg cactgtgact 30 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA4H_NT <400> 9 atggccatgg caaccaaagg aggtactgtc 30 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA6H_NT <400> 10 atggccaaac cagcacaggg tgccaagtac 30 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA11H_NT <400> 11 atgagctacc ctggctatcc cccgccccca 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> AA13H_NT <400> 12 atgggcaatc gtcatgctaa agcgagcagt 30
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| CN107417769A (en) * | 2016-05-19 | 2017-12-01 | 中国科学院过程工程研究所 | A kind of novel cell-penetrating peptide of mediate drug delivering and its application |
| WO2020130548A1 (en) * | 2018-12-19 | 2020-06-25 | 한국화학연구원 | Cell membrane penetrating domain derived from human gpatch4 protein |
| US11661464B2 (en) | 2020-05-13 | 2023-05-30 | Genesen Co., Ltd. | Protein transduction domain, fusion compound containing the same, and pharmaceutical composition containing the fusion compound |
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| AU2019269590A1 (en) * | 2018-05-15 | 2020-12-03 | Dnalite Therapeutics, Inc. | Mucus-penetrating peptides, delivery vehicles and methods of therapy |
| KR102067487B1 (en) | 2018-06-14 | 2020-01-17 | 한국과학기술연구원 | Novel cell penetrating peptide comprising beta-defensin dimer and uses thereof |
| WO2020235947A1 (en) * | 2019-05-22 | 2020-11-26 | 경북대학교 산학협력단 | Cacb1-derived peptide, variant of cacb1-derived peptide, and use thereof |
| KR102274877B1 (en) * | 2020-12-24 | 2021-07-08 | 주식회사 아임뉴런바이오사이언스 | Novel cell penetrating peptides and use thereof |
| KR102274876B1 (en) * | 2020-12-24 | 2021-07-08 | 주식회사 아임뉴런바이오사이언스 | Novel cell penetrating peptides and use thereof |
| US20240207162A1 (en) * | 2021-05-14 | 2024-06-27 | Remedi Co., Ltd. | Cargo molecule transduction domain rmad1, variant thereof, recombinant cargo molecule, and method for transducing cargo molecule using same |
| US20240252660A1 (en) * | 2021-06-03 | 2024-08-01 | Remedi Co., Ltd. | Cargo molecule transduction domain rmmr1, variant thereof, recombinant cargo molecule, and method for transducing cargo molecule using same |
| KR20230006405A (en) * | 2021-07-02 | 2023-01-10 | 주식회사 아임뉴런 | Novel cell penetrating peptides and use thereof |
| WO2023128501A1 (en) * | 2021-12-27 | 2023-07-06 | 아주대학교산학협력단 | Novel cell-permeable peptide and use thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107417769A (en) * | 2016-05-19 | 2017-12-01 | 中国科学院过程工程研究所 | A kind of novel cell-penetrating peptide of mediate drug delivering and its application |
| WO2020130548A1 (en) * | 2018-12-19 | 2020-06-25 | 한국화학연구원 | Cell membrane penetrating domain derived from human gpatch4 protein |
| US11661464B2 (en) | 2020-05-13 | 2023-05-30 | Genesen Co., Ltd. | Protein transduction domain, fusion compound containing the same, and pharmaceutical composition containing the fusion compound |
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