KR100492017B1 - Method for anticancer treatment using human mesenhymal stem cell - Google Patents

Method for anticancer treatment using human mesenhymal stem cell Download PDF

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KR100492017B1
KR100492017B1 KR10-2002-0036161A KR20020036161A KR100492017B1 KR 100492017 B1 KR100492017 B1 KR 100492017B1 KR 20020036161 A KR20020036161 A KR 20020036161A KR 100492017 B1 KR100492017 B1 KR 100492017B1
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stem cells
mesenchymal stem
cells
cmv
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KR20040000287A (en
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김영태
송준석
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학교법인고려중앙학원
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/861—Adenoviral vectors
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Abstract

본 발명은 아데노바이러스 벡터를 이용하여 인간 간엽 줄기 세포를 유전적으로 변형하여 암억제 단백질인 p53을 과량 분비하게 하여 이를 항암치료에 사용하는 용도에 관한 것이다. 구체적으로 본 발명은 아데노바이러스에 감염된 간엽 줄기 세포가 암억제 유전자 (tumor suppressor gene) 인 p53을 과량 분비한다는 사실에 착안해 이를 이용한 항암치료에 사용하는 용도에 관한 것으로, 본 발명의 p53 과량 분비 간엽 줄기 세포의 제조 방법은 종양 치료에 유용하게 사용 될 수 있다.The present invention relates to the use of adenovirus vectors for genetically modifying human mesenchymal stem cells to secrete excessive amounts of p53, a cancer suppressor protein, and use them for anticancer therapy. Specifically, the present invention relates to the use of adenovirus-induced mesenchymal stem cells to secrete an excess of p53, a tumor suppressor gene, and to use the same for anticancer therapy. The method for producing stem cells may be usefully used for the treatment of tumors.

Description

인간 간엽줄기 세포를 이용한 항암치료방법 {Method for anticancer treatment using human mesenhymal stem cell} Anticancer treatment using human mesenchymal stem cells {Method for anticancer treatment using human mesenhymal stem cell}

줄기 세포를 이용한 의료 연구는 기존의 의학적 한계를 뛰어넘는 새로운 형태의 의학으로 21 세기 의학기술에서 중요한 위치를 차지하리라 본다. 줄기 세포란 분화(differentiation)되지 않은 세포로써, 증식(proliferation)되고 분화되어 어떤 특정 조직(tissue)이나 기관(organ)을 형성할 수 있는 능력을 가진 세포를 말한다. 줄기 세포를 크게 구별하면 성인 골수(adult bone marrow)에서 얻는 간엽 줄기 세포 (mesenchymal stem cell; MSC) 와 같은 성인 줄기세포 (adult stem cell)와 배자 (embryo) 와 태아 (fetus) 에서 얻는 배자, 태아줄기 세포 (embryonic & fetal stem cell)로 대별된다. 현재까지의 연구 결과에 따르면 성인 줄기 세포에서 연골 (cartilage), 지방 (fat), 근육 (muscle), 조혈(hemstopoietic) 그리고 신경 세포인 성상 세포(astrocyte)를 얻을 수 있다.Medical research using stem cells is a new type of medicine that goes beyond existing medical limitations and will hold an important place in 21st century medical technology. Stem cells are cells that are not differentiated and that have the ability to proliferate and differentiate to form certain tissues or organs. Stem cells can be broadly divided into adult stem cells, such as mesenchymal stem cells (MSCs) from adult bone marrow, embryos from embryos and fetuses, and from embryos and fetuses. It is roughly classified into an embryonic & fetal stem cell. To date, studies have shown that adult stem cells can produce cartilage, fat, muscle, hematopoietic, and neuronal astrocytes.

유전자 치료 (gene therapy) 는 유전병과 암 등 각종 유전자 이상으로 유발되는 질병 치료를 위해 치료 유전자를 체내에 주입하는 방법으로 각종 치료 유전자에 의해 각종 질환의 치료 효과를 기대 할 수 있다. 유전자 치료의 가장 중요한 요건은 목적 유전자의 목표 세포에의 성공적 전달에 있다.이를 위해 리포좀 (liposome) 등의 운반체를 사용하여 유전자를 주입하거나, 아데노바이러스나 레트로 바이러스 등에 유전자를 실어 세포에 전달하게 되는데 이 두 방법 모두 세포에의 전달 효율이 극히 떨어지며 환자에게 불필요한 면역 반응을 유도해 생명을 잃기도 한다. 따라서 암세포 등 목적 세포에 특이적으로 작용하면서 동시에 그 발현 효율이 높은 새로운 유전자 전달 체계가 필요하다.Gene therapy is a method of injecting therapeutic genes into the body for the treatment of diseases caused by various genetic abnormalities such as genetic diseases and cancer, and can be expected to treat various diseases by various therapeutic genes. The most important requirement for gene therapy is the successful delivery of the target gene to the target cell, which involves injecting the gene using a carrier such as a liposome or loading the gene into an adenovirus or retrovirus. Both of these methods are extremely inefficient in cell delivery and can lead to unnecessary immune responses in patients, resulting in loss of life. Therefore, there is a need for a new gene delivery system that specifically acts on target cells such as cancer cells and has high expression efficiency.

p53 유전자는 암억제 유전자 (tumor suppressor gene) 로 대략 50~60 % 암환자의 p53 유전자가 변이 또는 결손되어 그 활성이 나타나지 않는다. 구체적으로 p53 단백질은 세포 내 DNA 손상 시 세포주기 G1 기에 멈추게 하여 DNA 손상 복구 과정을 통해 암세포 생성을 막는다. 또한 이미 성장한 암세포에는 세포 아폽토시스 (apoptosis)를 유발시켜 암세포를 사멸케 한다. 따라서 p53 유전자를 p53 변이가 있는 암세포에서 발현시키면 암세포의 사멸을 유도 할 수 있다.The p53 gene is a tumor suppressor gene, and its activity is not shown due to mutation or deletion of the p53 gene in approximately 50 to 60% of cancer patients. Specifically, p53 protein stops at the cell cycle G1 phase when intracellular DNA damage prevents the formation of cancer cells through DNA damage repair process. In addition, cancer cells that have already grown cause cellular apoptosis (apoptosis) to kill the cancer cells. Therefore, expression of p53 gene in cancer cells with p53 mutation can induce the death of cancer cells.

현재 까지 연구 결과에 따르면 간엽 줄기 세포는 자가 면역 반응을 일으키지 않는 것으로 알려져 있다. 즉, 간엽 줄기 세포는 주조직 적합성 복합체(MHC) 를 갖음에도 불구하고, 면역학적으로 중성인 성질을 갖으므로 다른 사람에게서 이식받은 간엽 줄기 세포는 면역 거부 반응을 일으키지 않는다. 따라서 조직 적합 항원이 서로 다른 환자에게도 간엽 줄기 세포의 이식이 가능하다. 또한 간엽 줄기 세포는 면역체계에서 회피되는 세포이다. 즉, 동종이형 T 림파구와 간엽 줄기 세포를 공배양시 T세포에 의한 면역 반응이 일어나지 않음을 확인하였다.To date, research has shown that mesenchymal stem cells do not cause an autoimmune response. In other words, the mesenchymal stem cells have immunohistochemical properties despite having a major histocompatibility complex (MHC), so that the mesenchymal stem cells transplanted from others do not cause an immune rejection response. Thus, mesenchymal stem cells can be transplanted to patients with different tissue-compatible antigens. Mesenchymal stem cells are also cells that are evaded in the immune system. That is, the co-culture of allogeneic T lymphocytes and mesenchymal stem cells was confirmed that the immune response by T cells does not occur.

또한, Snyder 등에 의하면 신경 줄기 세포에 자살 유전자인 사이토신 다아미나제 (CD)를 도입하고 이를 뇌종양 세포가 이식된 설치류에 주입하자 신경 줄기 세포가 뇌종양세포를 추적하여 대부분의 종양 세포를 괴사시켰음을 확인하였다. 본 발명에서는 간엽 줄기 세포도 신경 줄기세포와 같은 종양 특이적 부착능력이 있음을 확인하였다.In addition, according to Snyder et al., The introduction of a suicide gene called cytosine daaminase (CD) into neural stem cells and injecting them into rodents in which the brain tumor cells were transplanted neural stem cells followed the brain tumor cells to necrosis most tumor cells. Confirmed. In the present invention, it was confirmed that mesenchymal stem cells also have tumor-specific adhesion ability, such as neural stem cells.

본 발명의 목적은 인간 간엽 줄기 세포에 아데노바이러스 벡터를 감염시켜 p53 발현량이 증가된 유전적으로 변형된 간엽 줄기 세포를 이용하여 이를 항암 유전자 치료에 응용하는데 있다. 또한 간엽 줄기 세포에는 종양 추적능력이 있음으로 이렇게 유전적으로 변형된 간엽 줄기 세포는 종양을 추적해 종양 세포에 부착되어 과량의 p53을 분비함으로써 종양세포 만을 특이적으로 괴사 시킬 것으로 생각된다.An object of the present invention is to apply adenovirus vectors to human mesenchymal stem cells and to apply them to anticancer gene therapy using genetically modified mesenchymal stem cells with increased p53 expression. In addition, since mesenchymal stem cells have tumor tracking ability, these genetically modified mesenchymal stem cells are likely to specifically attach tumor cells by attaching to tumor cells and secreting excess p53.

본 발명은 p53을 과량 발현하는 인간 간엽 줄기 세포의 제조방법 및 이를 이용한 항암 치료 방법을 제공한다.The present invention provides a method for producing human mesenchymal stem cells overexpressing p53 and an anticancer treatment method using the same.

이하, 본 발명을 실시예에 의해 더욱 상세히 설명한다. 하기 실시예들은 본 발명을 예시하는 것으로, 본 발명의 내용이 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

실시예 1 세포주 배양Example 1 Cell Line Culture

정상 사람 간엽 줄기 세포는 미국 바이오 휘테커 사 (Bio-whittaker, USA)에서 분양 받았으며, 간엽줄기 세포 성장 배지 (MSCGM)을 사용하여 37℃ 5% CO2 의 조건에서 배양하였다. 인간 정상 피부 섬유 아세포 세포인 IMR90과 부유 (suspension) 위암 세포주인 SNU-16 세포주는 10% 소태아혈청을 포함하는 배지 RPMI1640 에서 배양하여 사용하였다. 종양 세포주는 전립선암 세포주 Du-145, LN-CAP, 난소암 세포주 Ovca3 등을 이용하였다. 간엽 줄기 세포주를 제외한 모든 세포주는 한국 세포주 은행에서 구입하였다.Normal human mesenchymal stem cells were distributed by Bio-whittaker, USA and cultured at 37 ° C. 5% CO 2 using mesenchymal stem cell growth medium (MSCGM). Human normal dermal fibroblast cells IMR90 and suspension gastric cancer cell line SNU-16 cell line were cultured in medium RPMI1640 containing 10% fetal bovine serum. Tumor cell lines used prostate cancer cell line Du-145, LN-CAP, ovarian cancer cell line Ovca3. All cell lines except mesenchymal stem cell lines were purchased from Korea Cell Line Bank.

실시예 2 p53 과량 분비하는 인간 간엽 줄기 세포의 제조Example 2 Preparation of p53 Oversecreting Human Mesenchymal Stem Cells

아데노바이러스 Ad5.CMV-Null, Ad5.CMV-LacZ, Ad5.CMV-hp53 바이러스는 미국 퀀탐사 (Quantum-Appligene,USA) 에서 구입하였다. 간엽 줄기 세포의 아데노바이러스에 의한 유전자 전달 효율을 알아보기 위해 Ad5.CMV-LacZ을 10, 100 moi 감염시킨 후 X-gal 염색법을 시행하였다. 그 결과 moi 에 관계없이 거의 100% 세포가 감염됨을 확인한다 (도 1). p53을 과량 분비하는 간엽 줄기 세포의 제조를 위해 각각 바이러스를 10, 100 moi 씩 간엽 줄기 세포에 1시간 감염 후 새 배지로 바꿔주었다. 그 후 매 5일간 15일 동안 ELISA (Enzyme-Linked lmmuno-Sorbent Assay) 방법을 이용해 상층액에서 p53의 발현량을 조사하였다. 그 결과 p53 발현 아데노바이러스 Ad5.CMV-hp53 에 비해 Ad5.CMV-Null, Ad5.CMV-LacZ 감염 간엽 줄기 세포에서 약 4배의 p53이 발현됨을 확인 한다 (도 2). 이는 아데노바이러스 감염에 의한 간엽 줄기 세포의 방어책으로 p53 이 급격히 증가한 것으로 생각된다. Ad5.CMV-hp53에 의한 p53 증가량이 증가하지 않는 이유는 p53 발현량 증가에 의한 세포 자체의 항상성 (homeostasis)유지를 위한 것으로 생각된다. 또한 100 moi Ad5.CMV-hp53 처리한 간엽 줄기 세포의 거의 대부분이 사멸함을 확인해서 과량의 p53 분비를 위한 간엽줄기 세포의 제조에는 Ad5.CMV-hp53 이 적절치 않고, Ad5.CMV-Null, Ad5.CMV-LacZ 등 p53을 발현하지 않는 아데노바이러스를 100 moi 정도로 감염시켜 사용하는 것이 유리하다. 이러한 현상은 일시적이지 않고 15일 이후에도 계속 지속됨을 확인한다.Adenovirus Ad5.CMV-Null, Ad5.CMV-LacZ, Ad5.CMV-hp53 viruses were purchased from Quantum-Appligene, USA. To examine the gene transfer efficiency of adenoviruses in mesenchymal stem cells, X-gal staining was performed after 10, 100 moi of Ad5.CMV-LacZ infection. As a result, it was confirmed that almost 100% of cells were infected regardless of moi (FIG. 1). For the production of mesenchymal stem cells oversecreting p53, the virus was infected with 10 and 100 moi of mesenchymal stem cells for 1 hour and then replaced with fresh medium. Then, the expression level of p53 in the supernatant was examined by ELISA (Enzyme-Linked lmmuno-Sorbent Assay) method for 15 days every 5 days. As a result, it was confirmed that the expression of p53 was approximately four-fold in Ad5.CMV-Null and Ad5.CMV-LacZ-infected mesenchymal stem cells compared to p53-expressing adenovirus Ad5.CMV-hp53 (FIG. 2). This is a defense against mesenchymal stem cells caused by adenovirus infection. The reason why the increase in p53 by Ad5.CMV-hp53 does not increase is thought to be to maintain homeostasis of the cell itself by increasing p53 expression. In addition, almost all of the mesenchymal stem cells treated with 100 moi Ad5.CMV-hp53 were killed, and Ad5.CMV-hp53 was not suitable for the production of mesenchymal stem cells for excessive p53 secretion. It is advantageous to use adenoviruses that do not express p53, such as .CMV-LacZ, for infection at about 100 moi. It is confirmed that this phenomenon is not temporary and persists after 15 days.

실시예 3 간엽 줄기 세포의 종양 특이적 부착성 확인Example 3 Confirmation of Tumor Specific Adhesion of Mesenchymal Stem Cells

간엽 줄기 세포의 종양 특이적 부착성을 알아보기 위해 간엽 줄기 세포와 정상 인간 섬유 아세포 IMR-90 세포주에 부유 종양 세포주인 SNU-16 세포주를 투여해 보았다. 그 다음날 관찰해 본 결과 부유 종양 세포주가 강하게 부착됨에 비해 정상 섬유 아세포주 IMR-90엔 부착되지 않음을 확인한다 (도 3 참조). 따라서 간엽 줄기 세포엔 종양 특이적 부착 능력이 있음을 확인한다. 따라서 이러한 종양 세포 특이적 부착 능력을 갖는 간엽 줄기 세포를 종양 유전자 치료에 이용한다면 종양 세포만을 선택적으로 사멸 시킬 수 있을것이다.In order to investigate the tumor specific adhesion of mesenchymal stem cells, SNU-16 cell line, which is a floating tumor cell line, was administered to mesenchymal stem cells and normal human fibroblast IMR-90 cell line. Observation of the next day confirms that the floating tumor cell line is not attached to the normal fibroblast line IMR-90 yen, compared with the strong attachment (see FIG. 3). Therefore, it is confirmed that mesenchymal stem cells have tumor-specific adhesion ability. Therefore, if the mesenchymal stem cells having such tumor cell specific adhesion capacity are used for tumor gene therapy, only tumor cells may be selectively killed.

실시예 4 아데노바이러스 감염시킨 간엽 줄기 세포에 의한 종양 세포 살상효과Example 4 Tumor Cell Killing Effect by Adenovirus Infected Mesenchymal Stem Cells

아데노바이러스에 의해 감염된 간엽 줄기세포에서 분비되는 p53 유전자에 의한 세포 살상 효과를 알아보기 위해 아데노바이러스 간염 간엽 줄기 세포의 상층액을 각 종양 세포주에 투여한 후 6일 후 미국 프로메가사 제품인 CytoTox 96 Non-Radioactive Cytotoxicity assay kit를 이용해 세포 살상도를 측정해 보았다. 그 결과 Ad5.CMV-Null 감염 간엽 줄기 세포의 상층액이 세포 살상 효과를 갖음을 알 수 있다 (도 4 참조). DU-145는 전립선암 세포주, LNCap-LN3는 전립선암 세포주, OVCAR-3은 난소암 세포주이다.To investigate the cell killing effect of p53 gene secreted from mesenchymal stem cells infected with adenovirus, supernatant of adenovirus hepatitis mesenchymal stem cells was administered to each tumor cell line 6 days after CytoTox 96, a US Promega company. Cell killing was measured using a non-radioactive cytotoxicity assay kit. As a result, it can be seen that the supernatant of Ad5.CMV-Null infected mesenchymal stem cells has a cytotoxic effect (see FIG. 4). DU-145 is a prostate cancer cell line, LNCap-LN3 is a prostate cancer cell line, and OVCAR-3 is an ovarian cancer cell line.

본 발명의 p53 과량 분비 간엽 줄기세포는 암세포를 추적해 부착해서 과량의 p53 분비함으로써 암세포를 사멸시키거나 성장을 억제시키는데 탁월한 효과를 나타낼 것으로 기대된다. 또한 상기 세포는 기존의 바이러스 유래 유전자 벡터의 부작용인 면역 반응이나 낮은 전달효율성, 제조 및 정제과정의 어려움 등을 일시에 해결이 가능하고, 항원 항체 반응을 일으키지 않아 암환자에게 직접 투여가 가능한 새로운 차원의 항암 유전자 요법으로 기대가 매우 크다.The p53 excess secretory mesenchymal stem cells of the present invention are expected to exhibit excellent effects in killing or inhibiting growth of cancer cells by tracking and attaching and secreting excess p53. In addition, the cells can solve the immune response, low delivery efficiency, difficulty in manufacturing and purification, which are side effects of the existing virus-derived gene vectors, and can be directly administered to cancer patients without causing antigen antibody response. Expectations of anticancer gene therapy are very high.

도 1은 간엽 줄기 세포의 아데노바이러스에 의한 유전자 전달 효율을 알아보기 위해 Ad5.CMV-LacZ 을 100 moi 감염시킨 후 X-gal 염색법을 시행한 결과이다.1 is a result of X-gal staining after 100 moi infection of Ad5.CMV-LacZ to determine the gene transfer efficiency of adenoviruses of mesenchymal stem cells.

도 2는 ELISA(Enzyme-Linked lmmuno-Sorbent Assay) 방법을 이용해 각 아데노바이러스 벡터 감염 후 15일 후 p53의 발현량을 조사한 결과이다.Figure 2 shows the results of investigating the expression level of p53 15 days after each adenovirus vector infection using the Enzyme-Linked lmmuno-Sorbent Assay (ELISA) method.

1:Ad5.CMV-Null 감염 간엽 줄기 세포 상층액의 p53 분비량1:53 p53 Secretion of Ad5.CMV-Null Infected Mesenchymal Stem Cell Supernatants

2.Ad5.CMV-hp53 감염 간엽 줄기 세포 상층액의 p53 분비량2.p53 secretion of Ad5.CMV-hp53 infected mesenchymal stem cell supernatant

3.목 (mock) 간엽 줄기 세포 상층액의 p53 분비량3.p53 secretion of mock mesenchymal stem cell supernatant

도 3은 간엽 줄기 세포엔 종양 특이적 부착성이 있음을 확인한 그림이다. Figure 3 shows that the mesenchymal stem cells have tumor-specific adhesion.

도4는 아데노바이러스 감염 간엽 줄기 세포의 세포 상층액을 이용한 세포사멸 효과를 나타낸 그림이다.Figure 4 is a diagram showing the apoptosis effect using the cell supernatant of adenovirus infected mesenchymal stem cells.

Claims (3)

인간 간엽줄기세포에 p53을 발현하지 않는 △E1/△E3 복제 결손 재조합 아데노바이러스 혈청형 5(replication-defective recombination Adenovirus serotype 5)로서, 사이토메갈로바이러스(CMV) 프로모터/인핸서의 제어를 받는 LacZ 유전자를 포함하는 Ad5.CMV-LacZ 또는 아무 유전자도 포함하지 않는 Ad5.CMV-Null를 감염시켜 제조되는 종양 추적 능력이 있으며 p53을 과량 분비하는 인간간엽줄기세포.ΔE1 / ΔE3 replication deficient recombinant adenovirus serotype 5, which does not express p53 in human mesenchymal stem cells, is a LacZ gene under the control of a cytomegalovirus (CMV) promoter / enhancer. Human mesenchymal stem cells that have a tumor-tracking ability produced by infecting Ad5.CMV-LacZ containing or Ad5.CMV-Null containing no genes and overexpress p53. 삭제delete 제1항에 있어서, 암을 치료하는데 사용되는 것을 특징으로 하는 인간간엽줄기세포. The human mesenchymal stem cell of claim 1, which is used for treating cancer.
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