KR102570201B1 - Synthetic mRNA comprising sequence encoding peptide derived from apoptin and uses thereof - Google Patents
Synthetic mRNA comprising sequence encoding peptide derived from apoptin and uses thereof Download PDFInfo
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- KR102570201B1 KR102570201B1 KR1020230064069A KR20230064069A KR102570201B1 KR 102570201 B1 KR102570201 B1 KR 102570201B1 KR 1020230064069 A KR1020230064069 A KR 1020230064069A KR 20230064069 A KR20230064069 A KR 20230064069A KR 102570201 B1 KR102570201 B1 KR 102570201B1
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- synthetic mrna
- cancer
- apoptin
- preventing
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Abstract
본 발명은 아폽틴 유래 펩타이드 코딩 서열을 포함하는 합성 mRNA 및 이의 용도에 관한 것으로, 서열번호 1의 염기서열로 이루어진 합성 mRNA는 암세포의 사멸 유도 효과가 우수하므로, 암 치료제 개발 분야에 유용하게 사용될 수 있을 것이다.The present invention relates to a synthetic mRNA comprising an apoptin-derived peptide coding sequence and its use. Since the synthetic mRNA composed of the nucleotide sequence of SEQ ID NO: 1 has an excellent effect of inducing death of cancer cells, it can be usefully used in the field of cancer treatment development. There will be.
Description
본 발명은 아폽틴 유래 펩타이드의 코딩 서열을 포함하는 합성 mRNA 및 이의 용도에 관한 것이다.The present invention relates to synthetic mRNA comprising the coding sequence of an apoptin-derived peptide and its use.
아폽틴(apoptin)은 121개의 아미노산으로 구성된 닭전염성빈혈바이러스(chicken infectious anemia virus) 유래 단백질로서, 암세포 특이적인 사멸 효능을 가진다고 알려져 있다. 정상적인 세포에서는 사멸 유도 효능이 없으나 간암, 폐암, 혈액암, 피부암 등 다양한 암세포에서 특이적인 사멸 유도 효능이 나타나기 때문에 사람 및 동물의 항암 치료제 개발을 위하여 아폽틴 단백질을 생산하거나 이를 전달하는 유전체 개발에 대한 연구가 다양하게 시도되고 있다. 그러나 환자에서 고농도의 아폽틴 단백질이 효과적으로 발현되지 않는 등의 문제점으로 인하여 아폽틴을 이용한 암 치료제 개발은 현재까지 보고된 바 없다.Apoptin is a protein derived from chicken infectious anemia virus composed of 121 amino acids, and is known to have a specific killing effect on cancer cells. Although there is no apoptosis-inducing effect in normal cells, specific apoptosis-inducing effects are shown in various cancer cells such as liver cancer, lung cancer, blood cancer, and skin cancer. Therefore, there is a need for development of genomes that produce or deliver apoptin proteins for the development of human and animal anticancer drugs. Research is being attempted in various ways. However, development of a cancer treatment using apoptin has not been reported to date due to problems such as the ineffective expression of a high concentration of apoptin protein in patients.
암 치료제 개발을 위하여 아폽틴과 같은 암세포 사멸 효능이 있는 단백질을 이용한 다양한 연구들이 보고되고 있다. 주로 바이러스 발현 벡터 또는 세균 발현 벡터를 이용하여 암세포 사멸 효능이 있는 단백질을 발현하는 방법 등에 대한 연구가 보고되고 있다. 그러나 단백질 형태로 발현할 경우 치료제로 활용할 정도로 발현량이 충분하지 못하고, 치료제로 개발되어 2차, 3차로 추가 사용시 1차 접종된 단백질에 대한 면역항체 형성으로 치료 효능 감소 등의 단점이 발생한다고 알려져 있다. For the development of cancer therapeutics, various studies using proteins having cancer cell killing effects such as apoptin have been reported. Research on a method for expressing a protein having cancer cell killing effect using a viral expression vector or a bacterial expression vector has been reported. However, when expressed in the form of a protein, the expression level is not sufficient to be used as a therapeutic agent, and when developed as a therapeutic agent and used for the second or third time, it is known that there are disadvantages such as reduced therapeutic efficacy due to the formation of immune antibodies against the first inoculated protein. .
한편, 한국등록특허 제0528755호에는 아데노바이러스 벡터를 이용한 '세포자멸사 유도 단백질 브이피2 및/또는 아폽틴을 발현시키는 유전자 전달 운반체'가 개시되어 있고, 한국등록특허 제0561985호에는 '종양 특이적 아폽틴 발현 카세트 및 이를 포함하는 아데노바이러스 발현 벡터'가 개시되어 있으나, 본 발명의 '아폽틴 유래 펩타이드 코딩 서열을 포함하는 합성 mRNA 및 이의 용도'에 대해서는 기재된 바가 없다.On the other hand, Korean Patent No. 0528755 discloses 'a gene delivery vehicle expressing apoptosis-inducing protein VP2 and/or apoptin' using an adenovirus vector, and Korean Patent No. 0561985 discloses 'a tumor-specific A poptin expression cassette and an adenovirus expression vector containing the same are disclosed, but there is no description of 'synthetic mRNA comprising a poptin-derived peptide coding sequence and its use' of the present invention.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 암세포 살해 효능을 높이기 위하여 아폽틴 단백질이 암세포 핵내로 효과적으로 이동될 수 있도록 아폽틴 단백질중 암세포 살해 효능이 있는 일부 펩타이드(아폽틴의 121개의 아미노산중 42개 아미노산)만을 발현할 수 있도록 하였다. 이에, 5'-캡, 5'-UTR(untranslated region), 아폽틴 유래 펩타이드 코딩 서열, 3'-UTR 및 폴리 A 테일(poly A tail) 서열이 순차적으로 연결된 합성 mRNA를 제조하여 사람 유래 암세포인 Hep-2 세포 또는 동물 유래 암세포인 DF-1 세포에 각각 처리한 결과, 본 발명에 따른 합성 mRNA를 처리했을 때의 세포 사멸 비율은 아무것도 처리하지 않은 대조군 대비 약 2~3배 이상 증가한 것을 확인함으로써, 본 발명을 완성하였다.The present invention has been derived from the above needs, and the present inventors have developed some peptides (121 of apoptin) having cancer cell killing effect among apoptin proteins so that apoptin proteins can be effectively transported into cancer cell nuclei in order to increase cancer cell killing efficacy. 42 amino acids among dog amino acids) were allowed to be expressed. Accordingly, a synthetic mRNA comprising a 5'-cap, 5'-UTR (untranslated region), apoptin-derived peptide coding sequence, 3'-UTR, and poly A tail sequence is sequentially linked to human-derived cancer cells. As a result of treating Hep-2 cells or animal-derived cancer cells, DF-1 cells, respectively, it was confirmed that the cell death rate when treated with the synthetic mRNA according to the present invention increased by about 2 to 3 times compared to the untreated control group. , completed the present invention.
상기 과제를 해결하기 위해, 본 발명은 서열번호 1의 염기서열로 이루어진 암세포 성장 억제 또는 사멸용 합성 mRNA를 제공한다.In order to solve the above problems, the present invention provides a synthetic mRNA for inhibiting or killing cancer cell growth consisting of the nucleotide sequence of SEQ ID NO: 1.
또한, 본 발명은 5'-캡, 5'-UTR(untranslated region), 아폽틴 유래 펩타이드 코딩 서열, 3'-UTR 및 폴리 A 테일(poly A tail) 서열이 순차적으로 연결되도록 합성하는 단계를 포함하는, 암세포 성장 억제 또는 사멸용 합성 mRNA의 제조방법을 제공한다.In addition, the present invention includes the steps of synthesizing a 5'-cap, 5'-UTR (untranslated region), apoptin-derived peptide coding sequence, 3'-UTR and poly A tail sequence to be sequentially linked To provide a method for producing synthetic mRNA for inhibiting or killing cancer cells.
또한, 본 발명은 상기 합성 mRNA를 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the synthetic mRNA as an active ingredient.
또한, 본 발명은 상기 합성 mRNA를 유효성분으로 포함하는 암의 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving cancer comprising the synthetic mRNA as an active ingredient.
본 발명에 따른 합성 mRNA는 암세포의 사멸 유도 효과가 우수하므로, 암 치료제 개발 분야에 유용하게 사용될 수 있을 것이다.Since the synthetic mRNA according to the present invention has an excellent effect of inducing death of cancer cells, it can be usefully used in the field of cancer therapeutics development.
도 1은 본 발명의 합성 mRNA의 모식도(A) 및 염기서열(B)을 나타낸 것이다.
도 2는 본 발명의 합성 mRNA에 포함된 아폽틴 유래 펩타이드 코딩 서열(KNU12014-P)에 의해 번역되는 아미노산 서열을 나타낸 것이다.
도 3은 본 발명의 합성 mRNA에 의한 사람 유래 암세포인 Hep-2 세포의 사멸 효과를 확인한 결과로, A는 아무것도 처리하지 않은 대조군의 세포 사멸 비율을 나타낸 것이고, B는 본 발명의 합성 mRNA를 처리한 실험군의 세포 사멸 비율을 나타낸 것이다.
도 4는 본 발명의 합성 mRNA에 의한 동물 유래 암세포인 DF-1 세포의 사멸 효과를 확인한 결과로, A는 아무것도 처리하지 않은 대조군의 세포 사멸 비율을 나타낸 것이고, B는 본 발명의 합성 mRNA를 처리한 실험군의 세포 사멸 비율을 나타낸 것이다.Figure 1 shows a schematic diagram (A) and nucleotide sequence (B) of the synthetic mRNA of the present invention.
Figure 2 shows the amino acid sequence translated by the apoptin-derived peptide coding sequence (KNU12014-P) included in the synthetic mRNA of the present invention.
Figure 3 is a result of confirming the killing effect of human-derived cancer cells Hep-2 cells by the synthetic mRNA of the present invention, A shows the cell death rate of the control group not treated with anything, B is the treatment with the synthetic mRNA of the present invention It shows the cell death rate of one experimental group.
Figure 4 is a result of confirming the killing effect of DF-1 cells, which are animal-derived cancer cells, by the synthetic mRNA of the present invention. It shows the cell death rate of one experimental group.
본 발명의 목적을 달성하기 위하여, 본 발명은 서열번호 1의 염기서열로 이루어진 암세포 성장 억제 또는 사멸용 합성 mRNA를 제공한다.In order to achieve the object of the present invention, the present invention provides a synthetic mRNA for inhibiting or killing cancer cell growth consisting of the nucleotide sequence of SEQ ID NO: 1.
본 발명에 따른 서열번호 1의 염기서열로 이루어진 합성 mRNA는 5'-캡, 5'-UTR(untranslated region), 아폽틴 유래 펩타이드 코딩 서열, 3'-UTR 및 폴리 A 테일(poly A tail) 서열이 순차적으로 연결된 것일 수 있으며, 상기 아폽틴 유래 펩타이드는 바람직하게는 서열번호 2의 아미노산 서열로 이루어진 것일 수 있으나, 이에 제한되지 않는다. The synthetic mRNA consisting of the nucleotide sequence of SEQ ID NO: 1 according to the present invention includes a 5'-cap, a 5'-UTR (untranslated region), an apoptin-derived peptide coding sequence, a 3'-UTR and a poly A tail sequence It may be sequentially linked, and the apoptin-derived peptide may preferably consist of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.
본 발명에 따른 서열번호 1의 염기서열로 이루어진 합성 mRNA 염기서열에서 U(uridine)는 N1-methlypseudouridine(m1ψ)으로 대체된 것일 수 있으나, 이에 제한되지 않는다. U (uridine) in the synthetic mRNA nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 1 according to the present invention may be replaced with N 1 -methlypseudouridine (m1ψ), but is not limited thereto.
본 발명은 또한, 5'-캡, 5'-UTR(untranslated region), 아폽틴 유래 펩타이드 코딩 서열, 3'-UTR 및 폴리 A 테일(poly A tail) 서열이 순차적으로 연결되도록 합성하는 단계를 포함하는, 암세포 성장 억제 또는 사멸용 합성 mRNA의 제조방법을 제공한다.The present invention also includes the step of synthesizing a 5'-cap, 5'-UTR (untranslated region), apoptin-derived peptide coding sequence, 3'-UTR and poly A tail sequence to be sequentially linked. To provide a method for producing synthetic mRNA for inhibiting or killing cancer cells.
본 발명의 합성 mRNA의 제조방법에 있어서, 상기 합성은 통상의 기술자에게 알려진 바에 따라 수행될 수 있으며 특별한 방법으로 한정되는 것은 아니다. In the method for producing synthetic mRNA of the present invention, the synthesis may be performed as known to those skilled in the art and is not limited to a specific method.
본 발명은 또한, 본 발명의 합성 mRNA를 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물을 제공한다. The present invention also provides a pharmaceutical composition for preventing or treating cancer comprising the synthetic mRNA of the present invention as an active ingredient.
본 발명의 약학 조성물에 있어서, 상기 합성 mRNA는 암세포의 세포사멸(apoptosis) 유도를 통해 항암 효과를 가지는 것이 특징이다. In the pharmaceutical composition of the present invention, the synthetic mRNA is characterized by having an anticancer effect by inducing apoptosis of cancer cells.
본 발명의 약학 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 또는 희석제를 더 포함할 수 있으며, 바람직하게는 리포좀과 같은 안정제를 추가로 포함할 수 있으나, 이에 제한되는 것은 아니다.The pharmaceutical composition of the present invention may further include suitable carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions, and preferably may further include a stabilizer such as liposome, but is not limited thereto.
본 발명에 따른 조성물의 약학적 투여 형태는 단독으로 또는 타 약학적 활성 화합물과 결합뿐만 아니라 적당한 집합으로 사용될 수 있다.The pharmaceutical dosage form of the composition according to the present invention may be used alone or in combination with other pharmaceutically active compounds as well as in suitable combinations.
본 발명에 따른 약학 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 상기 합성 mRNA를 포함하는 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 포함한 다양한 화합물 혹은 혼합물을 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 합성 mRNA에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트, 수크로오스 또는 락토오스, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이 외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition according to the present invention is formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions. can Carriers, excipients, and diluents that may be included in the pharmaceutical composition containing the synthetic mRNA include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium phosphate. various compounds or mixtures, including calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. there is. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., to the synthetic mRNA. mixed and prepared In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.
본 발명의 약학 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the patient, the severity of the disease, the drug type, the route and duration of administration, but can be appropriately selected by those skilled in the art.
본 발명은 또한, 본 발명의 합성 mRNA를 유효성분으로 포함하는 암의 예방 또는 개선용 건강기능식품 조성물을 제공한다. The present invention also provides a health functional food composition for preventing or improving cancer comprising the synthetic mRNA of the present invention as an active ingredient.
본 발명의 건강기능식품 조성물은 분말, 과립, 환, 정제, 캡슐, 캔디, 시럽 및 음료 중에서 선택된 어느 하나의 제형일 수 있으나, 이에 제한되지 않는다.The health functional food composition of the present invention may be any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto.
본 발명의 건강기능식품 조성물을 식품첨가물로 사용하는 경우, 상기 건강기능식품 조성물을 그대로 첨가하거나 다른 식품 또는 식품성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분은 그의 사용 목적(예방 또는 개선)에 따라 적절하게 사용될 수 있다. 일반적으로, 식품 또는 음료의 제조시 본 발명의 건강기능식품 조성물은 원료에 대하여 15 중량부 이하, 바람직하게는 10 중량부 이하의 양으로 첨가된다. 그러나 건강을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로 사용될 수 있다.When using the health functional food composition of the present invention as a food additive, the health functional food composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods. Active ingredients can be appropriately used depending on their purpose of use (prevention or improvement). In general, when preparing food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material. However, in the case of long-term intake for health purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.
상기 건강기능식품의 종류에 특별한 제한은 없다. 상기 건강기능식품 조성물을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There is no particular limitation on the type of health functional food. Examples of foods to which the health functional food composition can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea There are drinks, alcoholic beverages, and vitamin complexes, and includes all health foods in a conventional sense.
또한, 본 발명의 건강기능식품 조성물은 식품, 특히 기능성 식품으로 제조될 수 있다. 본 발명의 기능성 식품은 통상적으로 첨가되는 성분을 포함할 수 있다. 예를 들어, 단백질, 탄수화물, 지방, 영양소 및 조미제를 포함한다. 예컨대, 드링크제로 제조되는 경우에는 유효성분 이외에 천연 탄수화물 또는 향미제를 추가 성분으로서 포함시킬 수 있다. 상기 천연 탄수화물은 모노사카라이드(예컨대, 글루코오스, 프럭토오스 등), 디사카라이드(예컨대, 말토스, 수크로오스 등), 올리고당, 폴리사카라이드(예컨대, 덱스트린, 시클로덱스트린 등) 또는 당알코올(예컨대, 자일리톨, 소르비톨, 에리쓰리톨 등)인 것이 바람직하다. 상기 향미제는 천연 향미제(예컨대, 타우마틴, 스테비아 추출물 등)와 합성 향미제(예컨대, 사카린, 아스파르탐 등)를 이용할 수 있다.In addition, the health functional food composition of the present invention can be made into food, particularly functional food. The functional food of the present invention may contain ingredients that are commonly added. Examples include proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared as a drink, natural carbohydrates or flavors may be included as additional ingredients in addition to active ingredients. The natural carbohydrates are monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrins, cyclodextrins, etc.) or sugar alcohols (eg, maltose, sucrose, etc.) , xylitol, sorbitol, erythritol, etc.) are preferred. As the flavoring agent, natural flavoring agents (eg, thaumatin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used.
상기 건강기능식품 조성물 이외에 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 더 함유할 수 있다. 이러한 상기 첨가되는 성분의 비율은 크게 중요하진 않지만 본 발명의 건강기능식품 조성물 100 중량부에 대하여, 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the health functional food composition, various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonic acid It may further contain a carbonation agent used in beverages and the like. The ratio of the components to be added is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited to the following examples.
실시예 1. 합성 mRNA의 제조Example 1. Preparation of synthetic mRNA
5' 말단부터 5'-캡, 5'-UTR(untranslated region), 아폽틴 유래 펩타이드(KNU12014-P) 코딩 서열, 3'-UTR, 폴리 A 테일 서열이 순차적으로 연결되어 있는 IVT(in vitro transcribed) mRNA를 합성하였고, 상기 mRNA의 염기서열에서 모든 U(uridine)는 N1-methlypseudouridine(m1ψ)으로 대체하여 합성하였다(도 1). 5’-UTR은 사람 α-글로불린 mRNA 유래의 것을 사용하였고, 3’-UTR은 미토콘드리아 12S 리보좀 RNA 유래의 것을 사용하였으며, 아폽틴 유래 펩타이드(KNU12014-P) 코딩 서열은 본 발명자들이 보유하고 있는 닭전염성빈혈바이러스(chicken infectious anemia virus) 중 KNU12014주의 VP3 단백질을 암호화하는 유전자의 일부를 클로닝하여 사용하였다. 5' 말단부터 5'-캡, 5'-UTR(untranslated region), 아폽틴 유래 펩타이드(KNU12014-P) 코딩 서열, 3'-UTR 서열이 순차적으로 연결되어 있는 합성 DNA를 주형으로 사용하여 mRNA 합성 키트(HiScribe T7 mRNA Kit, NEB) 를 이용하여 mRNA를 제조하였다. 상기 아폽틴 유래 펩타이드 코딩 서열에 의해 번역되는 아미노산은 NLS 1(nuclear localization signal 1), NES(nuclear export signal), NLS 2(nuclear localization signal 2)가 포함되어 있는 42개 아미노산 서열로 구성되어 있다(도 2). In vitro transcribed (IVT) in which 5'-cap, 5'-UTR (untranslated region), apoptin-derived peptide (KNU12014-P) coding sequence, 3'-UTR, and poly A tail sequence are sequentially connected from the 5' end. ) mRNA was synthesized, and all U (uridine) in the nucleotide sequence of the mRNA was synthesized by replacing with N 1 -methlypseudouridine (m1ψ) (FIG. 1). The 5'-UTR was derived from human α-globulin mRNA, the 3'-UTR was derived from mitochondrial 12S ribosomal RNA, and the coding sequence of the apoptin-derived peptide (KNU12014-P) was obtained from chicken Part of the gene encoding the VP3 protein of the KNU12014 strain of the chicken infectious anemia virus was cloned and used. mRNA synthesis using synthetic DNA as a template, in which 5'-cap, 5'-UTR (untranslated region), apoptin-derived peptide (KNU12014-P) coding sequence, and 3'-UTR sequence are sequentially linked from the 5' end mRNA was prepared using a kit (HiScribe T7 mRNA Kit, NEB). The amino acid translated by the apoptin-derived peptide coding sequence consists of a 42 amino acid sequence including NLS 1 (nuclear localization signal 1), NES (nuclear export signal), and NLS 2 (nuclear localization signal 2) ( Fig. 2).
실시예 2. 합성 mRNA의 암세포 사멸 효과 분석Example 2. Analysis of cancer cell killing effect of synthetic mRNA
본 발명에 따른 합성 mRNA의 암세포 사멸 효과를 분석하기 위하여 사람 유래 암세포인 Hep-2 세포(ATCC, CCL-23) 및 동물 유래 암세포인 DF-1 세포(ATCC, CRL-12203)에 대한 사멸 효과를 유세포분석기(Flow cytometry)를 이용하여 각각 측정하였다. In order to analyze the cancer cell killing effect of the synthetic mRNA according to the present invention, the killing effect on human-derived cancer cells, Hep-2 cells (ATCC, CCL-23) and animal-derived cancer cells, DF-1 cells (ATCC, CRL-12203) Each was measured using flow cytometry.
구체적으로, 세포배양용 6-웰 플레이트에 Hep-2 세포 또는 DF-1 세포를 1X106 cell의 농도로 분주하고 24시간 동안 배양하여 단층세포를 형성하게한 후, 상기 실시예 1의 합성 mRNA 100 ng을 각각 처리하고 6~7일 동안 배양하였다. 그 후, 처리된 세포를 0.25% 트립신-EDTA를 사용하여 소화한 뒤 세포를 회수하고 세포 사멸 검출 키트(Annexin V-FITC Apoptosis Detection Kit, AB)를 이용하여 키트 매뉴얼에 따라 세포를 염색한 뒤 유세포분석기(Beckman Coulter)를 이용하여 세포 사멸을 확인하였다. Specifically, Hep-2 cells or DF-1 cells were dispensed in a 6-well plate for cell culture at a concentration of 1X10 6 cells and cultured for 24 hours to form monolayer cells, and the synthetic mRNA 100 of Example 1 ng each and cultured for 6-7 days. Then, the treated cells were digested with 0.25% trypsin-EDTA, the cells were collected, and the cells were stained using a cell death detection kit (Annexin V-FITC Apoptosis Detection Kit, AB) according to the kit manual, followed by flow cytometry. Apoptosis was confirmed using an analyzer (Beckman Coulter).
그 결과, 사람 유래 암세포인 Hep-2 세포에 본 발명에 따른 합성 mRNA를 처리했을 때의 세포 사멸 비율은 47.01%로, 아무것도 처리하지 않은 대조군 대비 약 2배 이상 증가한 것을 확인하였고(도 3), 동물 유래 암세포인 DF-1 세포에 본 발명에 따른 합성 mRNA를 처리했을 때의 세포 사멸 비율은 67.27%로, 아무것도 처리하지 않은 대조군 대비 약 3배 이상 증가한 것을 확인하였다(도 4).As a result, when human-derived cancer cells Hep-2 cells were treated with the synthetic mRNA according to the present invention, the cell death rate was 47.01%, which was about twice as high as that of the untreated control group (FIG. 3). When DF-1 cells, which are animal-derived cancer cells, were treated with the synthetic mRNA according to the present invention, the cell death rate was 67.27%, which was about 3 times higher than that of the untreated control group (FIG. 4).
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Citations (3)
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EP0924296A2 (en) * | 1997-12-03 | 1999-06-23 | Leadd B.V. | Methods and means for inducing apoptosis by interference in RNA processing |
KR100561985B1 (en) * | 2004-07-03 | 2006-03-22 | 고려대학교 산학협력단 | Cancer-specific apoptin-expression cassette and adenovirus expression vector comprising the same |
KR20230043111A (en) * | 2020-06-24 | 2023-03-30 | 사프렘 테크놀로지스 비.브이. | A combination comprising an ADC or AOC comprising VHH and a saponin or ligand-saponin conjugate |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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EP0924296A2 (en) * | 1997-12-03 | 1999-06-23 | Leadd B.V. | Methods and means for inducing apoptosis by interference in RNA processing |
KR100561985B1 (en) * | 2004-07-03 | 2006-03-22 | 고려대학교 산학협력단 | Cancer-specific apoptin-expression cassette and adenovirus expression vector comprising the same |
KR20230043111A (en) * | 2020-06-24 | 2023-03-30 | 사프렘 테크놀로지스 비.브이. | A combination comprising an ADC or AOC comprising VHH and a saponin or ligand-saponin conjugate |
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