KR20130107943A - Composite for delivering a gene into a cell and gene delivery system using the same - Google Patents
Composite for delivering a gene into a cell and gene delivery system using the same Download PDFInfo
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- KR20130107943A KR20130107943A KR1020120030145A KR20120030145A KR20130107943A KR 20130107943 A KR20130107943 A KR 20130107943A KR 1020120030145 A KR1020120030145 A KR 1020120030145A KR 20120030145 A KR20120030145 A KR 20120030145A KR 20130107943 A KR20130107943 A KR 20130107943A
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
Abstract
Description
본 발명은 세포내 유전자 전달용 복합체 및 이를 이용한 유전자 전달체에 관한 것으로, 보다 상세하게는 동물세포 내로의 유전자 전달효율을 증가시킬 수 있는 유전자 전달용 복합체, 이를 이용한 유전자 전달체 및 이를 포함하는 항암 조성물에 관한 것이다.The present invention relates to a complex for intracellular gene transfer and a gene carrier using the same, and more particularly, to a gene transfer complex capable of increasing gene transfer efficiency into an animal cell, a gene carrier using the same, .
유전자 치료는 질병을 유발하는 유전자를 대신할 수 있는 정상 유전자를 외부로부터 도입함으로써 유전자가 본래의 기능을 발휘할 수 있도록 하는 것을 말한다. 근래에는 유전적 질환뿐만 아니라, 암, 에이즈, 심혈관계 질환, 자가면역질환 등 다양한 후천성 질환의 치료와 예방을 위하여 유용하게 사용될 것으로 전망되고 있다. 그러나, 거대분자인 DNA, RNA 등을 수용액 상태로 세포내로 전달하는 경우 특정효소에 의해 급속하게 분해되고 세포전달률이 낮아, 이를 효율적으로 전달하는 기술개발을 위한 노력이 지속되고 있다.Gene therapy refers to the introduction of a normal gene, which can replace a disease-causing gene, so that the gene can perform its original function. In recent years, it is expected to be useful not only for genetic diseases but also for the treatment and prevention of various acquired diseases such as cancer, AIDS, cardiovascular diseases and autoimmune diseases. However, when macromolecules such as DNA and RNA are delivered into cells in an aqueous solution, they are rapidly degraded by specific enzymes and the cell delivery rate is low. Efforts are continuing to develop technologies for efficiently delivering them.
유전자를 시험관내 또는 생체내의 세포로 전달하는 방법에는 레트로바이러스, 아데노바이러스 등을 이용하는 바이러스성 벡터법과 지질, 고분자, 전기충격법 등을 이용하는 비바이러스성 벡터법이 포함된다. 바이러스성 벡터는 비바이러스성 벡터에 비하여 유전자 전달 효율이 높은 반면, 체내 적용 시 벡터 자체가 유발하는 면역반응이나 암 유발 등이 문제가 되며, 벡터에 삽입할 수 있는 유전자의 크기에도 제한이 있다. 비바이러스성 벡터는 바이러스성 벡터에 의한 생물학적 위험성이 낮고, 비면역원성이며, 변형 및 대량생산이 가능하고, 전달할 수 있는 유전자의 크기에도 비교적 제한이 없다는 장점 때문에 최근 많은 연구가 이루어지고 있다. Methods for delivering a gene into an in vitro or in vivo cell include a viral vector method using retrovirus, adenovirus, etc., and a nonviral vector method using lipid, polymer, electric shock, or the like. Viral vectors have higher gene transfer efficiency than nonviral vectors, while immunoreactions or cancer induction caused by vectors themselves are problematic in the body, and the size of genes that can be inserted into vectors is also limited. Numerous studies have been conducted recently on the nonviral vectors due to their low biological hazards due to viral vectors, their non-immunogenicity, their ability to be modified and mass produced, and their relatively small size.
그러나 비바이러스성 벡터는 바이러스성 벡터에 비해 유전자 전달 효율이 현저히 낮기 때문에, 비바이러스성 벡터의 광범위한 적용을 위해서는 안전하고 효율적인 유전자 전달을 위한 비바이러스성 벡터의 제공 및 확보가 요구되고 있다.However, since non-viral vectors have a significantly lower gene transfer efficiency than viral vectors, it is required to provide and secure non-viral vectors for safe and efficient gene delivery for wide application of non-viral vectors.
이에, 본 발명자들은 유전자 전달체(gene delivery system)의 동물세포 내로의 유전자 전달효율을 증가시킬 수 있는 방법을 개발하고자 노력한 결과, 핵산을 비펩티드성 링커를 통해 활성화된 폴리에틸렌이민에 세포투과 펩티드(cell penetrating peptide) 및 트리스-(2-카복실에틸)인산염(Tris(2-Carboxyethyl) phosphine, TCEP)을 포함하는 유전자 전달용 복합체와 병용하면, 유전자 전달효율을 크게 증가시킬 수 있음을 확인함으로써, 본 발명을 완성하게 되었다.The present inventors have made efforts to develop a method for increasing the gene delivery efficiency into a mammalian cell of a gene delivery system. As a result, the present inventors have found that when a nucleic acid is bound to a polyethyleneimine activated through a non-peptide linker (2-carboxyethyl) phosphine (TCEP), the inventors of the present invention have found that the use of the present invention in combination with a gene transfer complex comprising a tris- (2-carboxyethyl) phosphate and a tris- .
본 발명의 목적은 동물세포로의 유전자 전달 효율을 개선시키기 위한 세포내 유전자 전달용 복합체 및 이의 제조방법을 제공하는 것이다.It is an object of the present invention to provide an intracellular gene transfer complex for improving gene transfer efficiency into animal cells and a method for producing the same.
본 발명의 또 다른 목적은 상기 유전자 전달용 복합체를 포함하는 항종양 조성물을 제공하는 것이다.It is still another object of the present invention to provide an antitumor composition comprising the gene transfer complex.
본 발명은 상기 목적을 달성하기 위해, 동물세포로의 유전자 전달 효율을 개선시키기 위한 세포내 유전자 전달용 복합체 및 이의 제조방법을 제공한다.In order to achieve the above object, the present invention provides a complex for intracellular gene transfer for improving gene transfer efficiency into animal cells and a method for producing the same.
또한, 본 발명은 상기 유전자 전달용 복합체를 포함하는 항종양 조성물을 제공한다.The present invention also provides an antitumor composition comprising the gene transfer complex.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 SSMCC(succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate)가 결합되어 활성화된 폴리에틸렌이민; The present invention relates to a polyethylenimine which is activated by binding of succinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate (SSMCC);
상기 활성화된 폴리에틸렌이민에 세포투과 펩티드(cell penetrating peptide); 및 트리스-(2-카복실에틸)인산염(Tris(2-Carboxyethyl) phosphine, TCEP);를 포함하는 세포내 유전자 전달용 복합체를 제공한다.A cell penetrating peptide to the activated polyethyleneimine; And tris- (2-carboxyethyl) phosphate (TCE (2-Carboxyethyl) phosphine, TCEP).
본 발명에 있어서, 폴리에틸렌이민(poly(ethylenimine): PEI)은 에틸아민을 반복단위로 함유하고 있어 매 3번째 원소가 질소이어서 높은 양전하를 띄고 있고, 따라서 음전하를 띄는, 예를 들어 DNA와의 정전기적 상호작용에 의하여 나노미터크기 (50-200nm)의 polyelectolyte 복합체 형성이 용이하다. PEI는 직선형(linear) 또는 가지형(branched) 등의 형태로, 25 KDa, 75 KDa, 150 KDa 등 다양한 분자량을 갖는 것들이 제조되거나 시판되고 있다.In the present invention, poly (ethylenimine) (PEI) contains ethylamine as a repeating unit, and every third element is nitrogen, and thus has a high positive charge. Therefore, a negative charge, for example, an electrostatic Interaction facilitates formation of polyelectolyte complexes of nanometer size (50-200 nm). PEI has a variety of molecular weights, such as 25 KDa, 75 KDa, and 150 KDa, in the form of linear or branched, and are manufactured or marketed.
본 발명에 따른 폴리에틸렌이민(PEI)은 평균 분자량 100 내지 500,000 달톤을 가지는 직선형 또는 가지형 형태인 것으로, 보다 바람직하게는 상기 폴리에틸렌이민은 가지형의 폴리에틸렌이민(bPEI)인 것을 특징으로 한다.The polyethyleneimine (PEI) according to the present invention is a linear or branched type having an average molecular weight of 100 to 500,000 daltons. More preferably, the polyethyleneimine is a branched polyethyleneimine (bPEI).
본 발명에 있어서, 상기 세포투과 펩티드는 전이성 유방암 세포(4T1)에서의 세포질과 핵 주변에 많이 발현하는 것을 특징으로 하고, 보다 바람직하게는 서열번호 1로 이루어진 것을 특징으로 한다. In the present invention, the cell-penetrating peptide is highly expressed in the cytoplasm and nucleus around the metastatic breast cancer cell (4T1), and more preferably, it is composed of SEQ ID NO: 1.
본 발명에 따른 상기 세포는 혈관내피세포(HUVEC 세포), 전이성 유방암 세포(4T1 세포) 및 전립선암세포(PC-3 세포)로 이루어진 군에서 선택되는 것으로, 바람직하게는 전이성 유방암 세포(MDA-MB231 세포)인 것을 특징으로 한다.The cells according to the present invention are selected from the group consisting of vascular endothelial cells (HUVEC cells), metastatic breast cancer cells (4T1 cells) and prostate cancer cells (PC-3 cells), preferably metastatic breast cancer cells (MDA-MB231 cells ).
본 발명에 따른 유전자 전달용 복합체는 하기 방법으로 제조되는 것을 특징으로 한다.The gene transfer complex according to the present invention is characterized by being produced by the following method.
보다 상세하게는 a) 폴리에틸렌이민(PEI)에 SMCC(succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate)를 결합시켜 활성화시키는 단계; 및 b) 상기 활성화된 폴리에틸렌이민에 세포투과 펩티드(cell penetrating peptide) 및 트리스-(2-카복실에틸)인산염(Tris(2-Carboxyethyl) phosphine, TCEP)을 첨가하여 유전자 전달체를 만드는 단계;를 포함하는 세포내 유전자 전달용 복합체의 제조방법을 제공한다. 도 5를 참조한다.More particularly, the present invention relates to a method for producing a poly (ethyleneimine) (PEI) comprising the steps of: a) binding SMCC (succinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate) And b) preparing a gene carrier by adding a cell penetrating peptide and Tris (2-carboxyethyl) phosphate (TCEP) to the activated polyethyleneimine, There is provided a method for producing an intracellular gene transfer complex. Please refer to Fig.
본 발명에 따른 상기 폴리에틸렌이민은 가지형 형태의 폴리에틸렌이민(bPEI)이고, 상기 세포투과 펩티드는 서열번호 1로 이루어진 것을 특징으로 한다.The polyethyleneimine according to the present invention is a polyethyleneimine (bPEI) in the form of a branch, and the cell permeable peptide is characterized by comprising SEQ ID NO: 1.
본 발명의 세포내 유전자 전달용 복합체는 중선전하에 가까운 양전하를 갖고 있기 때문에 음전하를 갖는 유전자와 정전기적 상호작용으로 결합을 형성하여 유전자를 응축하고 외부 환경으로부터 보호할 뿐 아니라, 유전자를 목적하는 세포의 세포질 또는 세포핵으로 보다 효율적으로 전달한다.Since the intracellular gene transfer complex of the present invention has positive charge close to the neutral charge, it forms a bond by electrostatic interaction with a gene having a negative charge to condense the gene and protect it from the external environment, To the cytoplasm or nucleus of the cell.
본 발명은 상기 제조방법으로 제조된 세포내 유전자 전달용 복합체에 핵산을 결합시켜 제조되는 유전자 전달체를 제공한다.The present invention provides a gene carrier prepared by binding a nucleic acid to an intracellular gene transfer complex produced by the above production method.
본 발명에 따른 유전자 전달체는 입자의 크기가 대략 150 내지 200 nm이며, 표면전하는 중성에 가까운 양전하를 띠는 것을 특징으로 한다.The gene carrier according to the present invention is characterized in that the particle size is approximately 150 to 200 nm and the surface charge has a positive charge close to neutrality.
본 발명은 SSMCC(succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate)가 결합되어 활성화된 폴리에틸렌이민; The present invention relates to a polyethylenimine which is activated by binding of succinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate (SSMCC);
상기 활성화된 폴리에틸렌이민에 세포투과 펩티드(cell penetrating peptide); 및 트리스-(2-카복실에틸)인산염(Tris(2-Carboxyethyl) phosphine, TCEP);를 포함하는 세포내 유전자 전달용 복합체; 및 핵산;을 포함하는 항종양 조성물을 제공한다.A cell penetrating peptide to the activated polyethyleneimine; And tris- (2-carboxyethyl) phosphate (Tris (2-Carboxyethyl) phosphine, TCEP); And a nucleic acid.
본 발명에 있어서, 폴리에틸렌이민(PEI)을 유전자 전달체로 사용하는 경우, 유전자 발현효율은 폴리에틸렌이민과 유전자의 배합비율 즉 PEI/DNA complex의 Nitrogen/phosphate (N/P)에 따라 크게 영향을 받는 것으로 알려져 있다. N/P의 비(N/P ratio)가 증가하면 DNA를 더 응축시킬 수 있고, 전체적으로 양이온을 띄어 세포 내로 높은 효율로 전달된다. 하지만 N/P의 비가 증가하면 독성이 커지는 단점이 있다. 특히 in vivo 상에서 전달시킬 때 여러 단백질과 결합하여 발현효율이 떨어지고 독성이 높아진다.In the present invention, when polyethyleneimine (PEI) is used as a gene carrier, the gene expression efficiency is greatly influenced by the ratio of the polyethyleneimine to the gene, that is, Nitrogen / phosphate (N / P) of the PEI / DNA complex It is known. As the N / P ratio increases, the DNA can be further condensed, and the cation is released as a whole and delivered to the cell with high efficiency. However, if the ratio of N / P increases, toxicity increases. Especially, when it is delivered in vivo, it binds to various proteins, resulting in low expression efficiency and high toxicity.
본 발명의 유전자 전달체에 있어서, 유전자 전달체의 N/P의 비는 목적하는 유전자 전달체의 특성에 맞추어 조절이 가능하며, 0.1/1 내지 100/1의 범위, 바람직하게는 0.5/1 내지 50/1의 범위, 보다 바람직하게는 1/1 내지 20/1 범위이다.In the gene delivery vehicle of the present invention, the ratio of N / P of the gene delivery vehicle can be adjusted according to the characteristics of the desired gene delivery vehicle and is in the range of 0.1 / 1 to 100/1, preferably 0.5 / 1 to 50/1 , More preferably in the range of 1/1 to 20/1.
본 발명의 복합체가 전달하는 핵산은 DNA와 RNA 및 이들의 합성동종체를 포괄한다. 이의 예로서는 gDNA, cDNA, pDNA, mRNA, tRNA, rRNA, siRNA, miRNA, 안티센스뉴클레오티드 등을 들 수 있다. 이들은 자연에 존재하거나 합성될 수 있으며, 크기에 있어서 올리고뉴클레오티드에서 크로모좀까지 다양한 크기로 존재할 수 있다.Nucleic acids delivered by the complexes of the present invention encompass DNA and RNA and their synthetic dendritic cells. Examples thereof include gDNA, cDNA, pDNA, mRNA, tRNA, rRNA, siRNA, miRNA, antisense nucleotide, and the like. They can be present or synthesized in nature, and can vary in size, ranging from oligonucleotides to chromosomes.
이들 핵산은 인간, 동물, 식물, 박테리아, 바이러스 등으로부터 기원된다. 이들은 당 분야에 공지된 방법을 이용하여 획득될 수 있다.These nucleic acids originate from humans, animals, plants, bacteria, viruses and the like. These can be obtained using methods known in the art.
보다 상세하게는 본 발명에 따른 핵산은 전이성 유방암 세포(4T1)에서의 세포증식을 억제능을 가지는 것을 특징으로 하고, 보다 바람직하게는 miR-145인 것으로, 서열번호 2로 이루어진 것을 특징으로 한다.More particularly, the nucleic acid according to the present invention is characterized in that it has the ability to inhibit cell proliferation in metastatic breast cancer cells (4T1), more preferably miR-145, and is characterized by being composed of SEQ ID NO: 2.
표면에 양이온성을 갖는 본 발명의 유전자 전달체는 세포표면의 음이온과의 정전기적 작용으로 인해 엔도사이토시스 과정을 통해 세포내로 이입된다. 보통, 이입된 유전자 전달체는 엔도좀을 거쳐 라이소좀으로 가게 되는데, 라이소좀에는 다양한 분해 효소가 있어 생분해성 전달체인 경우 유전자와 전달체가 분해된다. 그러므로 효율적인 유전자 전달을 위해서는 유전자 전달체가 라이소좀으로 가기 전에 엔도좀을 나와 세포질로의 탈출(endosomalescaping)이 필수적이다. 엔도좀으로부터의 탈출을 촉진하여 유전자 전달효율을 높이는 방법으로는 화학적으로 lysosomal escaping signal peptide인 KALA(cationic fusogenic peptide)나 receptor mediated endocytosis를 일으키는 리간드를 결합시키는 방법 등이 연구되고 있다(이 등, Biotechnology and Bioprocess Engineering, Vol. 6, pp269-273, 2001).The gene carrier of the present invention having cationic surface is introduced into the cell through the endocytosis process due to the electrostatic action with the anion on the cell surface. Usually, the transferred gene transporter goes through the endosome to lysosomes. Lysosomes have various kinds of degrading enzymes, which cause degradation of genes and transporters in the case of biodegradation. Therefore, for effective gene transfer, endosomalescaping is essential for the gene carrier to exit the endosomes before it goes to lysosomes. Methods for enhancing the gene transfer efficiency by promoting the escape from the endosome include chemically binding a cationic fusogenic peptide (KALA), which is a lysosomal escaping signal peptide, or a ligand for causing receptor mediated endocytosis (see, for example, Biotechnology and Bioprocess Engineering, Vol. 6, pp. 269-273, 2001).
본 발명의 조성물은 종양과 관련된 다양한 질병 또는 질환, 예컨대 위암, 폐암, 유방암, 난소암, 간암, 기관지암, 비인두암, 후두암, 췌장암, 방광암, 결장암 및 자궁경부암 등의 치료에 이용될 수 있으며, 바람직하게는 전이성 유방암인 것을 특징으로 한다.The composition of the present invention can be used for treatment of various diseases or diseases related to tumors such as stomach cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer and cervical cancer, It is preferably a metastatic breast cancer.
본 명세서에서 용어 “치료”는 (ⅰ) 종양 세포 형성의 예방; (ⅱ) 종양 세포의 제거에 따른 종양과 관련된 질병 또는 질환의 억제; 및 (ⅲ) 종양 세포의 제거에 따른 종양과 관련된 질병 또는 질환의 경감을 의미한다.As used herein, the term " treatment " includes (i) prevention of tumor cell formation; (Ii) inhibition of tumor-related diseases or disorders associated with the removal of tumor cells; And (iii) relief of a tumor-associated disease or disorder associated with the removal of tumor cells.
따라서, 본 명세서에서 용어 “치료학적 유효량”은 상기한 약리학적 효과를 달성하는 데 충분한 양을 의미한다.Thus, as used herein, the term " therapeutically effective amount " means an amount sufficient to achieve the above pharmacological effect.
본 발명의 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다.The pharmaceutically acceptable carriers to be included in the composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, no.
본 발명의 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등 을 추가로 포함할 수 있다.The composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavors, emulsifiers, suspending agents, preservatives, etc. in addition to the above components.
본 발명의 조성물은 비경구 투여가 바람직하고, 예컨대 정맥내 투여, 복강내 투여, 근육내 투여, 피하투여, 또는 국부 투여를 이용하여 투여할 수 있다. 난소암에서 복강내로 투여하는 경우 및 간암에서 문맥으로 투여하는 경우에는 주사 방법(injection)으로 투여할 수 있고, 방광암의 경우에는 카테테르 내 직접 주사하여 투여할 수 있고, 유방암의 경우에는 종양 매스에 직접 주사하여 투여할 수 있으며, 결장암의 경우에는 관장으로 직접 주사하여 투여할 수 있다.The composition of the present invention is preferably parenterally administered, and can be administered, for example, by intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration. In case of intraperitoneal administration of ovarian cancer and injection of portal vein in liver cancer, injection can be performed. In case of bladder cancer, it can be administered by direct injection into catheter. In case of breast cancer, In the case of colon cancer, it can be administered by direct injection into the enema.
본 발명의 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 질병 증상의 정도, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련 의사는 목적하는 치료에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다.Suitable dosages of the compositions of the present invention will vary depending on factors such as formulation method, mode of administration, age, weight, sex, severity of disease symptoms, food, time of administration, route of administration, excretion rate and responsiveness of the patient, Usually, a skilled physician can easily determine and prescribe dosages that are effective for the desired treatment.
본 발명의 조성물은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화 됨으로써 단위 용량 형태로 제조되거나 또는 다용량 용기내에 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 composition of the present invention may be prepared in a unit dose form by being formulated using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. 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 조성물은 단독의 요법으로 이용될 수 있으나, 다른 통상적인 화학 요법 또는 방사 요법과 함께 이용될 수도 있으며, 이러한 병행 요법을 실시하는 경우에는 보다 효과적으로 암 치료를 할 수 있다. 본 발명의 조성물과 함께 이용될 수 있는 화학 요법제는 시스플라틴 (cisplatin), 카르보플라틴 (carboplatin), 프로카르바진 (procarbazine), 메클로레타민 (mechlorethamine), 시클로포스파미드 (cyclophosphamide), 이포스파미드 (ifosfamide), 멜팔란(melphalan), 클로라부실 (chlorambucil), 비술판 (bisulfan), 니트로소우레아(nitrosourea), 디악티노마이신 (dactinomycin), 다우노루비신 (daunorubicin), 독소루비신 (doxorubicin), 블레오마이신 (bleomycin), 플리코마이신 (plicomycin), 미토마이신 (mitomycin), 에토포시드 (etoposide), 탁목시펜 (tamoxifen), 택솔 (taxol), 트랜스플라티눔 (transplatinum), 5-플루오로우라실 (5-fluorouracil), 빈크리스틴 (vincristin), 빈블라스틴 (vinblastin) 및 메토트렉세이트 (methotrexate) 등을 포함한다. 본 발명의 조성물과 함께 이용될 수 있는 방사 요법은 X-선 조사 및 γ-선 조사 등이다.The composition of the present invention may be used alone, but may be used in combination with other conventional chemotherapy or radiotherapy, and cancer therapy can be more effectively performed when such concurrent therapy is carried out. Chemotherapeutic agents that can be used with the compositions of the present invention include, but are not limited to, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, But are not limited to, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, , Bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum, 5-fluoro 5-fluorouracil, vincristin, vinblastin and methotrexate, and the like. Radiation therapies that can be used with the compositions of the present invention include X-ray irradiation and gamma-ray irradiation.
본 발명에 따른 유전자 전달체는 세포내 유전자 전달용 복합체를 이용함으로써, 유전자를 종양 세포에 안전하고 효율적으로 전달할 수 있으며, 상기 세포내 유전자 전달용 복합체에 항암 효과가 있는 다양한 유전자를 결합시켜 사용함으로써 종양과 관련된 질병 또는 질환 치료에 유용하게 사용될 수 있을 것이다.The gene carrier according to the present invention can deliver the gene to the tumor cells safely and efficiently by using the intracellular gene transfer complex. By using various genes having anticancer effect to the intracellular gene transfer complex, May be useful for the treatment of diseases or diseases associated with < RTI ID = 0.0 >
도 1은 본 발명에 따른 유전자 전달용 복합체와 핵산의 결합 모식도를 나타낸 것이고,
도 2는 본 발명의 세포투과 펩티드에 대한 전이성 유방암 세포(4T1)에서의 세포질 내 발현을 확인할 결과이며,
(파랑: DAPI for nuclei staining; 빨강: F-actin for actin staining; 초록: FITC-labeled DS 4-3 peptide)
도 3은 본 발명의 세포투과 펩티드에 대한 전이성 유방암 세포(4T1)에서의 세포 내 도입 경로를 확인할 결과이고,
(파랑: DAPI for nuclei staining; 빨강: F-actin for actin staining; 초록: FITC-labeled DS 4-3 peptide)
도 4는 본 발명의 세포투과 펩티드에 대한 비전이성 유방암 세포(4T7)에서의 위치 발현을 확인할 결과이며,
(파랑: DAPI for nuclei staining; 빨강: F-actin for actin staining; 초록: FITC-labeled DS 4-3 peptide)
도 5는 화학 구조식으로 본 발명에 따른 유전자 전달용 복합체의 합성 모식도를 나타낸 것이고,
도 6은 본 발명에 따른 유전자 전달용 복합체의 NMR 분석 결과를 나타낸 것이며,
(A: bPEI peak, B: DS 4-3 peptide peak, C: SMCC-activated bPEI peak, D: D2O에 bPEI-SMCC-DS 4-3 peak)
도 7은 본 발명에 따른 유전자 전달용 복합체에 핵산을 결합시켜 제조되는 유전자 전달체의 표면전하 값을 측정한 결과이고,
(DS1: DS 4-3의 C 말단에 cysteine기를 도입하여 bPEI와 결합, DS2: DS 4-3의 N 말단에 cyteine기를 도입하여 bPEI와 결합, SCR: Scramble DS peptide(Control), bPEI: Only bPEI)
도 8은 본 발명에 따른 유전자 전달용 복합체에 핵산을 결합시켜 제조되는 유전자 전달체의 입자크기를 확인한 결과이며,
도 9는 본 발명에 따른 유전자 전달용 복합체에 핵산을 결합시켜 제조되는 유전자 전달체의 입자 형태를 확인한 결과이고,
도 10은 본 발명에 따른 유전자 전달용 복합체에 핵산의 결합을 아가로스 겔(agarose gel)을 통해 확인한 결과이며,
도 11은 본 발명에 따른 유전자 전달용 복합체에 핵산을 결합시켜 제조되는 유전자 전달체의 세포독성을 평가 결과를 나타낸 것이고,
(A: 전이성 유방암 세포(4T1), B: 비전이성 유방암 세포(4T7))
도 12는 본 발명에 따른 유전자 전달용 복합체에 핵산을 결합시켜 제조되는 유전자 전달체의 전이성 유방암 세포(4T1)에서의 유전자 전달 효율 평가 결과를 나타낸 것이며,
도 13은 본 발명에 따른 유전자 전달용 복합체에 핵산을 결합시켜 제조되는 유전자 전달체의 비전이성 유방암 세포(4T7)에서의유전자 전달 효율 평가 결과를 나타낸 것이고,
도 14는 본 발명에 따른 유전자 전달용 복합체에 핵산을 결합시켜 제조되는 유전자 전달체의 세포 특이성을 확인한 결과를 나타낸 것이며,
(A: 정상 섬유아세포(3T3), B: 자궁경부암 세포(HeLa))
도 15는 본 발명에 따른 유전자 전달용 복합체에 핵산을 결합시켜 제조되는 유전자 전달체의 전이성 유방암 세포(4T1)에서의 발현을 확인한 결과이고,
(파랑: DAPI for nuclei staining; 빨강: Flamma Fluor(FNR-675)-labeled DS-bPEI, SCR-bPEI, and bPEI; 초록: YOYO1-labeled miR-145)
도 16은 본 발명에 따른 유전자 전달용 복합체에 핵산을 결합시켜 제조되는 유전자 전달체의 전이성 유방암 세포에 대한 세포증식 억제효과를 확인한 결과이다.
(A: 전이성 유방암 세포(4T1), B: 비전이성 유방암 세포(4T7))Brief Description of the Drawings Fig. 1 is a schematic diagram showing the binding between a gene transfer complex according to the present invention and a nucleic acid,
2 is a result of confirming the expression in the cytoplasm of metastatic breast cancer cells (4T1) against the cytotoxic peptides of the present invention,
(Blue: DAPI for nuclei staining; red: F-actin for actin staining; green: FITC-labeled DS 4-3 peptide)
FIG. 3 shows the results of confirming the intracellular introduction pathway in metastatic breast cancer cells (4T1) to the cell penetrating peptides of the present invention,
(Blue: DAPI for nuclei staining; red: F-actin for actin staining; green: FITC-labeled DS 4-3 peptide)
FIG. 4 shows the results of confirming the positional expression in the non-metastatic breast cancer cells (4T7) of the cell-penetrating peptides of the present invention,
(Blue: DAPI for nuclei staining; red: F-actin for actin staining; green: FITC-labeled DS 4-3 peptide)
FIG. 5 is a schematic diagram showing synthesis of a gene transfer complex according to the present invention in a chemical structural formula,
6 shows NMR analysis results of the gene transfer complex according to the present invention,
(A: bPEI peak, B: DS 4-3 peptide peak, C: SMCC-activated bPEI peak, D: bPEI-SMCC-DS 4-3 peak in D 2 O)
FIG. 7 shows the results of measuring the surface charge value of a gene carrier prepared by binding a nucleic acid to a gene transfer complex according to the present invention,
(DS1: cysteine group introduced at the C-terminus of DS 4-3 to bind to bPEI, DS2: introduced at the N-terminus of DS 4-3 with cyteine group to bind bPEI, SCR: Scramble DS peptide (Control), bPEI: Only bPEI )
FIG. 8 is a result of confirming the particle size of a gene delivery vehicle prepared by binding a nucleic acid to a gene transfer complex according to the present invention,
FIG. 9 is a result of confirming the particle shape of a gene delivery body prepared by binding a nucleic acid to a gene transfer complex according to the present invention,
10 shows the result of confirming the binding of the nucleic acid to the gene transfer complex according to the present invention through an agarose gel,
FIG. 11 shows the results of evaluating cytotoxicity of a gene carrier prepared by binding a nucleic acid to a gene transfer complex according to the present invention,
(A: metastatic breast cancer cell (4T1), B: non-metastatic breast cancer cell (4T7))
FIG. 12 shows the results of gene transfer efficiency evaluation of a gene carrier prepared by binding a nucleic acid to a gene transfer complex according to the present invention in metastatic breast cancer cells (4T1)
FIG. 13 shows the results of gene transfer efficiency evaluation in a non-metastatic breast cancer cell (4T7) of a gene carrier prepared by binding a nucleic acid to a gene transfer complex according to the present invention,
FIG. 14 shows the result of confirming the cell specificity of a gene carrier prepared by binding a nucleic acid to a gene transfer complex according to the present invention,
(A: normal fibroblast (3T3), B: cervical cancer cell (HeLa))
FIG. 15 shows the results of confirming expression of a gene carrier prepared by binding a nucleic acid to a gene transfer complex according to the present invention in metastatic breast cancer cells (4T1)
(Blue: DAPI for nuclei staining; red: Flamma Fluor (FNR-675) -labeled DS-bPEI, SCR-bPEI, and bPEI; Abstract: YOYO1-labeled miR-145)
FIG. 16 shows the results of confirming the cell proliferation inhibitory effect of a gene carrier prepared by binding a nucleic acid to a gene transfer complex according to the present invention on metastatic breast cancer cells.
(A: metastatic breast cancer cell (4T1), B: non-metastatic breast cancer cell (4T7))
이하, 본 발명을 구체적인 실시예에 의해 보다 상세히 설명하고자 한다. 하지만, 본 발명은 하기 실시예에 의해 한정되는 것은 아니며, 본 발명의 아이디어와 범위 내에서 여러 가지 변형 또는 수정할 수 있음은 이 분야에 종사하는 업자에게는 명백한 것이다.Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited by the following examples, and it is apparent to those skilled in the art that various changes or modifications can be made within the idea and scope of the present invention.
이 때, 사용되는 기술용어 및 과학용어에 있어 다른 정의가 없다면, 이 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 일반적으로 이해하는 의미를 지닌다.Here, unless otherwise defined in the technical terms and scientific terms used, those having ordinary skill in the art to which the present invention belongs have a generally understood meaning.
또한, 종래와 동일한 기술적 구성 및 작용에 대한 반복되는 설명은 생략하기로 한다.Repeated descriptions of the same technical constitution and operation as those of the conventional art will be omitted.
[실시예 1] 세포투과 펩티드의 세포에서의 발현 분석[Example 1] Expression analysis of cell permeable peptides in cells
본 발명의 세포투과 펩티드(DS 4-3 펩티드) 자체에 대한 전이성 유방암 세포에서의 위치발현을 확인하였다.The expression of the cell-mediated peptides (DS 4-3 peptide) itself in the metastatic breast cancer cells was confirmed.
실험에 사용된 세포투과 펩티드(DS 4-3 peptide, 순도 95% 이상)는 애니젠(광주, 한국)에서 합성을 의뢰하였으며, 전이성 유방암 세포에 세포투과 펩티드 서열 특이성을 확인하기 위해 두 가지의 종류로 합성하였다. 또한, 세포투과 펩티드 아미노산 서열(서열번호 1: RIMRILRILKLAR)의 C-말단과 N-말단에 각각 시스테인(C) 잔기를 도입시키고, 아미노기(-NH2)를 결합시켰다. 각 명칭과 아미노산 서열은 다음과 같다.The cell-permeable peptide (DS 4-3 peptide, purity 95% or more) used in the experiment was synthesized in Anisen (Gwangju, Korea). Two types of cell permeable peptides . Also, the cysteine (C) residue was introduced at the C-terminal and the N-terminal of the cell-penetrating peptide amino acid sequence (SEQ ID NO: 1: RIMRILRILKLAR), respectively, and the amino group (-NH 2 ) was bound thereto. Each name and amino acid sequence is as follows.
C-말단 : DS 4-3C, CRIMRILRILKLAR-NH2 C-terminus: DS 4-3C, C RIMRILRILKLAR-NH 2
N-말단 : DS 4-3N, RIMRILRILKLARC-NH2
N-terminus: DS 4-3N, RIMRILRILKLAR C -NH 2
상기 전이성 유방암 세포는 쥐 유래의 전이성 유방암 세포(4T1)와 비전이성 유방암 세포(4T7)를 이용하였고, 상기 세포투과 펩티드(DS 4-3 펩티드)를 세포에 전달시켜 그 특이적인 세포 내 위치발현 및 이동 경로를 하기의 방법으로 확인하였다. The metastatic breast cancer cells were transfected with metastatic breast cancer cells (4T1) and non-metastatic breast cancer cells (4T7) derived from mice, and the cell permeable peptide (DS 4-3 peptide) The movement route was confirmed by the following method.
세포투과 펩티드(DS 4-3 펩티드)의 세포 위치발현 및 이동경로를 확인하기 위하여, 세포투과 펩티드를 녹색형 형광물질인 FITC(fluorescein isothiocyanate)로 표지화 한 후, 콜라겐 코팅 커버글라스에 1×104/well로 분주하여 24시간 동안 부착시킨 후 전이성 유방암 세포(4T1)와 비전이성 유방암 세포(4T7)에 처리하였으며, 세포 내 혹은 표면에 결합된 녹색 형광을 형광현미경으로 관찰하였다. Cell permeation peptides were labeled with fluorescein isothiocyanate (FITC), which is a green fluorescent substance, in order to confirm the cell location expression and migration pathway of the cell permeation peptide (DS 4-3 peptide), and then 1 × 10 4 / well and adhered for 24 hours. Then, the cells were treated with metastatic breast cancer cells (4T1) and non-metastatic breast cancer cells (4T7), and green fluorescence bound to the intracellular or surface was observed with a fluorescence microscope.
그 결과, 도 2에서 확인할 수 있듯이 전이성 유방암 세포(4T1)에서의 전이성 유방암 특이적 세포투과 펩티드는 세포질에 위치함을 확인할 수 있었다.As a result, as shown in FIG. 2, it was confirmed that the metastatic breast cancer-specific cytotoxic peptide in the metastatic breast cancer cell (4T1) is located in the cytoplasm.
또한, 세포 내로의 도입 경로(endocytosis pathway) 확인한 결과 도 3에서 확인할 수 있듯이, 클라트린 의존성 엔도사이토시스 억제제(clathrin dependent endocytosis inhibitor)인 클로르프로마진(Chlorpromazine, CPZ)을 처리했을 때 4T1 세포에 대한 결합이 현저히 억제됨을 확인함으로써 클라트린 의존성 신호전달 로(clathrin-dependent pathway)를 통하여 세포투과 펩티드(DS 4-3 펩티드)가 세포 내로 도입되는 것을 확인할 수 있었다. In addition, as shown in FIG. 3, when the endocytosis pathway into the cell was examined, it was found that when treated with chlorpromazine (CPZ), which is a clathrin dependent endocytosis inhibitor, (DS 4-3 peptide) was introduced into the cells through the clathrin-dependent pathway by confirming that the binding was significantly suppressed.
한편, 비전이성 유방암 세포(4T7)에서의 세포투과 펩티드(DS 4-3 펩티드)의 세포내 위치를 관찰한 결과 도 4에서 확인할 수 있듯이, 세포 내에 위치하지 않음을 확인하였다. Meanwhile, the intracellular location of the cell penetrating peptide (DS 4-3 peptide) in the non-metastatic breast cancer cell (4T7) was observed, and it was confirmed that it was not located in the cell as shown in FIG.
상기의 결과로부터 세포투과 펩티드인 DS 4-3가 전이성 유방암 세포(4T1)에서만 특이적인 세포투과가 일어남을 확인할 수 있었다.
From the above results, it was confirmed that DS 4-3, a cell-penetrating peptide, specifically penetrates into only metastatic breast cancer cells (4T1).
[[ 실시예Example 2] 유전자 전달용 복합체( 2] Complexes for gene transfer ( DSDS -- bPEIbPEI )의 합성) Synthesis of
아민기를 가진 bPEI(MW 25,000 Da)에 SMCC(succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate)를 반응시켜 결합하였다.BCHE (MW 25,000 Da) with an amine group was reacted with succinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate.
77 mg의 bPEI(MW 25,000 Da)을 3 mL의 PBS 버퍼(pH 7.2)에 녹인 후, 1M HCl 수용액으로 pH를 7.0인 용액을 제조하였다. 상기 bPEI 용액에 0.75 mL DMSO에 녹인 SMCC(41 mg) 용액을 첨가하여 1시간 동안 실온에서 반응시킨 후, PD-10 column(Sephadex G-25)를 이용하여 반응하지 않은 SMCC를 제거하여 SMCC가 기능화된 bPEI(SMCC-activated bPEI, 이하, ‘bPEI-SMCC’라 칭한다.)를 수득하였다.77 mg of bPEI (MW 25,000 Da) was dissolved in 3 mL of PBS buffer (pH 7.2), and then a solution of pH 7.0 was prepared with 1 M HCl aqueous solution. SMCC (41 mg) dissolved in 0.75 mL DMSO was added to the bPEI solution, and reacted at room temperature for 1 hour. Unreacted SMCC was removed using PD-10 column (Sephadex G-25) BpEI (SMCC-activated bPEI, hereinafter referred to as 'bPEI-SMCC') was obtained.
본 발명에 있어서, DS 펩티드는 DS1 펩티드(DS 4-3의 C 말단에 cysteine기를 도입한 펩티드), DS2(DS 4-3의 N 말단에 cysteine기를 도입한 펩티드), SCR(scramble DS 펩티드, control)를 말한다.In the present invention, the DS peptide includes a DS1 peptide (a peptide having a cysteine group introduced at the C terminus of DS 4-3), DS2 (a peptide having a cysteine group introduced at the N terminus of DS 4-3), SCR (scramble DS peptide, ).
19.4 mg의 Cystein기를 갖는 DS1 펩티드(DS 4-3의 C 말단에 cysteine기를 도입한 펩티드)를 정제된 bPEI-SMCC 수용액에 첨가하고, 과량의 트리스-(2-카복실에틸)인산염(Tris(2-Carboxyethyl) phosphine, TCEP) 존재하에서 12시간 동안 실온에서 반응시킨다. 이때 반응하지 않는 펩티드를 24 시간 동안 증류수에서 투석(MWCO 3,500)을 통하여 제거하고, 이를 동결건조하여 bPEI-SMCC-DS1을 획득하였다. DS2(DS 4-3의 N 말단에 cysteine기를 도입한 펩티드)와 SCR(scramble DS 펩티드, control)도 앞에서 기술한 DS1 펩티드와 동일한 방법으로 bPEI-SMCC에 도입하였다.The DS1 peptide (peptide with cysteine group introduced at the C-terminus of DS 4-3) with 19.4 mg of cystein group was added to the purified bPEI-SMCC aqueous solution and excess tris- (2-carboxylethyl) phosphate (Tris Carboxyethyl phosphine (TCEP) at room temperature for 12 hours. At this time, unreacted peptides were removed by dialysis (MWCO 3,500) in distilled water for 24 hours and lyophilized to obtain bPEI-SMCC-DS1. DS2 (a peptide in which a cysteine group was introduced at the N-terminus of DS 4-3) and SCR (scramble DS peptide, control) were introduced into bPEI-SMCC in the same manner as DS1 peptide described above.
상기 제조된 유전자 전달용 복합체(DS-bPEI)는 bPEI-SMCC-DS1, bPEI-SMCC-DS2, bPEI-SMCC-SCR를 제조하였다.
BPEI-SMCC-DS1, bPEI-SMCC-DS2, and bPEI-SMCC-SCR were prepared as the gene transfer complex (DS-bPEI)
[[ 실시예Example 3] 합성된 유전자 전달용 복합체( 3] Synthesized complexes for gene transfer ( DSDS -- bPEIbPEI )의 )of NMRNMR 분석 analysis
상기 실시예 2로부터 합성된 유전자 전달용 복합체(bPEI-SMCC-DS1)를 1H-NMR을 이용하여 bPEI에 DS 4-3 peptide가 결합되었는지 여부를 확인하였다. The bPEI gene binding conjugate (bPEI-SMCC-DS1) synthesized from Example 2 above was confirmed by 1 H-NMR to determine whether the DS 4-3 peptide was bound.
도면 6A는 bPEI를, 도면 6B는 세포투과 펩티드(DS 4-3 peptide)의 NMR 스펙트럼을 나타낸 것이다. bPEI와 비교했을 때, SMCC가 기능화 된 bPEI(bPEI-SMCC)는 2.5 내지 2.7 부근에서 나타나는 bPEI의 peak가 넓어지고, 2.0 이하 및 3.2 이상에서 peak이 나타나는 것으로 보아 SMCC가 효과적으로 도입되었음을 알 수 있었다. 6A shows the bPEI, and FIG. 6B shows the NMR spectrum of the cell permeation peptide (DS 4-3 peptide). Compared with bPEI, bPEI (bPEI-SMCC) in which SMCC was functionalized showed a broad peak of bPEI at around 2.5 to 2.7 and a peak at 2.0 or less and 3.2 or more, indicating that SMCC was effectively introduced.
또한 상기 bPEI-SMCC에 DS-1를 컨쥬게이션(bPEI-SMCC-DS1) 한 후에는 bPEI-SMCC의 peak 뿐 아니라 6B에서 관찰되었던 세포투과 펩티드(DS 4-3 peptide)의 peak이 함께 발견되었다. After DS-1 conjugation (bPEI-SMCC-DS1) to bPEI-SMCC, peaks of bPEI-SMCC and DS 4-3 peptides observed in 6B were also found.
상기의 결과를 통해, bPEI에 DS 4-3 펩티드가 효과적으로 결합되어 있는 것을 확인할 수 있었다.
From the above results, it was confirmed that DS 4-3 peptide was effectively bound to bPEI.
[[ 실시예Example 4] 유전자 전달체( 4] gene transporter ( DSDS -- bPEIbPEI /Of miRmiR -145 -145 pDNApDNA )의 자기적 성질 분석) Magnetic property analysis
(1) 유전자 전달체((1) gene delivery system DSDS -- bPEIbPEI /Of miRmiR -145 -145 pDNApDNA )의 제조Manufacturing
본 발명에서 물리화학적 특성을 분석할 목적으로 사용된 유전자는 진핵세포에서 gWIZ-Luciferase라는 luciferase 효소를 발현시키는 유전자를 이용하였고, 유전자 전달용 복합체(DS-bPEI)가 전달하는 핵산은 miR-145 유전자(miR-145 pDNA)를 사용하였다. 유전자 전달체는 상기 실시예 2의 유전자 전달용 복합체(bPEI-SMCC-DS1, bPEI-SMCC-DS2, bPEI-SMCC-SCR, bPEI-SMCC) 용액에 상기 uciferase 효소를 발현시키는 유전자 및 miR-145 유전자를 한 방울씩 떨어뜨린 후 상온에서 약 15분 정도 반응시켜 제조하였다. In the present invention, the gene used for the purpose of analyzing the physicochemical properties was a gene expressing a luciferase enzyme called gWIZ-Luciferase in eukaryotic cells, and the nucleic acid transferred by the gene transfer complex (DS-bPEI) was miR-145 gene (miR-145 pDNA) was used. The gene transporter was transformed with the gene for expressing the uciferase enzyme and the miR-145 gene in the gene transfer complex (bPEI-SMCC-DS1, bPEI-SMCC-DS2, bPEI-SMCC-SCR, bPEI- And then allowed to react at room temperature for about 15 minutes.
본 발명에 사용한 miR-145 pDNA는 진코피디아(로크빌, 미국)에서 합성하여 사용하였으며, miR-145 pDNA 염기서열은 다음과 같다. The miR-145 pDNA used in the present invention was synthesized and used in Jin-Kopia (Rockville, USA), and the sequence of miR-145 pDNA is as follows.
miR-145(서열번호 2) : AUUCCUGGAAAUACUGUUCUUGmiR-145 (SEQ ID NO: 2): AUUCCUGGAAAUACUGUUCUUG
상기 제조된 유전자 전달체(DS-bPEI/miR-145 pDNA)는 유전자 전달용 복합체에 따라 DS1-bPEI/miR-145 pDNA(DS1-NP), DS2-bPEI/miR-145 pDNA(DS2-NP), SCR-bPEI/miR-145 pDNA(SCR-NP), bPEI-SMCC/miR-145 pDNA(bPEI-NP)를 제조하였다.
The thus-prepared gene delivery vehicle (DS-bPEI / miR-145 pDNA) was transformed into DS1-bPEI / miR-145 pDNA (DS1-NP), DS2-bPEI / miR- SCR-bPEI / miR-145 pDNA (SCR-NP) and bPEI-SMCC / miR-145 pDNA (bPEI-NP).
(2) 유전자 전달체((2) gene delivery system DSDS -- bPEIbPEI /Of miRmiR -145 -145 pDNApDNA )의 )of 표면전하Surface charge 측정 Measure
상기 (1)에서 제조된 유전자 전달체(DS-bPEI/miR-145 pDNA) 즉 DS1-NP, DS2-NP, SCR-NP, bPEI-NP에 있어, 유전자 전달용 복합체(bPEI-SMCC-DS1, bPEI-SMCC-DS2, bPEI-SMCC-SCR, bPEI-SMCC)가 전달 핵산(miR-145 pDNA)과 안정적인 유전자 전달체를 형성할 수 있는지를, N/P 비(복합체의 질소원자/유전자의 인산염 원자 비)가 3, 5, 9인 유전자 전달체(DS-bPEI/pDNA)에서의 표면 전위를 측정하여 확인하였다.The gene transfer complexes (bPEI-SMCC-DS1, bPEI-NP1, bPEI-NP1 and bPEI-NP) in the gene carrier (DS-bPEI / miR-145 pDNA) (SNP-SMCC-DS2, bPEI-SMCC-SCR and bPEI-SMCC) can form a stable gene carrier with the transfected nucleic acid (miR-145 pDNA) (DS-bPEI / pDNA), which are 3, 5, and 9, respectively.
실험방법은 상기 실시예 1에서 같은 방법으로, 유전자 전달체(DS1-NP, DS2-NP, SCR-NP, bPEI-NP)를 1 mL의 PBS(완충용액)에 넣고, 제타사이저 나노 제트(말버른,영국)에 방법대로 표면 전하를 측정하였다. The experimental method was the same as in Example 1 except that the gene delivery vehicle (DS1-NP, DS2-NP, SCR-NP, bPEI-NP) was added to 1 mL of PBS (buffer solution) Burton, England).
그 결과 도 7에서도 확인할 수 있듯이, DS-bPEI/miR-145 pDNA의 표면전하의 평균 값은 2 내지 12 mV로 중성에 가까운 양전하를 띠는 것을 알 수 있었다.
As a result, as shown in FIG. 7, it was found that the average value of the surface charge of the DS-bPEI / miR-145 pDNA was in the range of 2 to 12 mV, which was close to neutrality.
[[ 실시예Example 5] 유전자 전달체( 5] gene carrier ( DSDS -- bPEIbPEI /Of miRmiR -145 -145 pDNApDNA )의 크기 및 형태를 확인Check the size and shape of
상기 실시예 4에서 제조된 유전자 전달체(DS-bPEI/miR-145 pDNA) 즉 DS1-NP, DS2-NP, SCR-NP, bPEI-NP의 크기 및 형태를 나입자분석기(Dynamic Light Scattering , DLS) 및 TEM 이미지를 통해 확인하였다.The size and shape of the gene carrier (DS-bPEI / miR-145 pDNA) prepared in Example 4, DS1-NP, DS2-NP, SCR-NP and bPEI-NP were analyzed by Dynamic Light Scattering (DLS) And TEM images.
그 결과, 도 8 및 도 9에서 확인할 수 있듯이 평균 입자의 크기가 150 내지 200 nm 정도 직경을 갖는 구름과 같은 형태를 보였다. As a result, as shown in FIGS. 8 and 9, the average particle size was in the form of a cloud having a diameter of about 150 to 200 nm.
상기 결과로부터 유전자 전달용 복합체인 DS-bPEI이 효과적으로 miR-145 유전자(miR-145 pDNA)와 결합하고, 결합된 유전자 전달체(DS-bPEI/miR-145 pDNA)는 세포 내에 유입되기 유리한 표면 전하와 크기 및 형태를 가짐을 확인할 수 있었다.
The result shows that DS-bPEI effectively binds miR-145 gene (miR-145 pDNA), and the bound gene carrier (DS-bPEI / miR-145 pDNA) Size and shape.
[[ 실시예Example 6] 유전자 전달체( 6] gene transporter ( DSDS -- bPEIbPEI /Of miRmiR -145 -145 pDNApDNA ) ) 형성여부Formation 확인 Confirm
유전자 전달용 복합체인 DS-bPEI와 miR-145 유전자(miR-145 pDNA)의 결합을 아가로스 겔(agarose gel)을 통해 확인하였다. 각각의 N/P ratio 별로 확인한 결과, 도 10에서 확인할 수 있듯이 결합이 되지 않는 Control miR-145 pDNA 경우, miR-145 pDNA 크기의 band가 내려왔으나 N/P 비가 3인 모든 sample의 경우에서는 DS-bPEI와 miR-145 pDNA이 결합되어 유전자 전달체(DS-bPEI/miR-145 pDNA)가 형성된 것을 확인할 수 있었다.
Binding of the miR-145 gene (miR-145 pDNA) with DS-bPEI, a gene transfer complex, was confirmed through agarose gel. As shown in FIG. 10, in the case of control miR-145 pDNA which does not bind, the miR-145 pDNA size band was lowered. However, in all samples with N / P ratio of 3, DS- bPEI and miR-145 pDNA were combined to form a gene carrier (DS-bPEI / miR-145 pDNA).
[[ 실시예Example 7] 유전자 전달체( 7] gene carrier ( DSDS -- bPEIbPEI /Of miRmiR -145 -145 pDNApDNA )의 세포독성 평가) ≪ / RTI >
상기 실시예 4에서 제조된 유전자 전달체(DS-bPEI/miR-145 pDNA) 즉 DS1-NP, DS2-NP, SCR-NP, bPEI-NP가 쥐 유래의 전이성 유방암 세포(4T1)와 비전이성 유방암 세포(4T7)에 미치는 독성을 세포 생존률로 평가하였다.(DS-bPEI / miR-145 pDNA), DS1-NP, DS2-NP, SCR-NP and bPEI-NP prepared in Example 4 were mixed with mouse metastatic breast cancer cells (4T1) and non-metastatic breast cancer cells (4T7) were evaluated for cell viability.
세포증식 및 생존능력을 측정하기 위해 다양한 테트라졸륨 염(tetrazolium salts; MTT, XTT, MTS 등의 kit 구성 품)을 이용한 방법이 사용되고 있다. MTS 키트(Promega, USA)는 생존하는 세포의 미토콘드리아 탈수소효소(dehydrogenase)인 석시네이트-테타라졸륨 리덕타아제(succinate-tetrazolium reductase; EC1.3.99.1)가 테트라졸륨 염인 MTS를 분해하여 수용성 산물인 포르마잔을 생성하는 원리를 적용하고 있어 살아있는 세포에서만 효과를 나타낸다. 따라서 포르마잔에 의한 발색 강도를 측정함으로써 살아있는 세포 수를 상대적으로 정량할 수 있다. Various tetrazolium salts (kit components such as MTT, XTT and MTS) have been used to measure cell proliferation and viability. The MTS kit (Promega, USA) was prepared by degrading MTS, a tetrazolium salt, of succinate-tetrazolium reductase (EC 1.3.99.1), a mitochondrial dehydrogenase of surviving cells, Applying the principle of creating for morphogen, it is effective only in living cells. Therefore, the number of living cells can be relatively determined by measuring the intensity of color development by formazan.
4T1과 4T7 세포를 96 well 플레이트에 세포 수 1×104/well로 분주한 후, 24시간 배양하여 안정화시켰다. 세포의 처리 조건은 아무것도 처리하지 않은 무처리 세포, N/P 3, 5, 9의 비율로 유전자 전달용 복합체인 DS-bPEI와 miR-145 pDNA를 섞어 15분 동안 착물을 형성시킨 후 무혈청 배지와 함께 세포에 각 조건대로 처리하여 4시간 동안 배양하고, 4시간 후 새로운 성장배지로 다시 교환하여 계속 배양하였다. 배양 24시간 후 MTS 시약을 세포 배양액에 첨가하여 4시간 동안 배양하여, 490 nm에서 흡광도를 측정하였다. 무처리 세포에 대한 활성을 100%로 하여 상대적인 세포 생존률을 평가하였다.4T1 and 4T7 cells were plated in 96-well plates at a density of 1 × 10 4 cells / well and cultured for 24 hours to stabilize them. Cells were treated with untreated cells, N /
도 11에서도 확인할 수 있듯이, N/P 비가 특히 3에서는 control인 bPEI에 비해 세포 독성이 낮았으며, N/P 비가 증가할수록 독성이 다소 증가하는 것을 확인할 수 있었다.
As can be seen in FIG. 11, the cytotoxicity was lower in the N / P ratio than in the control bPEI, especially at 3, and the toxicity was slightly increased as the N / P ratio increased.
[[ 실시예Example 8] 유전자 전달체( 8] gene carrier ( DSDS -- bPEIbPEI /Of miRmiR -145 -145 pDNApDNA )의 유전자 전달 효율 평가) Gene transfer efficiency evaluation
상기 실시예 4에서 제조된 유전자 전달체(DS-bPEI/miR-145 pDNA) 즉 DS1-NP, DS2-NP, SCR-NP, bPEI-NP를 쥐 유래의 전이성 유방암 세포(4T1)와 비전이성 유방암 세포(4T7)에 처리하여 Report 유전자인 루시터라아제가 발현된 정도를 비교하여 유전자 전달 효율을 평가하였다.(DS-bPEI / miR-145 pDNA), DS1-NP, DS2-NP, SCR-NP and bPEI-NP prepared in Example 4 were inoculated into mouse metastatic breast cancer cells (4T1) and non-metastatic breast cancer cells (4T7), and the gene transfer efficiency was evaluated by comparing the degree of expression of the Report gene, lucitolase.
각각의 세포를 24 well 플레이트에 세포 수 5×104/well로 분주한 후 24시간 배양하여 안정화시켰다. 세포의 N/P 3, 5, 9의 비율로 유전자 전달용 복합체인 DS-bPEI와 miR-145 pDNA를 섞어 15분 착물을 형성시킨 후, 무혈청 배지와 함께 세포에 각 조건대로 처리하여 4시간 동안 배양하였고, 4시간 후 새로운 성장배지로 다시 교환하여 계속 배양하였다. 24시간 후 세포용혈과정을 통해 루시터라아제 발현을 측정하였다. Each cell was subcultured in a 24-well plate at 5 × 10 4 cells / well and incubated for 24 hours. The complexes of DS-bPEI and miR-145 pDNA were mixed with each other at a ratio of N /
통상적으로 중성전하를 가지는 유전자 전달체의 경우 세포 내로의 효율이 낮으며, 양전하를 가지는 유전자 전달체의 경우 세포 내로의 효율이 높다. 우리는 상기 실시예 4의 표면전하 값을 확인한 결과에서도 확인 된 바, 본 발명의 유전자 전달체(DS-bPEI/miR-145 pDNA)의 경우 중성전하에 가까운 전하를 가지고 있다. Generally, a gene carrier having a neutral charge has low efficiency into a cell, and a gene carrier having a positive charge has a high efficiency into a cell. As a result of confirming the surface charge value of Example 4 above, the gene carrier (DS-bPEI / miR-145 pDNA) of the present invention has a charge close to neutral charge.
도 12 및 도 13에서 확인할 수 있듯이, 상기 실시예 4의 유전자 전달체(DS-bPEI/miR-145 pDNA)의 4T1 세포에서 세포 내 전달 효율을 확인한 결과, 특히 중성전하를 가진 N/P 비가 3에서의 세포 내 전달 효율이 가장 높은 것을 확인할 수 있었다.As can be seen from FIGS. 12 and 13, the intracellular delivery efficiency of 4T1 cells of the gene carrier (DS-bPEI / miR-145 pDNA) of Example 4 showed that the N / P ratio with neutral charge was 3 Was the highest in the cell.
일반적으로 고분자 복합체의 표면이 강한 양전하를 나타낼수록 세포로의 유전자 전달 효율이 증가하지만, 상기의 결과는 본 발명의 전이성 유방암에 특이적인 세포투과 펩티드의 영향으로 중성전하에 가까운 낮은 양전하에서도 높은 발현 효율을 나타내고 있음을 확인한 결과이다.
Generally, as the surface of the polymer complex exhibits a strong positive charge, the efficiency of gene transfer into cells is increased. However, the above results show that even in the case of a low positive charge near neutral charge due to the effect of the cell permeation peptide specific to the metastatic breast cancer of the present invention Of the total population.
[[ 실시예Example 9] 유전자 전달체( 9] gene carrier ( DSDS -- bPEIbPEI /Of miRmiR -145 -145 pDNApDNA )의 세포 특이성 평가) ≪ / RTI >
상기 실시예 8에서 전이성 유방암 세포의 특이적인 유전자 발현을 확인하였다. In Example 8, specific gene expression of metastatic breast cancer cells was confirmed.
상기의 결과에 대하여 상기 실시예 8과 동일한 실험방법으로 세포 종류 즉, 정상 섬유아세포(3T3) 및 자궁경부암 세포(HeLa)에서의 유전자 전달 효율을 평가하였다.Regarding the above results, the gene transfer efficiency in the cell types, that is, normal fibroblast (3T3) and cervical cancer cells (HeLa), was evaluated by the same experimental method as in Example 8. [
그 결과 도 14에서 확인할 수 있듯이, N/P 비가 3에서는 control인 bPEI 보다 오히려 유전자 전달 효율이 낮은 것을 확인할 수 있었다.As a result, as shown in FIG. 14, it was confirmed that the gene transfer efficiency was lower than the control bPEI at the N / P ratio of 3.
상기의 결과로부터 실시예 4의 유전자 전달체(DS-bPEI/miR-145 pDNA)가 전이성 유방암 세포에 대하여 높은 유전자 전달 효율을 가짐을 확인할 수 있었다.
From the above results, it was confirmed that the gene carrier (DS-bPEI / miR-145 pDNA) of Example 4 has high gene transfer efficiency for metastatic breast cancer cells.
[[ 실시예Example 10] 유전자 전달체( 10] gene carrier ( DSDS -- bPEIbPEI /Of miRmiR -145 -145 pDNApDNA )의 세포에서의 발현 분석) ≪ / RTI > expression in cells
상기 실시예 4에서 제조된 유전자 전달체(DS-bPEI/miR-145 pDNA) 즉 DS1-NP, DS2-NP, SCR-NP, bPEI-NP의 전이성 유방암 세포에서의 위치발현을 확인하였다.DS1-NP, DS2-NP, SCR-NP and bPEI-NP of the gene carrier (DS-bPEI / miR-145 pDNA) prepared in Example 4 were expressed in metastatic breast cancer cells.
그 결과 도 15에서도 확인할 수 있듯이, 유전자 전달체(DS-bPEI/miR-145 pDNA)가 전이성 유방암 세포(4T1)의 세포질과 핵 주변에 많이 존재하는 것을 확인할 수 있었다.
As a result, as shown in FIG. 15, it was confirmed that a gene carrier (DS-bPEI / miR-145 pDNA) is present in many cytoplasm and nucleus of metastatic breast cancer cells (4T1).
[[ 실시예Example 11] 유전자 전달체( 11] gene carrier ( DSDS -- bPEIbPEI /Of miRmiR -145)의 전이성 유방암에 대한 세포 증식률 분석-145) to metastatic breast cancer
상기 실시예 4에서 제조된 유전자 전달체(DS-bPEI/miR-145 pDNA) 즉 DS1-NP, DS2-NP, SCR-NP, bPEI-NP의 전이성 유방암 세포에 대한 세포증식 억제효과를 확인한 결과이다.
DS1-NP, DS2-NP, SCR-NP, and bPEI-NP of the gene carrier (DS-bPEI / miR-145 pDNA) prepared in Example 4 were examined for inhibiting cell proliferation of metastatic breast cancer cells.
4T1와 4T7 세포를 96 well 플레이트에 세포수 1×104/well로 분주한 후 24시간 배양하여 안정화시켰다. 세포의 처리 조건은 아무것도 처리하지 않은 무처리 세포, N/P 3, 5, 9의 비율로 유전자 전달용 복합체인 DS-bPEI와 miR-145 pDNA를 섞어 15분 착물을 형성시킨 후, 무혈청 배지와 함께 세포에 각 조건대로 처리하여 4시간 동안 배양하고, 4시간 후 새로운 성장배지로 다시 교환하여 계속 배양하였다. 48시간 후 MTS 시약을 세포 배양액에 첨가하여 4시간 동안 배양한 후 490 nm에서 흡광도를 측정하였다. 무처리 세포에 대한 활성을 100%로 하여 상대적인 세포 생존률을 평가하였다.4T1 and 4T7 cells were seeded in 96-well plates at 1 × 10 4 cells / well and cultured for 24 hours to stabilize them. Cells were treated with untreated cells, N /
그 결과, 도 16에서도 확인할 수 있듯이, 유전자 전달체(DS-bPEI/miR-145)는 전이성 유방암 세포(4T1)의 세포 증식이 억제되는 것을 확인할 수 있었고, 특히 유방암 특이성을 가지는 DS-1에서 세포의 증식 억제 효과가 현저히 있음을 확인할 수 있었다.As shown in FIG. 16, it was confirmed that the gene delivery vehicle (DS-bPEI / miR-145) inhibited the cell proliferation of metastatic breast cancer cells (4T1), and in particular, in DS-1 having breast cancer specificity, It was confirmed that the effect of inhibiting proliferation was remarkable.
특히, 유전자 전달체의 N/P 비가 3에서 전이성 유방암 세포에 대한 세포 증식 효율이 control에 비해 약 40%정도 증식이 억제되는 것을 확인할 수 있었고, 이는 결국, 전이성 유방암세포에서 약 40%정도의 암 억제 발현효과가 나타내는 것을 확인한 결과이다.
In particular, when the N / P ratio of the gene delivery vehicle is 3, the cell proliferation efficiency of the metastatic breast cancer cell is inhibited by about 40% compared with the control, and it is confirmed that the cell proliferation of the metastatic breast cancer cell is about 40% And the expression effect was confirmed.
상기의 결과들을 통해 유전자 전달용 복합체에 핵산을 결합시켜 제조되는 유전자 전달체(DS-bPEI/miR-145)는 전이성 유방암 세포에 안전하고 효율적으로 유전자 전달할 수 있고, 세포에 대한 독성이 낮아 전이성 유방암을 효과적으로 치료할 수 있음을 확인할 수 있었다.(DS-bPEI / miR-145), which is prepared by binding nucleic acid to a gene delivery complex, can transfer genes to metastatic breast cancer cells safely and efficiently, and has low toxicity to cells, It can be effectively treated.
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Claims (14)
상기 활성화된 폴리에틸렌이민에 세포투과 펩티드(cell penetrating peptide); 및 트리스-(2-카복실에틸)인산염(Tris(2-Carboxyethyl) phosphine, TCEP);를 포함하는 세포내 유전자 전달용 복합체.Polyethyleneimine activated by conjugation of SMCC (succinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate);
A cell penetrating peptide to the activated polyethyleneimine; And tris- (2-carboxyethyl) phosphate (Tris (2-Carboxyethyl) phosphine, TCEP).
상기 폴리에틸렌이민은 평균 분자량 100 내지 500,000 달톤을 가지는 직선형 또는 가지형 형태인 특징으로 하는 복합체.The method of claim 1,
Wherein said polyethyleneimine is in a linear or branched configuration with an average molecular weight of 100 to 500,000 daltons.
상기 세포투과 펩티드는 서열번호 1로 이루어진 것을 특징으로 하는 복합체.The method of claim 1,
The cell penetrating peptide is characterized in that consisting of SEQ ID NO: 1.
상기 세포는 혈관내피세포(HUVEC 세포), 전이성 유방암 세포(4T1 세포) 및 전립선암세포(PC-3 세포)로 이루어진 군에서 선택되는 것을 특징으로 하는 복합체.4. The compound according to any one of claims 1 to 3,
Wherein said cells are selected from the group consisting of vascular endothelial cells (HUVEC cells), metastatic breast cancer cells (4T1 cells) and prostate cancer cells (PC-3 cells).
상기 세포는 전이성 유방암 세포(MDA-MB231 세포)인 것을 특징으로 하는 복합체.5. The method of claim 4,
Wherein the cell is a metastatic breast cancer cell (MDA-MB231 cell).
b) 상기 활성화된 폴리에틸렌이민에 세포투과 펩티드(cell penetrating peptide) 및 트리스-(2-카복실에틸)인산염(Tris(2-Carboxyethyl) phosphine, TCEP)을 첨가하여 유전자 전달체를 만드는 단계;
를 포함하는 세포내 유전자 전달용 복합체의 제조방법.a) activating the polyethylenimine by binding succinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate to SMCC; And
b) adding a cell penetrating peptide and tris (2-carboxyethyl) phosphate (TCE (2-Carboxyethyl) phosphine (TCEP) to the activated polyethyleneimine to form a gene carrier;
Method for producing a complex for intracellular gene delivery comprising a.
상기 폴리에틸렌이민은 가지형 형태의 폴리에틸렌이민(bPEI)인 것을 특징으로 하는 제조방법.The method according to claim 6,
Wherein said polyethyleneimine is polyethyleneimine (bPEI) in the form of a branch.
상기 세포투과 펩티드는 서열번호 1로 이루어진 것을 특징으로 하는 제조방법.The method according to claim 6,
The cell penetrating peptide is characterized in that consisting of SEQ ID NO: 1.
상기 핵산은 gDNA, cDNA, pDNA, mRNA, tRNA, rRNA, siRNA, miRNA 및 안티센스뉴클레오티드로 이루어진 군으로부터 선택되는 것을 특징으로 하는 조성물.The method of claim 9,
Wherein said nucleic acid is selected from the group consisting of gDNA, cDNA, pDNA, mRNA, tRNA, rRNA, siRNA, miRNA and antisense nucleotides.
상기 핵산은 miR-145인 것을 특징으로 하는 조성물.The method of claim 10,
The nucleic acid composition is characterized in that miR-145.
상기 핵산은 서열번호 2로 이루어진 것을 특징으로 하는 조성물.12. The method of claim 11,
The nucleic acid composition is characterized in that consisting of SEQ ID NO: 2.
상기 종양은 위암, 폐암, 유방암, 난소암, 간암, 기관지암, 비인두암, 후두암, 췌장암, 방광암, 결장암 및 자궁경부암으로부터 선택되는 것을 특징으로 하는 조성물.The method according to any one of claims 9 to 12,
Wherein said tumor is selected from gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer and cervical cancer.
상기 종양은 전이성 유방암인 것을 특징으로 하는 조성물.The method of claim 13,
Wherein the tumor is metastatic breast cancer.
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