KR20230055751A - hepatocellular carcinoma specific targeting exosome composition and use thereof - Google Patents
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
Description
본 발명은 재조합 발현벡터로부터 형질전환된 엑소좀을 포함하는 간세포암 특이적 표적 엑소좀 조성물 및 이의 용도에 관한 것이다.The present invention relates to a hepatocellular carcinoma-specific target exosome composition comprising exosomes transformed from a recombinant expression vector and uses thereof.
간(liver)은 생명유지를 위한 각종 대사의 중심장기로서 인체에서 일어나는 대부분의 화학반응에 관여하는 인체 내 가장 큰 장기에 해당한다. 한국인의 7% 이상이 간염보균자로 OECD국가 중 간암 발병률 1위, 40-50대 남성 사망 원인 3위로 최근 alcohol, 각종 독성 약제, 바이러스 감염, 종양 및 이로 인한 수술 등으로 인해 간부전 발병이 국내외에서 지속적으로 증가하고 있다. 현재까지 소라페닙(sorafenib)을 제외하고는 간암에 대한 치료효능이 입증된 항암제가 없는 실정이다.The liver is the central organ of various metabolisms for life support and is the largest organ in the human body involved in most chemical reactions occurring in the human body. More than 7% of Koreans are carriers of hepatitis, which ranks first in the incidence of liver cancer among OECD countries and is the third leading cause of death in men in their 40s and 50s. is increasing with To date, there is no anticancer agent that has been proven to have therapeutic efficacy against liver cancer, except for sorafenib.
한편, 간세포암은 다른 부위에서 간으로 전이된 경우가 아닌 간세포 자체에서 발생하는 암으로, 간에서 발생하는 원발성 간암을 의미한다. 이는 전체 간암의 90％ 이상을 차지하며, 종류로는 1형 고분화형과 2형, 중등도 분화형, 3형 저분화형, 4형 미분화형 등 4종류가 있다. 치료를 하더라도 환자의 40~80％는 재발하고, 폐와 림프절, 복강을 둘러싸고 있는 안쪽 벽과 종격동에서 나타날 수도 있으며, 남성과 여성 환자의 비율은 5 대 1 정도이고, 대부분 중년 이후에 발생하는 특징이 있다.On the other hand, hepatocellular carcinoma is a cancer that occurs in hepatocytes themselves, not when they have metastasized to the liver from other sites, and refers to primary liver cancer that occurs in the liver. It accounts for more than 90% of all liver cancer, and there are four types:
간세포암이 간암으로 경과할 경우, 가장 효과적인 치료 방법은 간암의 수술적인 절제이나 진단 시에 수술적인 절제가 난해한 경우가 많으며, 이 경우 경동맥 화학색전술(transarterial chemoembolization, TACE), 경피적 에탄올 주입술(percutaneous ethanol injection therapy, PEIT), 고주파 열치료(radiofrequency ablation, RFA) 등의 국소치료를 해야하는 한계가 존재한다.When hepatocellular carcinoma progresses to liver cancer, the most effective treatment method is surgical resection of liver cancer or surgical resection is often difficult at the time of diagnosis. In this case, transarterial chemoembolization ( TACE) or percutaneous ethanol injection There is a limit to local treatment such as injection therapy, PEIT) and radiofrequency ablation,RFA.
이에, 본 발명은 인체에 무해한 biological vehicle인 외소포체(extracellular vesicle)를 이용한 간암표적 치료제로서 miRNA를 이용한 biologic therapy를 개발하기 위하여 예의 노력한 결과, 지방유래줄기세포로부터 추출된 엑소좀이 간세포암 표적 항암제의 기능이 있음을 확인함으로써, 본 발명을 완성하였다.Accordingly, the present invention has made diligent efforts to develop biologic therapy using miRNA as a liver cancer-targeted therapeutic agent using extracellular vesicles, a biological vehicle harmless to the human body. By confirming that there is a function of, the present invention was completed.
본 발명의 목적은 줄기세포의 세포외소포체로 발현되는 유전자 내에 간세포암 세포의 수용체와 결합하는 펩타이드를 발현시키는 유전자가 삽입된 재조합 발현벡터로 형질전환된 세포외소포체를 포함하는 간세포암의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.An object of the present invention is to prevent or prevent hepatocellular carcinoma including extracellular vesicles transformed with a recombinant expression vector into which a gene expressing a peptide that binds to a receptor of hepatocellular carcinoma cells is inserted into a gene expressed in extracellular vesicles of stem cells. It is to provide a pharmaceutical composition for treatment.
본 발명의 또 다른 목적은 (a) 지방유래줄기세포의 세포외소포체로 발현되는 유전자 내에 간세포암 세포의 수용체와 결합하는 펩타이드를 발현시키는 유전자를 도입하는 단계; (b) 상기 유전자가 도입된 세포를 배지에서 배양하여 세포외소포체를 수득하는 단계; 및 (c) 상기 수득된 세포외소포체에 간세포암 유발 유전자의 발현을 억제시킬 수 있는 핵산을 형질주입시키는 단계;를 포함하는 간세포암 예방 또는 치료용 약학적 조성물 제조방법을 제공하는 것이다.Another object of the present invention is to introduce a gene expressing a peptide that binds to a hepatocellular cancer cell receptor into a gene expressed in the extracellular vesicle of adipose-derived stem cells; (b) obtaining extracellular vesicles by culturing the cells into which the gene has been introduced in a culture medium; and (c) transfecting the obtained extracellular vesicle with a nucleic acid capable of suppressing the expression of a hepatocellular carcinoma-inducing gene.
본 출원에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 출원에서 개시된 다양한 요소들의 모든 조합이 본 출원의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 출원의 범주가 제한된다고 볼 수 없다.Each description and embodiment disclosed in this application can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not to be construed as being limited by the specific descriptions described below.
본 발명의 일 측면은 줄기세포의 세포외소포체로 발현되는 유전자 내에 간세포암 세포의 수용체와 결합하는 펩타이드를 발현시키는 유전자가 삽입된 재조합 발현벡터로 형질전환된 세포외소포체를 포함하는 간세포암의 예방 또는 치료용 약학적 조성물을 제공한다.One aspect of the present invention is to prevent hepatocellular carcinoma including extracellular vesicles transformed with a recombinant expression vector into which a gene expressing a peptide that binds to a receptor of hepatocellular carcinoma cells is inserted into a gene expressed in extracellular vesicles of stem cells. Or it provides a pharmaceutical composition for treatment.
본 발명에서의 "발현벡터"는 선형 DNA 벡터, 플라스미드 DNA 벡터 및 재조합 바이러스 벡터로 이루어진 군에서 선택될 수 있으나, 이에 제한되는 것은 아니며 당업계에서 형질전환을 위해 사용되는 통상적인 벡터들은 모두 본 발명의 방법에 사용될 수 있다. 본 발명의 일 구체예에 있어서, 상기 발현벡터는 pDisplay 벡터일 수 있다.The "expression vector" in the present invention may be selected from the group consisting of a linear DNA vector, a plasmid DNA vector, and a recombinant viral vector, but is not limited thereto, and conventional vectors used for transformation in the art are all of the present invention. can be used in the method of In one embodiment of the present invention, the expression vector may be a pDisplay vector.
본 발명에서의 "세포외소포체"는 세포에서 유래되는 막으로 둘러싸인 작은 구체를 의미하는 것으로, 상기 세포외소포체는 자연계, 예컨대 식물, 동물, 미생물로부터 유래된 세포외 소포체 또는 인공적으로 제조된 세포외소포체일 수 있다. 또한, 상기 세포는 자연계 생물 개체로부터 분리된 세포인 것일 수 있다.In the present invention, "extracellular vesicle" refers to a small sphere surrounded by a membrane derived from a cell, and the extracellular vesicle is an extracellular vesicle derived from nature, such as plants, animals, and microorganisms, or artificially prepared extracellular It may be an endoplasmic reticulum. In addition, the cells may be cells isolated from natural organisms.
상기 "세포외소포체"는 엑소좀(exosome), 엑토솜(ectosome) 또는 미세소포(microvesicle)일 수 있으나, 본 발명의 일 구체예에 있어서, 상기 "세포외소포체"는 엑소좀일 수 있다.The "extracellular vesicles" may be exosomes, ectosomes or microvesicles, but in one embodiment of the present invention, the "extracellular vesicles" may be exosomes.
본 발명에서의 "엑소좀"은 다양한 세포들로부터 분비되는 막 구조의 작은 소낭으로서, 다낭체와 원형질막의 융합이 일어나 세포 밖 환경으로 방출되는 소낭을 의미한다. 본 발명의 일 구체예에 있어서, 상기 엑소좀은 지방유래줄기세포로부터 유래 또는 분리한 것일 수 있다.In the present invention, "exosome" is a small membrane-structured vesicle secreted from various cells, and refers to a vesicle released into the extracellular environment after fusion of the polycystic body and the plasma membrane. In one embodiment of the present invention, the exosomes may be derived from or isolated from adipose-derived stem cells.
본 발명의 일 구체예에 있어서, 상기 엑소좀은 50 내지 150 nm의 직경을 갖는 것일 수 있다. 또한, 상기 엑소좀은 1 내지 50 μL/ml의 농도로 포함될 수 있다.In one embodiment of the present invention, the exo-some may have a diameter of 50 to 150 nm. In addition, the exosomes may be included at a concentration of 1 to 50 μL/ml.
본 발명에서의 "줄기세포(stem cells)"는 미분화 상태에서 적절한 조건을 맞춰주면 다양한 조직 세포로 분화될 수 있는 만능세포로서 다양한 유래의 세포를 의미한다."Stem cells" in the present invention refers to cells of various origins as pluripotent cells capable of differentiating into various tissue cells when appropriate conditions are met in an undifferentiated state.
상기 줄기세포는 골수유래줄기세포, 제대혈유래줄기세포 또는 지방유래줄기세포일 수 있다. 본 발명의 일 구체예에 있어서, 상기 줄기세포는 지방유래줄기세포일 수 있다. 또한, 상기 골수유래줄기세포, 제대혈유래줄기세포 또는 지방유래줄기세포는 인체 또는 동물유래 줄기세포인 것일 수 있다.The stem cells may be bone marrow-derived stem cells, cord blood-derived stem cells or adipose-derived stem cells. In one embodiment of the present invention, the stem cells may be adipose-derived stem cells. In addition, the bone marrow-derived stem cells, cord blood-derived stem cells, or adipose-derived stem cells may be human or animal-derived stem cells.
본 발명에서의 "지방유래줄기세포"는 지방 조직으로부터 유래한 줄기세포로서, 다분화능 및 자기증식능을 가진 세포를 의미하며, 본 발명의 지방유래줄기세포는 지방흡입술 및 다양한 외과적 수술을 통해 수득할 수 있으나, 이에 한정되지 않는다."Adipose-derived stem cells" in the present invention are stem cells derived from adipose tissue, and refer to cells having pluripotency and self-renewal ability, and the adipose-derived stem cells of the present invention are obtained through liposuction and various surgical operations. It can, but is not limited to this.
본 발명의 일 구체예에 있어서, 상기 엑소좀은 줄기세포 등 호스트세포의 엑소좀으로 발현하는 유전자 내에 간세포암 세포의 수용체와 결합하는 펩타이드를 발현시키도록 형질전환되어 간세포암 세포에 표적으로 작용할 수 있는 것을 의미한다.In one embodiment of the present invention, the exosome is transformed to express a peptide that binds to a receptor of hepatocellular carcinoma cells within a gene expressed by exosomes of host cells such as stem cells, and can act as a target for hepatocellular carcinoma cells. means there is
또한, 본 발명의 일 구체예에 있어서, 상기 엑소좀은 간세포암 유발 유전자의 발현을 억제시킬 수 있는 핵산이 형질주입된 것일 수 있다.Also, in one embodiment of the present invention, the exosome may be transfected with a nucleic acid capable of suppressing the expression of a hepatocellular carcinoma-inducing gene.
본 발명에서의 "형질주입"은 동물세포에 DNA 또는 RNA를 직접 도입하여 세포의 유전형질을 변이시키는 방법을 의미하며, 이는 당해 기술분야에 공지된 방법, 예를 들어, 칼슘 인산염 형질주입법(calcium phosphate transfection), 리포펙션법(lipofection), 전기천공법(electroporation), 미량주사법(microinjection), 마이크로프로젝틸법(microprojectile)을 이용할 수 있으나, 이에 제한되지 않는다. 진핵생물의 경우 DNA인산칼슘침전법 또는 리포펙션, PEI 등의 상품화된 시약과 DNA를 혼합하여 세포를 처리하는 방법을 이용할 수 있다."Transfection" in the present invention refers to a method of directly introducing DNA or RNA into animal cells to mutate the genetic traits of cells, which is a method known in the art, for example, calcium phosphate = transfection (calcium phosphate transfection) Phosphate transfection), lipofection, electroporation, microinjection, and microprojectile may be used, but are not limited thereto. In the case of eukaryotes, a method of treating cells by mixing DNA with commercially available reagents such as DNA calcium phosphate precipitation, lipofection, or PEI may be used.
본 발명에서의 "핵산"은 줄기세포의 세포외소포체로 발현되는 유전자 내에 삽입되어 세포외소포체로 발현 시 간세포암 세포의 수용체와 결합하는 펩타이드를 코딩할 수 있는 유전자를 포함하는 것을 의미한다.In the present invention, "nucleic acid" means a gene that can encode a peptide that is inserted into a gene expressed in the extracellular vesicle of stem cells and binds to a receptor of hepatocellular carcinoma cells when expressed in the extracellular endoplasmic reticulum.
본 발명에서의 "핵산"은 간세포암 유발 유전자의 발현을 억제시킬 수 있는 핵산으로서, 발암 유전자를 발현하는 핵산과 결합 또는 발암 유전자와 직접 결합하여 발현을 억제시키는 방식으로 간세포암 유발 유전자의 발현을 억제시킬 수 있다."Nucleic acid" in the present invention is a nucleic acid capable of suppressing the expression of a hepatocellular carcinoma-inducing gene, and inhibits the expression of a hepatocellular carcinoma-inducing gene by combining with a nucleic acid expressing an oncogene or by directly binding to an oncogene to inhibit expression. can suppress.
본 발명에서의 "핵산"은 miRNA, siRNA, shRNA, 안티센스 RNA 및 리보자임(ribozyme)으로 이루어진 군으로부터 선택된 하나 이상의 핵산일 수 있으나, 본 발명의 일 구체예에 있어서, 상기 "핵산"은 miRNA 및 siRNA로 이루어진 군으로부터 선택된 하나 이상의 핵산을 포함한다."Nucleic acid" in the present invention may be one or more nucleic acids selected from the group consisting of miRNA, siRNA, shRNA, antisense RNA and ribozyme, but in one embodiment of the present invention, the "nucleic acid" is miRNA and It includes one or more nucleic acids selected from the group consisting of siRNAs.
또한, 본 발명의 또 다른 측면은 (a) 지방유래줄기세포의 세포외소포체로 발현되는 유전자 내에 간세포암 세포의 수용체와 결합하는 펩타이드를 발현시키는 유전자를 도입하는 단계; (b) 상기 유전자가 도입된 세포를 배지에서 배양하여 세포외소포체를 수득하는 단계; 및 (c) 상기 수득된 세포외소포체에 간세포암 유발 유전자의 발현을 억제시킬 수 있는 핵산을 형질주입시키는 단계;를 포함하는 간세포암 예방 또는 치료용 발현벡터 제조방법을 제공한다.Another aspect of the present invention is (a) introducing a gene expressing a peptide that binds to a receptor of hepatocellular carcinoma cells into a gene expressed in the extracellular vesicles of adipose-derived stem cells; (b) culturing the cells into which the gene has been introduced in a medium to obtain extracellular vesicles; and (c) transfecting the obtained extracellular vesicle with a nucleic acid capable of suppressing the expression of a hepatocellular carcinogenic gene.
본 발명에서의 "세포외소포체"는 엑소좀(exosome), 엑토솜(ectosome) 또는 미세소포(microvesicle)일 수 있으나, 본 발명의 일 구체예에 있어서, 상기 "세포외소포체"는 엑소좀일 수 있다."Extracellular vesicles" in the present invention may be exosomes, ectosomes or microvesicles, but in one embodiment of the present invention, the "extracellular vesicles" may be exosomes there is.
본 발명에서의 "배지"는 지방유래줄기세포 배양배지인 것일 수 있다. 본 발명의 일 구체예에 있어서, 상기 지방유래줄기세포 배양배지는 지방유래줄기세포를 FBS(inactivated fetal bovine serum) 및 P/S(penicillin-streptomycin)을 첨가한 DMEM high low glucose 배지 또는 FBS-Free DMEM low glucose로 배지일 수 있다.The "medium" in the present invention may be an adipose-derived stem cell culture medium. In one embodiment of the present invention, the adipose-derived stem cell culture medium is DMEM high-low glucose medium or FBS-Free to which FBS (inactivated fetal bovine serum) and P/S (penicillin-streptomycin) are added. The medium can be DMEM low glucose.
본 발명에서의 “예방”은 본 발명에 따른 약학적 조성물의 투여에 의해 간세포암을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다."Prevention" in the present invention refers to all activities that inhibit or delay the onset of hepatocellular carcinoma by administration of the pharmaceutical composition according to the present invention.
본 발명에서의 “치료”는 본 발명에 따른 약학적 조성물의 투여에 의해 간세포암에 대한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다."Treatment" in the present invention refers to all activities that improve or beneficially change the symptoms of hepatocellular carcinoma by administration of the pharmaceutical composition according to the present invention.
본 발명에서의 "간세포암(hepatocellular carcinoma, HCC)"은 간세포에서 유래하는 일차성 간암을 의미하는 것으로, 구체적으로, 알콜 남용, 바이러스성 간염 및 대상성 간질환과 같은 위험인자를 갖는 환자에서 발생하는 간조직 자체에서 발생하는 원발성 악성 종양을 의미한다.In the present invention, "hepatocellular carcinoma (HCC)" refers to primary liver cancer derived from hepatocytes, and specifically, occurs in patients with risk factors such as alcohol abuse, viral hepatitis, and compensated liver disease. It refers to a primary malignant tumor arising from the liver itself.
본 발명의 줄기세포의 세포외소포체로 발현되는 유전자 내에 간세포암 세포의 수용체와 결합하는 펩타이드를 발현시키는 유전자가 삽입된 재조합 발현벡터로 형질전환된 세포외소포체를 포함하는 약학적 조성물은 세포외소포체로서 엑소좀을 약물전달물질로서 활용하여 표적 세포인 간세포암 세포에 선별적으로 결합하여 간세포암에 특이적으로 작용할 수 있다. 따라서, 상기 표적 엑소좀은 간세포암 치료 분야, 특히 임상적 적용 기술로 응용될 수 있다.A pharmaceutical composition comprising extracellular vesicles transformed with a recombinant expression vector into which a gene expressing a peptide that binds to a receptor of hepatocellular carcinoma cells is inserted into a gene expressed in the extracellular vesicle of stem cells of the present invention. As such, exosomes can be used as a drug delivery material to selectively bind to hepatocellular carcinoma cells, which are target cells, and act specifically on hepatocellular carcinoma. Therefore, the target exosome can be applied to the field of hepatocellular carcinoma treatment, in particular, as a clinical application technology.
도 1의 A는 본 발명의 재조합 단백질의 발현을 위한 pDisplay에서의 벡터 구성이고, B는 벡터의 발현에 따른 양상을 나타낸 것이다.
도 2는 본 발명의 발현벡터 도입 이 후, 엑소좀 마커인 CD9의 발현으로서 펩타이드 발현을 확인한 결과이다.
도 3은 본 발명의 발현벡터 도입 이 후, 엑소좀의 FACs을 확인한 결과이다.
도 4는 본 발명의 발현벡터 도입 이 후, 엑소좀의 TEM을 확인한 결과이다.
도 5는 본 발명에 따른 표적 엑소좀의 간암세포주인 HepG2, Hep3B에서의 PIN1의 발현 분석을 나타낸 것이다.
도 6은 본 발명에 따른 표적 엑소좀의 간암세포주인 Hep3B, Huh7 및 HepG2에서의 각 인자 별 발현 분석을 나타낸 것이다.
도 7은 본 발명에 따른 표적 엑소좀의 간암세포주인 Hep3B 및 Huh7에서의 각 인자 별 유세포 분석(FACs) 분석을 나타낸 것이다.
도 8은 본 발명에 따른 표적 엑소좀의 간암세포주인 Hep3B 및 Huh7에서의 각 인자 별 유세포 분석(FACs) 분석을 나타낸 것이다.
도 9는 본 발명에 따른 표적 엑소좀의 간암세포주인 Hep3B 및 Huh7에서의 wound healing assay에 따른 cell migration을 나타낸 결과이다.
도 10은 BALB/C Nude mouse에 간암세포주 Huh7 이식하여 구축된 간암 동물 모델에서의 외형 관찰 결과를 나타낸 것이다.
도 11은 BALB/C Nude mouse에 간암세포주 Huh7 이식하여 이식한 간암 동물 모델에서의 각 인자 별 발현 분석을 나타낸 것이다.1A is a vector composition in pDisplay for expression of the recombinant protein of the present invention, and B shows an aspect according to the expression of the vector.
Figure 2 is the result of confirming the peptide expression as the expression of CD9, an exosome marker, after introduction of the expression vector of the present invention.
Figure 3 is the result of confirming the FACs of exosomes after introduction of the expression vector of the present invention.
Figure 4 is the result of confirming the TEM of the exosome after introduction of the expression vector of the present invention.
5 shows analysis of PIN1 expression in HepG2 and Hep3B, which are liver cancer cell lines of target exosomes according to the present invention.
Figure 6 shows the expression analysis of each factor in Hep3B, Huh7, and HepG2, which are the target exosomes according to the present invention, which are liver cancer cell lines.
7 shows flow cytometry (FACs) analysis of each factor in Hep3B and Huh7, which are target exosomes according to the present invention, which are liver cancer cell lines.
8 shows flow cytometry (FACs) analysis of each factor in Hep3B and Huh7, which are liver cancer cell lines, of target exosomes according to the present invention.
9 is a result showing cell migration according to wound healing assay in liver cancer cell lines Hep3B and Huh7 using target exosomes according to the present invention.
10 shows the external appearance observation results in a liver cancer animal model constructed by transplanting the liver cancer cell line Huh7 into a BALB/C Nude mouse.
11 shows expression analysis of each factor in a liver cancer animal model transplanted by transplanting the liver cancer cell line Huh7 into a BALB/C Nude mouse.
이하, 실시예를 통하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. 지방유래줄기세포 유래의 표적 엑소좀 제작 및 확인Example 1. Adipose-derived stem cell-derived "target exosome" production and confirmation
본 발명의 간세포암에 특이적으로 작용하는 표적 엑소좀을 제작하기 위하여 지방유래줄기세포를 사용하여 엑소좀을 추출하였다. 참고적으로, 본 발명에서는 표적 엑소좀을 생산하기 위한 donor cells로서 지방유래줄기세포를 사용하였지만, NK세포와 같은 면역세포도 가능하므로, 이에 한정하지 않는다.In order to prepare target exosomes that specifically act on hepatocellular carcinoma of the present invention, exosomes were extracted using adipose-derived stem cells. For reference, in the present invention, adipose-derived stem cells were used as donor cells for producing target exosomes, but immune cells such as NK cells are also possible, so it is not limited thereto.
지방유래줄기세포를 제조사의 지침에 따라 형질감염시약(lipofectamine, invitrogen)을 사용하여 pDisplay 벡터(invitrogen)에 간암 세포 표적 펩타이드를 인코딩하는 아미노산 서열(TATCATTGGTACGGATATACCCCCCAGAATGTTATA)로 형질도입(transduction)시켰다. 상기 벡터를 클로닝한 후 해당 벡터가 클로닝된 결과를 확인하였으며(도 1), 지방유래줄기세포에 삽입하여 표적 엑소좀을 제작하였다.Adipose-derived stem cells were transduced with an amino acid sequence (TATCATTGGTACGGATATACCCCCCAGAATGTTATA) encoding a liver cancer cell-targeting peptide in a pDisplay vector (invitrogen) using a transfection reagent (lipofectamine, invitrogen) according to the manufacturer's instructions. After cloning the vector, the cloned result of the vector was confirmed (FIG. 1), and target exosomes were prepared by inserting into adipose-derived stem cells.
제작된 표적 엑소좀을 추출하기 위하여 지방유래줄기세포를 10% FBS(inactivated fetal bovine serum) 및 1% P/S(penicillin-streptomycin)을 첨가한 DMEM low glucose 배지에서 37℃, 5% CO2조건으로 배양하였다.To extract the prepared target exosome, adipose-derived stem cells were prepared in DMEM low glucose medium supplemented with 10% FBS (inactivated fetal bovine serum) and 1% P/S (penicillin-streptomycin) at 37°C and 5% CO 2 condition. cultured with.
24시간 경과 후 상기 배지를 FBS-Free DMEM low glucose로 배지를 교환하였으며, 배지 교환 후 24시간 경과 후 지방유래줄기세포의 배양 배지를 걷어 분별원심분리(differential centrifugation) 방법으로 1000rpm조건으로 원심분리하여 세포를 제거하였다.After 24 hours, the medium was exchanged with FBS-Free DMEM low glucose, and after 24 hours after the medium exchange, the culture medium of adipose-derived stem cells was removed and centrifuged at 1000 rpm by differential centrifugation. cells were removed.
상기 세포가 제거된 배양 배지를 thermo exosome 추출 kit(Thermo Fisher scientific)을 사용하여 제조사의 지침에 따라 간암 표적 엑소좀을 확보하였다.Liver cancer target exosomes were obtained from the culture medium from which the cells were removed using a thermo exosome extraction kit (Thermo Fisher scientific) according to the manufacturer's instructions.
여기에, 상기 과정을 통하여 확보한 표적 엑소좀에 Exo-Fect kit(SBI System biosciences)을 사용하여 PIN-siRNA를 표적 엑소좀 내에 형질주입하였으며 이를 통해 간암 수용체 및 미토콘드리아에 이중 표적이 가능한 엑소좀을 추가적으로 제작하였다.Here, PIN-siRNA was transfected into target exosomes using the Exo-Fect kit (SBI System biosciences) in the target exosomes obtained through the above process, and through this, exosomes capable of dual targeting to liver cancer receptors and mitochondria were obtained. made additionally.
실시예 2. 지방유래줄기세포 유래의 표적 엑소좀의 특성 분석Example 2. Analysis of characteristics of target exosomes derived from adipose-derived stem cells
본 발명의 지방유래 줄기세포에서 생성된 간암표적 펩타이드를 장착한 표적 엑소좀을 확보하고, 간암에 특이적으로 작용하는 표적 엑소좀에 대한 구조 분석을 수행하였다.A target exosome loaded with the liver cancer-targeting peptide generated from the adipose-derived stem cells of the present invention was secured, and structural analysis of the target exosome specifically acting on liver cancer was performed.
도 2를 통하여, 본 발명의 줄기세포에 엑소좀 표면에 간암 표적 마커를 장착할 수 있는 플라스미드를 도입하여 엑소좀 표면 마커인 CD9의 발현을 western blot으로 확인할 수 있었다(EV(ASC Exosome), cv-EV(pDisplay only) 및 tEV(cloning-pDisplay)). 2, the expression of CD9, an exosome surface marker, was confirmed by western blot by introducing a plasmid capable of mounting a liver cancer target marker on the surface of the exosome to the stem cell of the present invention (EV (ASC Exosome), cv -EV (pDisplay only) and tEV (cloning-pDisplay)).
지방유래 줄기세포(ASC)에서 추출한 extracellular vesicle(ASC-EV)과 ASC에 pDisplay를 형질 도입하여 추출한 extracellular vesicle(ASC-tEXO)을 Exo-view 장비인 투과전자현미경(TEM) 및 유세포분석기 (FACS)을 이용하여 분석하였다. 이에 대한, 엑소좀의 FACs, TEM 분석 결과를 도 3 및 도 4에 나타내었다.Extracellular vesicles (ASC-EV) extracted from adipose-derived stem cells (ASC) and extracellular vesicles (ASC-tEXO) extracted by transducing pDisplay into ASC were exo-viewed using transmission electron microscopy (TEM) and flow cytometry (FACS) was analyzed using . For this, the results of FACs, TEM analysis of exosomes are shown in FIGS. 3 and 4 .
실시예 3. 간암세포주인 HepG2 및 Hep3B에서의 표적 엑소좀의 PIN1 발현량 확인Example 3. Confirmation of PIN1 expression level of target exosomes in liver cancer cell lines HepG2 and Hep3B
간암세포주 별 발현에 차이를 확인하기 위하여, 간암세포주 및 간암 표적 엑소좀에 장착할 유전자를 선별하였다. 간암세포주로서 HepG2 및 Hep3B를 선별하고, 표적 엑소좀에 장착할 유전자는 간암에서 EMT를 촉진한다고 알려져 있는 PIN1을 선정하였다. 한편, 암세포는 EMT(Epithelial to Mesenchymal Transition)과정을 통하여 상피세포(Epithelial)가 간엽성세포(Mesenchymal)로 세포의 성질을 변형하여 이동 및 침윤을 진행하는 것으로 알려져 있다.In order to confirm the difference in expression of each liver cancer cell line, a gene to be loaded into the liver cancer cell line and the liver cancer target exosome was selected. HepG2 and Hep3B were selected as liver cancer cell lines, and PIN1, which is known to promote EMT in liver cancer, was selected as a gene to be loaded into target exosomes. On the other hand, it is known that cancer cells undergo migration and invasion by transforming the properties of epithelial cells into mesenchymal cells through an EMT (Epithelial to Mesenchymal Transition) process.
간 정상세포인 LO2와 간암세포주인 HepG2, Hep3B에서 RNA를 추출하여 cDNA를 합성하여 PCR을 진행하고 PIN1의 발현을 확인하였다(도 5). 간암세포주인 Hep3B에서 PIN1의 발현이 상당하게 증가함을 확인할 수 있었다.RNA was extracted from LO2, which is a normal liver cell, and HepG2 and Hep3B, which are liver cancer cell lines, and cDNA was synthesized, PCR was performed, and expression of PIN1 was confirmed (FIG. 5). It was confirmed that the expression of PIN1 was significantly increased in Hep3B, a liver cancer cell line.
실시예 4. 간암세포주인 Hep3B, Huh7 및 HepG2에서의 표적 엑소좀의 효능 평가Example 4. Efficacy evaluation of target exosomes in liver cancer cell lines Hep3B, Huh7 and HepG2
본 발명에 따른 재조합 벡터가 클로닝된 간암 표적 엑소좀의 항암 효능을 평가하기 위하여 간암세포주인 Hep3B, Huh7 및 HepG2에 각 엑소좀 조성물을 처리하고 PIN1, 세포사멸 기전에 관여하는 anti-apoptotic effect에 관여하는 인자인 Mcl-1 및 pro-apoptotic effect에 관여하는 인자인 PUMA의 발현 수준을 비교함으로써, 변화를 관찰하였다(도 6). 이를 위하여, 엑소좀(Ev), 표적 엑소좀(tEv)에 siNeg(Negative siRNA) 군, siPin(PIN-siRNA)을 장착한 군을 제작하였다. In order to evaluate the anticancer efficacy of the liver cancer target exosomes cloned with the recombinant vector according to the present invention, liver cancer cell lines Hep3B, Huh7 and HepG2 were treated with each exosome composition, and PIN1, involved in the anti-apoptotic effect involved in apoptosis mechanism Changes were observed by comparing the expression levels of Mcl-1, a factor involved in pro-apoptotic effects, and PUMA, a factor involved in pro-apoptotic effects (FIG. 6). To this end, siNeg (Negative siRNA) group and siPin (PIN-siRNA) group equipped with exosome (Ev) and target exosome (tEv) were prepared.
그 결과, 간암세포주인 Hep3B, Huh7과 HepG2에 Ct-siRNA 와 PIN1siRNA를 ASC 엑소좀과 간암 표적엑소좀에 장착하여 처리하였을 때 PIN1의 발현이 감소함을 확인할 수 있었다. As a result, it was confirmed that the expression of PIN1 was decreased when Ct-siRNA and PIN1siRNA were loaded into ASC exosomes and liver cancer target exosomes and treated with Hep3B, Huh7 and HepG2, which are liver cancer cell lines.
또한, PIN1siRNA 장착한 표적엑소좀의 경우 비표적엑소좀의 경우보다 MCL-1의 발현이 더욱 감소하는 것을 확인할 수 있었으며, PUMA의 발현은 더욱 증가하는 경향을 확인할 수 있었다.In addition, in the case of PIN1siRNA-loaded target exosomes, it was confirmed that the expression of MCL-1 was more decreased than in the case of non-targeted exosomes, and the expression of PUMA was confirmed to be more increased.
실시예 5. 간암세포주인 Hep3B 및 Huh7에서의 표적 엑소좀의 특성 분석Example 5. Characteristic analysis of target exosomes in liver cancer cell lines Hep3B and Huh7
지방유래줄기세포 엑소좀 및 본 발명의 지방유래줄기세포 간암표적 엑소좀의 세포사멸기전을 비교하기 위해서 PINsiRNA를 장착한 상기 엑소좀을 간암세포주에 처리하고 AnnexinV/PI로 염색하여 유세포 분석(FACs)을 수행하였다.In order to compare the apoptosis mechanism of the adipose-derived stem cell exosome and the adipose-derived stem cell liver cancer-targeted exosome of the present invention, the exosome equipped with PINsiRNA was treated with liver cancer cell lines, stained with AnnexinV/PI, and flow cytometric analysis (FACs) was performed.
FACs 수행 결과, 표적엑소좀에 PINsiRNA를 장착한 군에서 Apoptosis가 증가하는 경향을 확인할 수 있었다(도 7 및 도8).As a result of performing FACs, it was confirmed that apoptosis increased in the group in which PINsiRNA was attached to target exosomes (FIGS. 7 and 8).
실시예 6. 지방유래줄기세포 유래의 표적 엑소좀의 종양생성 억제능 평가Example 6. Evaluation of tumorigenesis inhibitory ability of target exosomes derived from adipose-derived stem cells
비표적 엑소좀과 표적엑소좀에 의한 세포의 전이능을 관찰하기 위하여 Hep3B 및 Huh7 간암세포주에 처리하여 wound healing assay를 진행하였다. 먼저, 대조군(Control)으로서 무처리(NC), 비표적 엑소좀(Ct-siRNA+tExo) 및 PIN-siRNA를 장착한(siPIN) 표적 엑소좀(siPin+tExo)을 준비하였다.In order to observe the metastatic ability of cells by non-targeting exosomes and targeting exosomes, Hep3B and Huh7 liver cancer cell lines were treated and wound, healing, and assays were performed. First, as a control (Control), untreated (NC), non-targeting exosomes (Ct-siRNA+tExo), and PIN-siRNA-equipped (siPIN) target exosomes (siPin+tExo) were prepared.
실험결과, wound healing assay(0hr 및 48hr)를 수행했을 때, PINsiRNA 장착 표적엑소좀에 의해서 migration이 유의하게 cell migration이 억제됨을 확인할 수 있었다(도 9).As a result of the experiment, when the wound healing assay (0 hr and 48 hr) was performed, it was confirmed that cell migration was significantly inhibited by the target exosomes loaded with PINsiRNA (FIG. 9).
실시예 7. 간암세포주 Huh7 이식에 따른 간암 동물 모델을 이용한 지방유래줄기세포 유래 표적 엑소좀의 외형 관찰 평가Example 7. Observation and evaluation of the appearance of adipose-derived stem cell-derived target exosomes using liver cancer animal models following transplantation of liver cancer cell line Huh7
BALB/C Nude mouse(in vivo)에 간암세포주 Huh7을 5x105으로 이식하여 간암을 유도한 뒤, 본 발명의 표적 엑소좀을 이용한 간암의 치료 효능을 확인하고자 하였다.After inducing liver cancer by transplanting the liver cancer cell line Huh7 into a BALB/C Nude mouse (in vivo) at 5x10 5 , the efficacy of liver cancer treatment using the target exosome of the present invention was confirmed.
대조군(Control)으로서 무처리(Ct), 엑소좀(EV), 표적 엑소좀(tEV) 및 PIN-siRNA를 장착한(si-PIN1) 엑소좀(tEV+PINsiRNA) 각각을 상기 구축된 종양모델에 Tail vein injection 방식으로 2주간 주입한 후, 마우스를 희생하여 간암의 항 종양효과에 엑소좀이 미치는 영향을 확인하였다.As a control (Control), untreated (Ct), exosomes (EV), target exosomes (tEV), and PIN-siRNA-equipped (si-PIN1) exosomes (tEV + PINsiRNA), respectively, to the constructed tumor model After injection for 2 weeks by tail vein injection method, the mouse was sacrificed to confirm the effect of exosomes on the anti-tumor effect of liver cancer.
그 결과, 구축된 간암 동물 모델에서의 외형 관찰 결과에 의하여 PIN1siRNA를 장착한 표적엑소좀을 처리한 군에서 종양 증식이 억제됨을 확인할 수 있었다(도 10).As a result, it was confirmed that tumor growth was suppressed in the group treated with the target exosome equipped with PIN1siRNA according to the external observation results in the constructed liver cancer animal model (FIG. 10).
실시예 8. 간암세포주 Huh7 이식에 따른 간암 동물 모델을 이용한 지방유래줄기세포 유래 표적 엑소좀의 효능 평가Example 8. Efficacy evaluation of adipose-derived stem cell-derived target exosomes using liver cancer animal models following transplantation of liver cancer cell line Huh7
BALB/C Nude mouse(in vivo)에 간암세포주 Huh7을 5x105으로 이식하여 간암을 유도한 뒤, 본 발명의 표적 엑소좀을 이용한 간암의 치료 효능을 확인하고자 하였다.After inducing liver cancer by transplanting the liver cancer cell line Huh7 into a BALB/C Nude mouse (in vivo) at 5x10 5 , the efficacy of liver cancer treatment using the target exosome of the present invention was confirmed.
대조군(Control)으로서 무처리(Ct), 엑소좀(EV), 표적 엑소좀(tEV) 및 PIN-siRNA를 장착한(si-PIN1) 엑소좀(tEV+PINsiRNA) 각각을 상기 구축된 종양모델에 Tail vein injection 방식으로 2주간 주입한 후, 마우스를 희생하여 간암의 항 종양효과에 엑소좀이 미치는 영향을 확인하였다.As a control (Control), untreated (Ct), exosomes (EV), target exosomes (tEV), and PIN-siRNA-equipped (si-PIN1) exosomes (tEV + PINsiRNA), respectively, to the constructed tumor model After injection for 2 weeks by tail vein injection method, the mouse was sacrificed to confirm the effect of exosomes on the anti-tumor effect of liver cancer.
그 결과, 구축된 종양 조직에서 단백질 수준에서의 Apoptosis 관련인자의 발현을 확인한 결과, PIN1siRNA장착한 표적엑소좀에서 anti-apoptotic 인자의 발현은 감소하고, pro-apoptotic 인자의 발현은 증가함을 확인할 수 있었다(도 11).As a result, as a result of confirming the expression of apoptosis-related factors at the protein level in the constructed tumor tissue, it was confirmed that the expression of anti-apoptotic factors decreased and the expression of pro-apoptotic factors increased in the PIN1siRNA-equipped target exosomes. There was (FIG. 11).
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.
<110> The Catholic University of Korea Industry-Academic Cooperation Foundation <120> Hepatocellular carcinoma specific targeting exosome composition and use thereof <130> DPC213410 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> incoding sequence <400> 1 tatcattggt acggatatac cccccagaat gttata 36 <110> The Catholic University of Korea Industry-Academic Cooperation Foundation <120> Hepatocellular carcinoma specific targeting exosome composition and use it <130> DPC213410 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 36 <212> DNA <213> artificial sequence <220> <223> encoding sequence <400> 1 tatcattggt acggatatac cccccagaat gttata 36
Claims (10)
(b) 상기 유전자가 도입된 세포를 배지에서 배양하여 세포외소포체를 수득하는 단계; 및
(c) 상기 수득된 세포외소포체에 간세포암 유발 유전자의 발현을 억제시킬 수 있는 핵산을 형질주입시키는 단계;를 포함하는 간세포암 예방 또는 치료용 발현벡터 제조방법.(a) introducing a gene expressing a peptide that binds to a hepatocellular cancer cell receptor into a gene expressed in the extracellular endoplasmic reticulum of adipose-derived stem cells;
(b) culturing the cells into which the gene has been introduced in a medium to obtain extracellular vesicles; and
(c) transfecting the obtained extracellular vesicle with a nucleic acid capable of suppressing the expression of a hepatocellular carcinoma-inducing gene;
10. The method of preparing an expression vector for preventing or treating hepatocellular carcinoma according to claim 9, wherein the extracellular vesicles are exosomes.
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