WO2023063739A1 - Mesenchymal stem cell-derived extracellular vesicles to which ace2 is attached, and use thereof - Google Patents

Mesenchymal stem cell-derived extracellular vesicles to which ace2 is attached, and use thereof Download PDF

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WO2023063739A1
WO2023063739A1 PCT/KR2022/015485 KR2022015485W WO2023063739A1 WO 2023063739 A1 WO2023063739 A1 WO 2023063739A1 KR 2022015485 W KR2022015485 W KR 2022015485W WO 2023063739 A1 WO2023063739 A1 WO 2023063739A1
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ace2
extracellular vesicles
mesenchymal stem
infection
coronavirus
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Korean (ko)
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백문창
예경무
박준국
허종익
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재단법인대구경북과학기술원
경북대학교 산학협력단
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  • the present invention relates to ACE2-attached mesenchymal stem cell-derived extracellular vesicles and uses thereof.
  • Coronavirus is a virus composed of a single piece of the (+) strand RNA genome of about 27-32 kb, distributed in humans and other mammals. It is known that coronaviruses produce 6 to 8 subgenomic RNAs that have a common mRNA at the 3' end of genome-RNA through replication and transcription processes. In most people, coronavirus infection causes mild symptoms but is highly contagious, with SARS (Severe Respiratory Syndrome, 10% mortality rate) coronavirus and MERS (Middle East Respiratory Syndrome, 37% mortality) coronavirus affecting more than 10,000 people over the past 20 years. has been infected
  • COVID-19 a recently discovered new coronavirus (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2) infection, was discovered in China on December 1, 2019 and first reported on December 12, 2019 as an acute respiratory syndrome. , symptoms include fever, cough, shortness of breath, and atypical pneumonia. Since January 2020, it has spread widely outside of China, and it is developing into a situation of concern, such as a rapid transmission around the Lunar New Year holiday in China, a rapid increase in the number of infected people, and the paralysis of the entire city of Wuhan.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV2 binds to the angiotensin-converting enzyme 2 (hACE2) protein, which is mainly present in human lung epithelial cells, invades cells and reproduces the virus by replicating its genetic material inside human cells. Finding strategies to prevent the coronavirus from entering human cells is a fundamental way to treat the COVID-19 pandemic.
  • the spike protein S1-RBD (Receptor Binding Domain) present on the surface of the corona virus binds to the hACE2 protein, and the three-dimensional structure of the binding at this time has been revealed by recent studies.
  • An object of the present invention is to provide mesenchymal stem cell-derived extracellular vesicles (EV) expressing ACE2.
  • Another object of the present invention is an antiviral composition for coronavirus containing the extracellular vesicles as an active ingredient, a pharmaceutical composition for preventing or treating coronavirus infection, or for preventing or treating acute respiratory distress syndrome (ARDS) It is to provide a pharmaceutical composition.
  • ARDS acute respiratory distress syndrome
  • the present invention provides mesenchymal stem cell-derived extracellular vesicles (EV) expressing ACE2.
  • the present invention provides an antiviral composition for coronavirus comprising the extracellular vesicles as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection comprising the extracellular vesicles as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating acute respiratory distress syndrome (ARDS) comprising the extracellular vesicles as an active ingredient.
  • ARDS acute respiratory distress syndrome
  • the present invention relates to ACE2-attached mesenchymal stem cell-derived extracellular vesicles and uses thereof, and more particularly, to mesenchymal stem cells (MSC) by lentivirus infection to induce ACE2
  • MSC mesenchymal stem cells
  • EVs extracellular vesicles
  • the ACE2-attached mesenchymal stem of the present invention Since it was confirmed that cell-derived extracellular vesicles can prevent infection of SARS-CoV2 through spike protein blocking, it is expected to be useful as an early-stage infection blocker of coronavirus infection.
  • Figure 2 shows the results of the virus infection blocking test analysis of extracellular vesicles (Extracellular vesicle; EV).
  • FIG 3 shows the results of regeneration analysis (MTS) of cells damaged by LPS.
  • Figure 4 shows the results of regeneration analysis (cell count) of cells damaged by LPS.
  • the present invention provides mesenchymal stem cell-derived extracellular vesicles (EVs) expressing ACE2.
  • EVs mesenchymal stem cell-derived extracellular vesicles
  • the ACE2-expressing mesenchymal stem cells may be those that express the ACE2 on the surface through lentivirus infection, but are not limited thereto.
  • extracellular vesicle (EV) refers to nano-sized vesicles derived from cells, and depending on the secretion type and size, exosomes, microvesicles, and ectosomes ), microparticles, membrane vesicles, nanovesicles, and outer membrane vesicles.
  • Extracellular endoplasmic reticulum is a major means of communication between cells, including nucleic acids and proteins, which are the main components of cells.
  • ACE2 of the present invention is angiotensin-converting enzyme 2 (Angiotensin-converting enzyme 2), NCBI accession no. It may be BAB40370, but is not limited thereto.
  • the present invention provides an antiviral composition for coronavirus comprising the extracellular vesicles as an active ingredient.
  • the coronavirus may be SARS-CoV2, but is not limited thereto.
  • the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection comprising the extracellular vesicles as an active ingredient.
  • the coronavirus infection may be COVID-19, but is not limited thereto.
  • COVID-19 refers to a novel coronavirus infection, and represents variants of SARS and MERS as RNA viruses. COVID-19 shares about 77.5% sequence identity with SARS and about 50% with MERS. However, in contrast to SARS and MERS, the spike glycoprotein of COVID-19 forms a structure in which one RBD domain protrudes upward, which causes the target receptor ACE2 (angiotensin) and It shows 100 to 1,000 times stronger binding force. This strong binding force facilitates penetration into cells and serves as a cause of increasing infectivity.
  • ACE2 angiotensin
  • the present invention provides a pharmaceutical composition for preventing or treating acute respiratory distress syndrome (ARDS) comprising the extracellular vesicles as an active ingredient.
  • ARDS acute respiratory distress syndrome
  • the ARDS may be due to a coronavirus infection, but is not limited thereto.
  • the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is one commonly used in formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, and starch. , acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, stear including, but not limited to, acid magnesium and mineral oil, and the like.
  • the pharmaceutically acceptable carrier is one commonly used in formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, and starch.
  • acacia gum calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenz
  • composition for preventing or treating cancer metastasis of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.
  • composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc. can be administered with
  • parenteral administration intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc.
  • oral compositions can be formulated to coat the active agent or protect it from degradation in the stomach, and the composition of the present invention can be used in any device through which the active agent can move to target cells. can be administered by
  • a suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity, usually This allows the skilled physician to readily determine and prescribe dosages effective for the desired treatment or prophylaxis.
  • the pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art, or Or it can be prepared by incorporating into a multi-dose container.
  • the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.
  • Example 1 mesenchymal stem cells ( Mesenchymal stem cells; MSc )at ACE2 expression confirmation
  • a lentivirus was prepared by transferring the known human ACE2 DNA sequence into a lentivirus vector. This lentivius was infected with mesenchymal stem cells (MSC), resulting in the expression of ACE2 on its surface. Then, FACS analysis was performed to confirm the expression of ACE2 in MSC. Facs analysis of the 610 nm emission wavelength confirmed mcherry tagged with ACE2. As a result, mcherry fluorescent signal was confirmed in most of the cells, indicating that ACE2 was successfully expressed in MSC (Fig. 1).
  • MSC mesenchymal stem cells
  • Example 2 extracellular endoplasmic reticulum ( Extracellular vesicle; EV) viral infection blocking test analysis
  • EVs were extracted from MSC cells expressing ACE2 verified in Example 1.
  • HEK-293T-hACE2 cells (NR-52511, BEI resources) were treated with various concentrations of EV (0ng/ml, 0.5ng/ml, 5ug/ml).
  • SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike-Pseudotyped Lentiviral Kit (NR-52948, BEI resources) were infected. Cells were lysed at 48 hpi and Luciferase activity was measured with Bright-GloTM (E2610, Promega, Madison, WI, USA) according to the manufacturer's instructions.
  • MSC-EVs Cell experiments using MTS were conducted to confirm that the efficacy of MSC-EVs to regenerate cells damaged by LPS due to cell engineering was not impaired.
  • Cells used for the assay were Human Bronchial Epithelial Cell Line (16HBE14o-, Sigma. SSC150), seeded in a 96 well plate, and cultured for 24 h. Then, LPS (Sigma, #L2880) was pre-treated with 1 ug/ml for 1 hour, and EVs were treated with various concentrations (0 ng/ml, 1 ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml).
  • the reagent Promega, CellTiter 96® AQueous One Solution Cell Proliferation Assay, G3582
  • a 2-hour reaction 37°C, 5% CO 2
  • absorbance absorbance at 470 nm. measured.
  • LPS Long Term Evolution
  • FITC-Dextran Sigma, #53379 was treated and reacted (15 min, RT), and the basal media of the bottom plate was measured at (em/ex 485 nm/530 nm) wavelengths did As a result, it was confirmed that the EV of the present invention restores cells and reduces cell layer permeability (FIG. 5).

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Abstract

The present invention relates to mesenchymal stem cell-derived extracellular vesicles to which ACE2 is attached, and a use thereof. More specifically, from the results that ACE2 is expressed in mesenchymal stem cells (MSCs) by using lentivirus infection, and extracellular vesicles (EVs) are extracted from MSCs in which ACE2 is expressed so that the effects of the extracellular vesicles on pseudo-virion entry are investigated, it has been identified that the mesenchymal stem cell-derived extracellular vesicles to which ACE2 is attached, of the present invention, can prevent the infection of SARS-CoV2 through spike protein blocking, and thus are expected to be effectively usable as an infection blocking agent in the early stages of coronavirus infection.

Description

ACE2가 부착된 중간엽 줄기세포 유래 세포외소포체 및 이의 용도ACE2-attached mesenchymal stem cell-derived extracellular vesicles and uses thereof
본 발명은 ACE2가 부착된 중간엽 줄기세포 유래 세포외소포체 및 이의 용도에 대한 것이다.The present invention relates to ACE2-attached mesenchymal stem cell-derived extracellular vesicles and uses thereof.
코로나바이러스 (Coronavirus)는 약 27-32 kb 정도의 (+) strand RNA genome 단일 조각으로 구성된 바이러스로서 사람과 다른 포유동물들에 분포한다. 코로나바이러스는 복제와 전사과정을 거쳐서 게놈-RNA (genome-RNA)와 3' 말단에 공통의 mRNA를 가지는 서브게놈 RNA (subgenomic RNA) 6~8개를 생산한다고 알려져 있다. 대부분의 사람에서 코로나바이러스 감염은 가벼운 증상을 나타내나 감염력이 높아 지난 20여 년간 10,000명 이상의 사람에 SARS (중증호흡기증후군, 치사율 10%) 코로나바이러스 및 MERS (중동호흡기증후군, 치사율 37%) 코로나바이러스가 감염되었다.Coronavirus is a virus composed of a single piece of the (+) strand RNA genome of about 27-32 kb, distributed in humans and other mammals. It is known that coronaviruses produce 6 to 8 subgenomic RNAs that have a common mRNA at the 3' end of genome-RNA through replication and transcription processes. In most people, coronavirus infection causes mild symptoms but is highly contagious, with SARS (Severe Respiratory Syndrome, 10% mortality rate) coronavirus and MERS (Middle East Respiratory Syndrome, 37% mortality) coronavirus affecting more than 10,000 people over the past 20 years. has been infected
최근에 발견된 신종 코로나바이러스 (SARS-CoV-2, Severe acute respiratory syndrome coronavirus 2) 감염증인 COVID-19는 2019년 12월 1일 중국에서 발견되어 2019년 12월 12일 최초 보고된 급성 호흡기 증후군으로, 발열과 기침, 호흡곤란, 비정형 폐렴 등의 증세를 보인다. 2020년 1월부터는 중국 국외로도 광범위하게 전파되었으며, 중국내 춘절 연휴를 전후하여 빠른 전염으로 감염자 급증, 우한시 도시 기능 전체가 마비되는 등 우려할 사태로 발전하고 있다.COVID-19, a recently discovered new coronavirus (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2) infection, was discovered in China on December 1, 2019 and first reported on December 12, 2019 as an acute respiratory syndrome. , symptoms include fever, cough, shortness of breath, and atypical pneumonia. Since January 2020, it has spread widely outside of China, and it is developing into a situation of concern, such as a rapid transmission around the Lunar New Year holiday in China, a rapid increase in the number of infected people, and the paralysis of the entire city of Wuhan.
SARS-CoV2는 인간 폐 상피세포에 주로 존재하는 안지오텐신 변환 효소 2(Angiotensin-converting enzyme 2; hACE2) 단백질에 결합하여 세포 내로 침입하게 되며 바이러스의 유전물질을 인간 세포 내부에서 복제시켜 바이러스를 재생산한다. 코로나 바이러스가 인간 세포로 침입하는 것을 방지하는 전략을 찾는 것이 COVID-19 감염병을 치료하는 근본적인 방법이다. 코로나 바이러스의 표면에 존재하는 스파이크(Spike) 단백질 S1-RBD(Receptor Binding Domain)은 hACE2 단백질에 결합하며, 이때 결합된 3차원 구조에 대해서는 최근 연구들에 의하여 밝혀져 있다.SARS-CoV2 binds to the angiotensin-converting enzyme 2 (hACE2) protein, which is mainly present in human lung epithelial cells, invades cells and reproduces the virus by replicating its genetic material inside human cells. Finding strategies to prevent the coronavirus from entering human cells is a fundamental way to treat the COVID-19 pandemic. The spike protein S1-RBD (Receptor Binding Domain) present on the surface of the corona virus binds to the hACE2 protein, and the three-dimensional structure of the binding at this time has been revealed by recent studies.
SARS-CoV2와 hACE2의 결합을 차단할 수 있으면 근본적으로 코로나 바이러스의 세포 침입을 막을 수 있을 것으로 기대되고 있으며, COVID-19인 SARS-CoV2에 의한 감염증을 예방 또는 치료하기 위해 SARS-CoV2와 hACE2의 결합을 차단할 수 있는 새로운 수단에 대한 연구가 지속되고 있다.If the combination of SARS-CoV2 and hACE2 can be blocked, it is expected to fundamentally prevent the cell invasion of coronavirus, and the combination of SARS-CoV2 and hACE2 to prevent or treat infections caused by SARS-CoV2, COVID-19. Research on new means to block
본 발명의 목적은 ACE2를 발현하는 중간엽 줄기세포 유래 세포외소포체(Extracellular vesicle; EV)를 제공하는 데에 있다.An object of the present invention is to provide mesenchymal stem cell-derived extracellular vesicles (EV) expressing ACE2.
본 발명의 다른 목적은 상기 세포외소포체를 유효성분으로 포함하는 코로나바이러스에 대한 항바이러스 조성물, 코로나바이러스 감염증 예방 또는 치료용 약학조성물 또는 급성호흡곤란증후군(Acute respiratory distress syndrome; ARDS) 예방 또는 치료용 약학조성물을 제공하는 데에 있다. Another object of the present invention is an antiviral composition for coronavirus containing the extracellular vesicles as an active ingredient, a pharmaceutical composition for preventing or treating coronavirus infection, or for preventing or treating acute respiratory distress syndrome (ARDS) It is to provide a pharmaceutical composition.
상기 목적을 달성하기 위하여, 본 발명은 ACE2를 발현하는 중간엽 줄기세포 유래 세포외소포체(Extracellular vesicle; EV)를 제공한다.In order to achieve the above object, the present invention provides mesenchymal stem cell-derived extracellular vesicles (EV) expressing ACE2.
또한, 본 발명은 상기 세포외소포체를 유효성분으로 포함하는 코로나바이러스에 대한 항바이러스 조성물을 제공한다.In addition, the present invention provides an antiviral composition for coronavirus comprising the extracellular vesicles as an active ingredient.
또한, 본 발명은 상기 세포외소포체를 유효성분으로 포함하는 코로나바이러스 감염증 예방 또는 치료용 약학조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection comprising the extracellular vesicles as an active ingredient.
또한, 본 발명은 상기 세포외소포체를 유효성분으로 포함하는 급성호흡곤란증후군(Acute respiratory distress syndrome; ARDS) 예방 또는 치료용 약학조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating acute respiratory distress syndrome (ARDS) comprising the extracellular vesicles as an active ingredient.
본 발명은 ACE2가 부착된 중간엽 줄기세포 유래 세포외소포체 및 이의 용도에 관한 것으로서, 더욱 상세하게는, 중간엽줄기세포(Mesenchymal stem cell; MSC)에 렌티바이러스 감염(Lentivirus infection)을 이용해 ACE2를 발현시켰고, ACE2를 발현하는 MSC 세포에서 세포외소포체(Extracellular vesicle; EV)를 추출하여, Pseudo-Virion 진입에 대한 상기 세포외소포체의 효과를 검증한 결과, 본 발명의 ACE2가 부착된 중간엽 줄기세포 유래 세포외소포체가 스파이크 단백질 차단(Spike protein blocking)을 통해 SARS-CoV2의 감염을 방지할 수 있다는 것을 확인하였으므로, 코로나바이러스 감염증의 초기 단계 감염 차단제로 유용하게 활용될 수 있을 것으로 예상된다.The present invention relates to ACE2-attached mesenchymal stem cell-derived extracellular vesicles and uses thereof, and more particularly, to mesenchymal stem cells (MSC) by lentivirus infection to induce ACE2 As a result of verifying the effect of the extracellular vesicles on Pseudo-Virion entry by extracting extracellular vesicles (EVs) from MSC cells expressing ACE2, the ACE2-attached mesenchymal stem of the present invention Since it was confirmed that cell-derived extracellular vesicles can prevent infection of SARS-CoV2 through spike protein blocking, it is expected to be useful as an early-stage infection blocker of coronavirus infection.
도 1은 중간엽줄기세포(Mesenchymal stem cell; MSC)에서 ACE2 발현 확인 결과를 나타낸다.1 shows the result of confirming the expression of ACE2 in mesenchymal stem cells (MSC).
도 2는 세포외소포체(Extracellular vesicle; EV)의 바이러스 감염 차단 시험 분석 결과를 나타낸다.Figure 2 shows the results of the virus infection blocking test analysis of extracellular vesicles (Extracellular vesicle; EV).
도 3은 LPS로 데미지를 입은 세포의 재생(regeneration) 분석 (MTS) 결과를 나타낸다.3 shows the results of regeneration analysis (MTS) of cells damaged by LPS.
도 4는 LPS로 데미지를 입은 세포의 재생(regeneration) 분석 (cell count) 결과를 나타낸다.Figure 4 shows the results of regeneration analysis (cell count) of cells damaged by LPS.
도 5는 LPS로 데미지를 입은 세포의 재생(regeneration) 분석 (Trans-well permeability assay) 결과를 나타낸다.5 shows the results of trans-well permeability assay of cells damaged by LPS.
본 발명은 ACE2를 발현하는 중간엽 줄기세포 유래 세포외소포체(Extracellular vesicle; EV)를 제공한다.The present invention provides mesenchymal stem cell-derived extracellular vesicles (EVs) expressing ACE2.
바람직하게는, 상기 ACE2를 발현하는 중간엽 줄기세포는 렌티바이러스(Lentivirus) 감염을 통해 표면에 상기 ACE2를 발현시키는 것일 수 있으나, 이에 한정되는 것은 아니다.Preferably, the ACE2-expressing mesenchymal stem cells may be those that express the ACE2 on the surface through lentivirus infection, but are not limited thereto.
본 발명의 "세포외소포체(Extracellular vesicle; EV)"는 세포로부터 유래된 나노 사이즈의 소포체를 의미하며, 분비 형태 및 크기에 따라 엑소좀(exosomes), 마이크로베시클(microvesicles), 엑토좀(ectosomes), 마이크로파티클(microparticles), 막 소포체(membrane vesicles), 나노베시클(nanovesicles), 외막 소포체(outer membrane vesicles) 등으로 분류된다. 세포외소포체는 세포의 주요 성분인 핵산 및 단백질을 포함하여 세포간 소통의 주요 수단이다.The "extracellular vesicle (EV)" of the present invention refers to nano-sized vesicles derived from cells, and depending on the secretion type and size, exosomes, microvesicles, and ectosomes ), microparticles, membrane vesicles, nanovesicles, and outer membrane vesicles. Extracellular endoplasmic reticulum is a major means of communication between cells, including nucleic acids and proteins, which are the main components of cells.
본 발명의 "ACE2"는 안지오텐신 변환 효소 2(Angiotensin-converting enzyme 2)로서, NCBI accession no. BAB40370일 수 있으나, 이에 한정되는 것은 아니다."ACE2" of the present invention is angiotensin-converting enzyme 2 (Angiotensin-converting enzyme 2), NCBI accession no. It may be BAB40370, but is not limited thereto.
또한, 본 발명은 상기 세포외소포체를 유효성분으로 포함하는 코로나바이러스에 대한 항바이러스 조성물을 제공한다.In addition, the present invention provides an antiviral composition for coronavirus comprising the extracellular vesicles as an active ingredient.
바람직하게는, 상기 코로나바이러스는 SARS-CoV2일 수 있으나, 이에 한정되는 것은 아니다.Preferably, the coronavirus may be SARS-CoV2, but is not limited thereto.
또한, 본 발명은 상기 세포외소포체를 유효성분으로 포함하는 코로나바이러스 감염증 예방 또는 치료용 약학조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection comprising the extracellular vesicles as an active ingredient.
바람직하게는, 상기 코로나바이러스 감염증은 COVID-19일 수 있으나, 이에 한정되는 것은 아니다.Preferably, the coronavirus infection may be COVID-19, but is not limited thereto.
본 발명의, "COVID-19"는, 신종 코로나바이러스 감염증을 지칭하는 것으로서, RNA 바이러스로서 사스와 메르스의 변종을 나타낸다. COVID-19는 사스와 약 77.5%의 서열 동일성을, 메르스와 약 50% 공유한다. 하지만, 사스와 메르스와는 대조적으로, COVID-19의 스파이크 당 단백질(spike glycoprotein)은 1 개의 RBD domain 이 위로 돌출된 형태의 구조를 형성하며, 이로 인해 타겟 리셉터(receptor)인 ACE2(angiotensin)과 100~1,000 배 더 강력한 결합력을 나타낸다. 이러한 강력한 결합력은 세포 내로 침투를 더욱 용이하게 하여 전염력을 높이는 원인으로 작용한다.In the present invention, "COVID-19" refers to a novel coronavirus infection, and represents variants of SARS and MERS as RNA viruses. COVID-19 shares about 77.5% sequence identity with SARS and about 50% with MERS. However, in contrast to SARS and MERS, the spike glycoprotein of COVID-19 forms a structure in which one RBD domain protrudes upward, which causes the target receptor ACE2 (angiotensin) and It shows 100 to 1,000 times stronger binding force. This strong binding force facilitates penetration into cells and serves as a cause of increasing infectivity.
또한, 본 발명은 상기 세포외소포체를 유효성분으로 포함하는 급성호흡곤란증후군(Acute respiratory distress syndrome; ARDS) 예방 또는 치료용 약학조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating acute respiratory distress syndrome (ARDS) comprising the extracellular vesicles as an active ingredient.
바람직하게는, 상기 ARDS는 코로나바이러스 감염증으로 인한 것일 수 있으나, 이에 한정되는 것은 아니다.Preferably, the ARDS may be due to a coronavirus infection, but is not limited thereto.
본 발명의 약학 조성물은 약제학적으로 허용되는 담체를 추가로 포함할 수 있으며, 상기 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로오스, 폴리비닐피롤리돈, 셀룰로오스, 물, 시럽, 메틸셀룰로오스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 암 전이 예방 또는 치료용 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is one commonly used in formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, and starch. , acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, stear including, but not limited to, acid magnesium and mineral oil, and the like. The composition for preventing or treating cancer metastasis of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.
본 발명의 약학 조성물은 경구 또는 비경구로 투여할 수 있고, 비경구 투여인 경우에는 정맥내 주입, 피하주입, 근육 주입, 복강 주입, 내피 투여, 국소 투여, 비내 투여, 폐내 투여 및 직장 내 투여 등으로 투여할 수 있다. 경구 투여시, 단백질 또는 펩타이드는 소화가 되기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화 될 수 있으며, 본 발명의 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc. can be administered with When administered orally, since proteins or peptides are digested, oral compositions can be formulated to coat the active agent or protect it from degradation in the stomach, and the composition of the present invention can be used in any device through which the active agent can move to target cells. can be administered by
본 발명의 약학 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성별, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다. A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity, usually This allows the skilled physician to readily determine and prescribe dosages effective for the desired treatment or prophylaxis.
본 발명의 약학 조성물은 당해 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화하여 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 산제, 좌제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art, or Or it can be prepared by incorporating into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
<< 실시예Example 1> 1> 중간엽줄기세포mesenchymal stem cells (( MesenchymalMesenchymal stem cell; stem cells; MSCMSc )에서 )at ACE2ACE2 발현 확인 expression confirmation
알려진 human ACE2 DNA 서열을 lentivirus vector로 옮겨 lentivirus를 제작하였다. 이 lentivius는 중간엽 줄기세포(Mescenchtmal stem cell; MSC)에 감염되었고, 결과적으로 표면에 ACE2가 발현되었다. 이후, MSC에서 ACE2 발현을 확인하기 위해 FACS 분석을 진행하였다. 610nm emission 파장을 Facs 분석하여 ACE2에 태깅된(tagging) mcherry를 확인하였다. 결과적으로, 대부분의 세포에서 mcherry fluorescent signal을 확인하였고 이는 MSC에서 ACE2가 성공적으로 발현되었음을 나타낸다(도 1).A lentivirus was prepared by transferring the known human ACE2 DNA sequence into a lentivirus vector. This lentivius was infected with mesenchymal stem cells (MSC), resulting in the expression of ACE2 on its surface. Then, FACS analysis was performed to confirm the expression of ACE2 in MSC. Facs analysis of the 610 nm emission wavelength confirmed mcherry tagged with ACE2. As a result, mcherry fluorescent signal was confirmed in most of the cells, indicating that ACE2 was successfully expressed in MSC (Fig. 1).
<< 실시예Example 2> 2> 세포외소포체extracellular endoplasmic reticulum (( ExtracellularExtracellular vesicle; EV)의 바이러스 감염 차단 시험 분석 vesicle; EV) viral infection blocking test analysis
실시예 1에서 검증된 ACE2를 발현하는 MSC 세포에서 EV를 추출하였다. Pseudo-Virion 진입에 대한 EV의 효과를 검증하기 위해, HEK-293T-hACE2 세포(NR-52511, BEI resources)를 다양한 농도의 EV (0ng/ml, 0.5ng/ml, 5ug/ml)으로 처리하였다. 또한, SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike-Pseudotyped Lentiviral Kit (NR-52948, BEI resources)을 감염시켰다. 세포를 48hpi에서 용해하고 제조사의 지침에 따라 Bright-Glo™ (E2610, Promega, Madison, WI, USA)로 Luciferase 활성을 측정하였다. Perkinelmer EnVision microplate reader를 사용하여 Luciferase 활성의 정량화를 수행하였다. 실험의 프로토콜은 Crawford, K. H. D., et al. "Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays." Viruses 12 (ZOZOI: E513. PubMed: 32384820)을 기반으로 진행하였다. 실험 결과 control EV(ACE2가 발현되지 않은 MSC-EV)에 비해 ACE2 발현 EV가 처리되었을 때 바이러스(virus) 감염이 감소하였다(도 2). 상기 실험 결과, 본 발명의 EV가 스파이크 단백질 차단(Spike protein blocking)을 통해 SARS-CoV2의 감염을 방지할 수 있다는 것을 의미한다.EVs were extracted from MSC cells expressing ACE2 verified in Example 1. To verify the effect of EV on pseudo-virion entry, HEK-293T-hACE2 cells (NR-52511, BEI resources) were treated with various concentrations of EV (0ng/ml, 0.5ng/ml, 5ug/ml). . In addition, SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike-Pseudotyped Lentiviral Kit (NR-52948, BEI resources) were infected. Cells were lysed at 48 hpi and Luciferase activity was measured with Bright-Glo™ (E2610, Promega, Madison, WI, USA) according to the manufacturer's instructions. Quantification of Luciferase activity was performed using a Perkinelmer EnVision microplate reader. The protocol of the experiment is described in Crawford, KHD, et al. "Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays." Viruses 12 (ZOZOI: E513. PubMed: 32384820). As a result of the experiment, virus infection was reduced when ACE2-expressing EVs were treated compared to control EVs (MSC-EVs without ACE2 expression) (FIG. 2). As a result of the above experiment, it means that the EV of the present invention can prevent infection of SARS-CoV2 through Spike protein blocking.
<< 실시예Example 3> 3> LPS로with LPS 데미지를damage 입은 세포의 재생(regeneration) 분석 (MTS) Mouth cell regeneration assay (MTS)
세포 Engineering으로 인해 LPS로 인해 데미지를 입은 세포를 재생(regeneration) 시키는 MSC-EV의 효능이 손상되지 않았음을 확인하기 위해 MTS를 이용한 세포 실험을 진행하였다. Assay에 사용된 세포는 Human Bronchial Epithelial Cell Line (16HBE14o-, Sigma. SSC150)로서, 96 well plate에 접종(seeding)되었고, 24h 배양(incubation) 되었다. 이후 LPS (Sigma, #L2880)를 1ug/ml로 1시간 전처리하고 EV를 다양한 농도로 (0ng/ml, 1ng/ml, 5ng/ml, 10ng/ml, 20ng/ml) 처리하였다. 이후 24hpt에서 세포의 생존능(viability)을 확인하기 위해 시약 (Promega, CellTiter 96® AQueous One Solution Cell Proliferation Assay, G3582)을 처리 후 2시간 반응(37℃, 5% CO2) 진행하고 470nm에서 흡광도를 측정하였다. 결과적으로, 본 발명의 EV가 세포의 생존능(viability)을 회복시킨다는 것을 확인하였다(도 3).Cell experiments using MTS were conducted to confirm that the efficacy of MSC-EVs to regenerate cells damaged by LPS due to cell engineering was not impaired. Cells used for the assay were Human Bronchial Epithelial Cell Line (16HBE14o-, Sigma. SSC150), seeded in a 96 well plate, and cultured for 24 h. Then, LPS (Sigma, #L2880) was pre-treated with 1 ug/ml for 1 hour, and EVs were treated with various concentrations (0 ng/ml, 1 ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml). Then, in order to check the viability of the cells at 24 hpt, the reagent (Promega, CellTiter 96® AQueous One Solution Cell Proliferation Assay, G3582) was treated, followed by a 2-hour reaction (37°C, 5% CO 2 ) and absorbance at 470 nm. measured. As a result, it was confirmed that the EV of the present invention restores cell viability (FIG. 3).
<< 실시예Example 4> 4> LPS로with LPS 데미지를damage 입은 세포의 재생(regeneration) 분석 (cell count) Analysis of regeneration of worn-out cells (cell count)
세포 Engineering으로 인해 LPS로 인해 데미지를 입은 세포를 재생(regeneration) 시키는 MSC-EV의 효능이 손상되지 않았음을 확인하기 위해 cell count를 측정하는 실험을 진행하였다. Assay에 사용된 세포는 Human Bronchial Epithelial Cell Line (16HBE14o-, Sigma. SSC150)로서, 6 well plate에 접종(seeding) 되었고, 24h 배양(incubation) 되었다. 이후 LPS (Sigma, #L2880)를 1ug/ml로 1시간 전처리하고 EV를 다양한 농도로 (0ng/ml, 1ng/ml, 5ng/ml, 10ng/ml, 20ng/ml) 처리하였다. 이후 24hpt에서 세포의 증식(proliferation)을 확인하기 위해 trypsin-EDTA를 처리하고 세포 숫자를 측정하였다. 결과적으로, 본 발명의 EV가 세포의 증식(proliferation)을 회복시킨다는 것을 확인하였다(도 4).In order to confirm that the efficacy of MSC-EVs to regenerate cells damaged by LPS due to cell engineering was not impaired, an experiment was conducted to measure cell count. Cells used in the assay were Human Bronchial Epithelial Cell Line (16HBE14o-, Sigma. SSC150), seeded in a 6 well plate, and cultured for 24 h. Thereafter, LPS (Sigma, #L2880) was pre-treated with 1 ug/ml for 1 hour, and EVs were treated with various concentrations (0 ng/ml, 1 ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml). Thereafter, trypsin-EDTA was treated and the number of cells was measured to confirm cell proliferation at 24 hpt. As a result, it was confirmed that the EV of the present invention restores cell proliferation (FIG. 4).
<< 실시예Example 5> 5> LPS로with LPS 데미지를damage 입은 세포의 재생(regeneration) 분석 (Trans-well permeability assay) Trans-well permeability assay
세포 Engineering으로 인해 LPS로 인해 데미지를 입은 세포를 재생(regeneration)시키는 MSC-EV의 효능이 손상되지 않았음을 확인하기 위해 trans-well permeability를 측정하는 실험을 진행하였다. Assay에 사용된 세포는 Human Bronchial Epithelial Cell Line (16HBE14o-, Sigma. SSC150)로서, collagen coating된 Insert (Falcon®, Permeable Support for 24-well Plate with 0.4 μm Transparent PET Membrane, #353095)에 접종(seeding) 되었고, monolayer를 형성할 때까지 bottom plate와 함께 48h-72h 배양(incubation) 되었다. 이후 LPS (Sigma, #L2880)를 1ug/ml로 1시간 전처리하고 EV를 다양한 농도로(0ng/ml, 1ng/ml, 5ng/ml) 처리하였다. 이후 24hpt에서 세포의 투과성(permeability)을 측정하기 위해 FITC-Dextran (Sigma, #53379)을 처리하고 반응(15min, RT) 시킨 뒤 bottom plate의 basal media를 (em/ex 485nm/530nm)파장에서 측정하였다. 결과적으로, 본 발명의 EV가 세포를 회복시켜 cell layer의 투과성(permeability)를 감소시킨다는 것을 확인하였다(도 5).In order to confirm that the efficacy of MSC-EVs to regenerate cells damaged by LPS due to cell engineering was not impaired, an experiment was conducted to measure trans-well permeability. Cells used in the assay are Human Bronchial Epithelial Cell Line (16HBE14o-, Sigma. SSC150), seeded on collagen coated Insert (Falcon®, Permeable Support for 24-well Plate with 0.4 μm Transparent PET Membrane, #353095) ) and incubated with the bottom plate for 48h-72h until a monolayer was formed. Thereafter, LPS (Sigma, #L2880) was pre-treated with 1 ug/ml for 1 hour, and EVs were treated with various concentrations (0 ng/ml, 1 ng/ml, 5 ng/ml). Then, in order to measure the permeability of cells at 24 hpt, FITC-Dextran (Sigma, #53379) was treated and reacted (15 min, RT), and the basal media of the bottom plate was measured at (em/ex 485 nm/530 nm) wavelengths did As a result, it was confirmed that the EV of the present invention restores cells and reduces cell layer permeability (FIG. 5).
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Having described specific parts of the present invention in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (8)

  1. ACE2를 발현하는 중간엽 줄기세포 유래 세포외소포체(Extracellular vesicle; EV).Extracellular vesicles (EVs) derived from mesenchymal stem cells expressing ACE2.
  2. 제1항에 있어서, 상기 ACE2를 발현하는 중간엽 줄기세포는 렌티바이러스(Lentivirus) 감염을 통해 표면에 상기 ACE2를 발현시키는 것을 특징으로 하는 세포외소포체.The extracellular vesicle according to claim 1, wherein the ACE2-expressing mesenchymal stem cells express the ACE2 on the surface through Lentivirus infection.
  3. 제1항 또는 제2항의 세포외소포체를 유효성분으로 포함하는 코로나바이러스에 대한 항바이러스 조성물. An antiviral composition against coronavirus comprising the extracellular vesicles of claim 1 or 2 as an active ingredient.
  4. 제3항에 있어서, 상기 코로나바이러스는 SARS-CoV2인 것을 특징으로 하는 항바이러스 조성물.The antiviral composition according to claim 3, wherein the coronavirus is SARS-CoV2.
  5. 제1항 또는 제2항의 세포외소포체를 유효성분으로 포함하는 코로나바이러스 감염증 예방 또는 치료용 약학조성물. A pharmaceutical composition for preventing or treating coronavirus infection comprising the extracellular vesicles of claim 1 or 2 as an active ingredient.
  6. 제5항에 있어서, 상기 코로나바이러스 감염증은 COVID-19인 것을 특징으로 하는 코로나바이러스 감염증 예방 또는 치료용 약학조성물.The pharmaceutical composition for preventing or treating coronavirus infection according to claim 5, wherein the coronavirus infection is COVID-19.
  7. 제1항 또는 제2항의 세포외소포체를 유효성분으로 포함하는 급성호흡곤란증후군(Acute respiratory distress syndrome; ARDS) 예방 또는 치료용 약학조성물.A pharmaceutical composition for preventing or treating acute respiratory distress syndrome (ARDS) comprising the extracellular vesicles of claim 1 or 2 as an active ingredient.
  8. 제7항에 있어서, 상기 ARDS는 코로나바이러스 감염증으로 인한 것을 특징으로 하는 ARDS 예방 또는 치료용 약학조성물. The pharmaceutical composition for preventing or treating ARDS according to claim 7, wherein the ARDS is caused by a coronavirus infection.
PCT/KR2022/015485 2021-10-14 2022-10-13 Mesenchymal stem cell-derived extracellular vesicles to which ace2 is attached, and use thereof WO2023063739A1 (en)

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