KR20230055388A - Complexes for nucleic acid delivery and uses thereof - Google Patents
Complexes for nucleic acid delivery and uses thereof Download PDFInfo
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- KR20230055388A KR20230055388A KR1020220134366A KR20220134366A KR20230055388A KR 20230055388 A KR20230055388 A KR 20230055388A KR 1020220134366 A KR1020220134366 A KR 1020220134366A KR 20220134366 A KR20220134366 A KR 20220134366A KR 20230055388 A KR20230055388 A KR 20230055388A
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- nucleic acid
- complex
- cell
- acid delivery
- mrna
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- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
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- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
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- A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
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Abstract
Description
본 발명은 핵산 전달용 복합체 및 이를 포함하는 핵산 전달에 관한 것이다.The present invention relates to a nucleic acid delivery complex and a nucleic acid delivery comprising the same.
마이크로베시클(microvesicles)은 일반적으로 0.03~1㎛의 크기를 가지는 세포 소기관의 일종으로, 거의 모든 종류의 세포에서 세포막으로부터 자연적으로 유리되어, 세포막의 구조인 이중 인지질(phospholipid) 막 형태를 가지고 있는 것을 특징으로 한다. 이와 같은 베시클은 물질 대사, 대사물질의 운송, 효소 저장, 화학 반응 등을 위한 세포의 기본 도구로서, 세포 간에 mRNA, miRNA 및 단백질 등을 전달함으로써 세포 간 신호전달의 매개 역할을 수행하는 것으로 알려져 있다.Microvesicles are a type of organelle with a size of 0.03 to 1 μm, which is naturally separated from the cell membrane in almost all types of cells and has a double phospholipid membrane structure, which is the structure of the cell membrane. characterized by Such vesicles are a basic tool of cells for metabolism, transportation of metabolites, storage of enzymes, chemical reactions, etc., and are known to mediate cell-to-cell signaling by transferring mRNA, miRNA, and proteins between cells. there is.
한편, 폴리에틸렌이민의 세포독성을 줄이기 위하여 텍스트란 설페이트(dextran sulfate), 인간 혈청 알부민(human serum albumin), 폴리에틸렌 글리콜(polyethylene glycol) 등으로 개질하는 방법이 많이 시도되었으나, 개질된 PEI는 공통적으로 PEI 자체 보다 낮은 유전자전달 효율을 보였다. PEI를 폴리에틸렌 글리콜로 개질하고 이를 유전자와 복합체를 형성시킨 상태로 생체 내에 투여한 경우, 복합체 표면의 폴리에틸렌글리콜 부분에 의한 "brush" layer에 의한 공간적 안정 효과로 복합체를 형성하여 투여된 유전자의 혈류 순환시간을 증가시켰으나, 이 역시 유전자 전달효율은 개질하지 않은 것 보다 매우 낮은 것을 관찰하였다 On the other hand, in order to reduce the cytotoxicity of polyethyleneimine, many attempts have been made to modify it with dextran sulfate, human serum albumin, polyethylene glycol, etc., but modified PEIs are commonly PEI showed a lower gene transfer efficiency than itself. When PEI is modified with polyethylene glycol and administered into a living body in a state in which it forms a complex with a gene, the complex is formed by the spatial stabilization effect of the "brush" layer by the polyethylene glycol part on the surface of the complex, and the administered gene circulates in the bloodstream Although the time was increased, it was also observed that the gene transfer efficiency was much lower than that without modification.
따라서, 세포 독성이 적으면서도 핵산을 효율적으로 세포내로 전달하는 기술은 당 분야에서 필요한 기술이다.Therefore, a technique for efficiently delivering nucleic acids into cells with less cytotoxicity is a technique required in the art.
이에 본 발명자들은 전술한 바와 같은 종래기술의 문제점을 해결하기 위하여 핵산 전달용 복합체를 개발함으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors have completed the present invention by developing a nucleic acid delivery complex in order to solve the problems of the prior art as described above.
따라서 본 발명의 목적은, 양이온성 폴리머, 핵산 및 세포 유래 베시클(cell-drived vesicle)을 함유하는 핵산 전달용 복합체를 제공하는 것이다.Accordingly, an object of the present invention is to provide a complex for nucleic acid delivery containing a cationic polymer, a nucleic acid, and a cell-driven vesicle.
본 발명의 다른 목적은, 양이온성 폴리머, 핵산 및 세포 유래 베시클을 함유하는 핵산 전달용 복합체를 포함하는 핵산 전달용 시약 조성물을 제공하는 것이다.Another object of the present invention is to provide a reagent composition for nucleic acid delivery comprising a complex for nucleic acid delivery containing a cationic polymer, a nucleic acid and a cell-derived vesicle.
본 발명의 또 다른 목적은, 양이온성 폴리머, 핵산 및 세포 유래 베시클을 함유하는 핵산 전달용 복합체를 포함하는, 약물 전달체를 제공하는 것이다.Another object of the present invention is to provide a drug delivery system comprising a nucleic acid delivery complex containing a cationic polymer, a nucleic acid and a cell-derived vesicle.
본 발명의 또 다른 목적은, 양이온성 폴리머, 핵산 및 세포 유래 베시클을 함유하는 핵산 전달용 복합체를 개체에 투여하는 단계;를 포함하는 핵산 전달 방법을 제공하는 것이다.Another object of the present invention is to provide a nucleic acid delivery method comprising administering to a subject a nucleic acid delivery complex containing a cationic polymer, a nucleic acid and a cell-derived vesicle.
상기 목적을 달성하기 위하여, 본 발명은 양이온성 폴리머, 핵산 및 세포 유래 베시클을 함유하는 핵산 전달용 복합체를 제공한다.In order to achieve the above object, the present invention provides a nucleic acid delivery complex containing a cationic polymer, a nucleic acid and a cell-derived vesicle.
또한 본 발명은 양이온성 폴리머, 핵산 및 세포 유래 베시클을 함유하는 핵산 전달용 복합체를 포함하는 핵산 전달용 시약 조성물을 제공한다.In addition, the present invention provides a reagent composition for nucleic acid delivery comprising a complex for nucleic acid delivery containing a cationic polymer, a nucleic acid and a cell-derived vesicle.
또한 본 발명은 양이온성 폴리머, 핵산 및 세포 유래 베시클을 함유하는 핵산 전달용 복합체를 포함하는 약물 전달체를 제공한다.In addition, the present invention provides a drug delivery system comprising a nucleic acid delivery complex containing a cationic polymer, a nucleic acid, and a cell-derived vesicle.
또한 본 발명은 양이온성 폴리머, 핵산 및 세포 유래 베시클을 함유하는 핵산 전달용 복합체를 개체에 투여하는 단계;를 포함하는 핵산 전달 방법을 제공한다.In addition, the present invention provides a nucleic acid delivery method comprising administering to a subject a nucleic acid delivery complex containing a cationic polymer, a nucleic acid and a cell-derived vesicle.
본 발명에 따른 핵산 전달용 복합체는 세포 유래 베시클을 사용하여 기존 PEI 기반의 복합체 대비 세포 독성을 나타내지 않으면서도, 핵산의 전달 효율 및 단백질 발현이 향상되는 것을 확인하였다. 따라서 본 발명의 핵산 전달용 복합체는 생체 적합하며 외부 물질에 의한 면역반응을 일으키지 않는 효율적인 약물 전달체로 활용될 수 있다.It was confirmed that the nucleic acid delivery complex according to the present invention uses cell-derived vesicles and improves nucleic acid delivery efficiency and protein expression without exhibiting cell toxicity compared to conventional PEI-based complexes. Therefore, the nucleic acid delivery complex of the present invention is biocompatible and can be used as an efficient drug delivery system that does not cause an immune response by external substances.
도 1은 bPEI/mRNA/CDV 복합체 및 lPEI/mRNA/CDV 복합체 제조를 위한 N/P ratio 계산식을 나타낸 도이다.
도 2는 jetPEI/mRNA/CDV 복합체 제조를 위한 N/P ratio 계산식을 나타낸 도이다.
도 3A는 형광 현미경 관찰을 통해 CDV에 의한 mRNA 전달 및 단백질 발현 향상 효과를 확인한 결과를 나타낸 도이다.
도 3B는 상기 도 3A의 형광 현미경 관찰 결과를 기반으로, 형광 세기를 정량한 결과를 나타낸 도이다.
도 4A는 형광 현미경 관찰을 통해 CDV 용량에 따른 bPEI/mRNA/CDV 복합체의 transfection 효율 평가한 결과를 나타낸 도이다.
도 4B는 상기 도 4A의 형광 세기를 정량한 결과; 및 제타 전위 측정 결과;를 나타낸 도이다.
도 5A는 CCK-8 assay를 통해 HEK293 세포에서 bPEI 및 bPEI/mRNA/CDV 복합체의 세포 독성을 확인한 결과를 나타낸 도이다.
도 5B는 N/P ratio 16 및 CDV 1x108 조건으로 제조된 bPEI/mRNA/CDV 처리군의 세포 독성을 확인한 결과를 나타낸 도이다.
도 6은 qPCR을 통해 bPEI/mRNA; 또는 bPEI/mRNA/CDV 1x108 complex; 처리에 따른 세포 내 mRNA level을 확인한 결과를 나타낸 도이다.
도 7은 유세포 분석을 통해 linear PEI(lPEI) 2.5kDa를 이용한 lPEI/mRNA/CDV; 및 Branched PEI 25kDa를 이용한 bPEI/mRNA/CDV;의 transfection efficiency를 확인한 결과를 나타낸 도이다.
도 8A는 jetPEI를 이용한 jetPEI/mRNA/CDV의 세포 독성을 확인한 결과를 나타낸 도이다.
도 8B는 jetPEI를 이용한 jetPEI/mRNA/CDV의 transfection efficiency를 확인한 결과를 나타낸 도이다.Figure 1 is a diagram showing the N / P ratio calculation formula for preparing bPEI / mRNA / CDV complex and lPEI / mRNA / CDV complex.
Figure 2 is a diagram showing the N / P ratio calculation formula for the preparation of jetPEI / mRNA / CDV complex.
Figure 3A is a diagram showing the results of confirming the effect of mRNA delivery and protein expression enhancement by CDV through fluorescence microscopic observation.
Figure 3B is a diagram showing the result of quantifying the fluorescence intensity based on the fluorescence microscope observation result of Figure 3A.
Figure 4A is a diagram showing the results of evaluating the transfection efficiency of the bPEI/mRNA/CDV complex according to the CDV dose through fluorescence microscopy.
Figure 4B is the result of quantifying the fluorescence intensity of Figure 4A; And zeta potential measurement results; is a diagram showing.
5A is a diagram showing the result of confirming the cytotoxicity of bPEI and bPEI/mRNA/CDV complex in HEK293 cells through CCK-8 assay.
5B is a diagram showing the result of confirming the cytotoxicity of the bPEI/mRNA/CDV-treated group prepared under N/
Figure 6 bPEI / mRNA through qPCR; or bPEI/mRNA/CDV 1x10 8 complex; It is a diagram showing the result of confirming the intracellular mRNA level according to the treatment.
Figure 7 shows lPEI/mRNA/CDV using linear PEI (lPEI) 2.5 kDa through flow cytometry; and bPEI/mRNA/CDV using Branched PEI 25kDa;
8A is a diagram showing the result of confirming the cytotoxicity of jetPEI/mRNA/CDV using jetPEI.
8B is a diagram showing the results of confirming the transfection efficiency of jetPEI/mRNA/CDV using jetPEI.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 양태에 따르면, 본 발명은 양이온성 폴리머, 핵산 및 세포 유래 베시클(cell-drived vesicle)을 함유하는 핵산 전달용 복합체를 제공한다.According to an aspect of the present invention, the present invention provides a complex for nucleic acid delivery comprising a cationic polymer, a nucleic acid and a cell-driven vesicle.
본 발명의 구체예에서, 상기 양이온성 폴리머는 폴리에틸렌이민(PEI), 폴리프로필렌이민(PPI), 펜타에틸렌헥사민, N,N'-비스(2-아미노에틸)-1,3-프로판디아민, N-(2-아미노에틸)-1,3-프로판디아민, N-(2-아미노프로필)-1,3-프로판디아민, 스퍼민(spermine), 스퍼미딘(spermidine), 1,4-비스(3-아미노프로필)피페라진, 1-(2-아미노에틸)피페라진, 트리(2-아미노에틸)아민, 폴리아미도아민(PAMAM), 폴리(L-라이신)(PLL) 및 키토산으로 이루어진 군에서 선택된 1종 이상일 수 있다.In an embodiment of the present invention, the cationic polymer is polyethyleneimine (PEI), polypropyleneimine (PPI), pentaethylenehexamine, N, N'-bis (2-aminoethyl) -1,3-propanediamine, N-(2-aminoethyl)-1,3-propanediamine, N-(2-aminopropyl)-1,3-propanediamine, spermine, spermidine, 1,4-bis(3 -aminopropyl)piperazine, 1-(2-aminoethyl)piperazine, tri(2-aminoethyl)amine, polyamidoamine (PAMAM), poly(L-lysine) (PLL) selected from the group consisting of chitosan It may be one or more.
본 발명의 구체예에서, 상기 양이온성 폴리머는 branched-폴리에틸렌이민(bPEI), linear-폴리에틸렌이민(lPEI) 또는 jet-폴리에틸렌이민(jetPEI)일 수 있으나, 이에 본 발명의 범위가 제한되지 않는다.In an embodiment of the present invention, the cationic polymer may be branched-polyethyleneimine (bPEI), linear-polyethyleneimine (lPEI) or jet-polyethyleneimine (jetPEI), but the scope of the present invention is not limited thereto.
본 발명의 구체예에서, 상기 핵산은 DNA, RNA, 또는 백본(backbone), 당 또는 염기가 화학적으로 변형되거나 말단이 수식된 폴리뉴클레오타이드 유도체 등의 모든 종류의 단일가닥 또는 이중가닥 형태의 핵산일 수 있다. 구체적으로, 상기 핵산은 플라스미드 유전자, 안티센스 올리고뉴클레오티드, siRNA, rRNA, RNA, DNA, cDNA, mRNA, shRNA, lncRNA, miRNA 및 tRNA로 이루어진 군에서 선택되는 1종 이상일 수 있다. In an embodiment of the present invention, the nucleic acid may be any kind of single-stranded or double-stranded nucleic acid, such as DNA, RNA, or polynucleotide derivatives in which backbones, sugars or bases are chemically modified or ends are modified. there is. Specifically, the nucleic acid may be one or more selected from the group consisting of plasmid gene, antisense oligonucleotide, siRNA, rRNA, RNA, DNA, cDNA, mRNA, shRNA, lncRNA, miRNA, and tRNA.
본 발명에 있어서, 세포 유래 베시클은 세포로부터 발생한 베시클을 말하며, 일반적으로 세포 소기관의 일종으로, 거의 모든 종류의 세포에서 세포막으로부터 유리되어, 세포막의 구조인 이중 인지질(phospholipid) 막 형태를 가지고 있는 것을 말한다. In the present invention, the cell-derived vesicle refers to a vesicle generated from a cell, and is generally a kind of organelle. It is separated from the cell membrane in almost all types of cells and has a double phospholipid membrane structure, which is the structure of the cell membrane. say what is
본 발명의 세포 유래 베시클은 마이크로미터 크기, 예컨대 0.03~1㎛를 가질 수 있다. 본 발명의 세포 유래 베시클은 유래한 세포의 세포막 성분으로 이루어진 지질 이중막에 의해 내부와 외부가 구분되며, 세포의 세포막 지질과 세포막 단백질, 핵산 및 세포 성분 등을 가지고 있으며, 원래 세포보다 크기가 작은 것을 의미하지만, 이에 제한되는 것은 아니다.The cell-derived vesicles of the present invention may have a micrometer size, such as 0.03-1 μm. The cell-derived vesicle of the present invention is divided into inside and outside by a lipid bilayer composed of cell membrane components of the derived cell, has cell membrane lipids, cell membrane proteins, nucleic acids, and cell components, and is larger than the original cell. It means small, but is not limited thereto.
본 발명의 베시클은 세포를 포함하는 현탁액을 압출, 초음파 분해, 세포 용해, 균질화, 냉동-해동, 전기천공, 화학 물질 처리, 기계적 분해 및 외부적으로 세포에 힘을 가한 물리적 자극의 처리로 이루어진 군으로부터 선택된 방법을 사용하여 제조할 수 있으나, 이에 제한되는 것은 아니다. 본원 발명에서는 세포를 포함하는 현탁액에 압력을 가해, 공극의 크기가 큰 필터로부터 공극의 크기가 작은 필터로 순차적으로 이행하여 압출하는, 미세공극으로 압출하는 방식의 세포압출기를 사용하여 본 발명의 세포 유래 베시클을 제조하였다. The vesicle of the present invention consists of extruding a suspension containing cells, sonication, cell lysis, homogenization, freezing-thawing, electroporation, chemical treatment, mechanical degradation, and treatment of a physical stimulus applied externally to the cells. It can be prepared using a method selected from the group, but is not limited thereto. In the present invention, by applying pressure to a suspension containing cells, the cell extruder of the method of extruding into micropores is used to sequentially transfer from a filter having large pores to a filter having small pores and extruding the cells of the present invention. Derived vesicles were prepared.
본 발명의 구체예에서, 상기 세포 유래 베시클은 세포를 압출하여 제조된 것으로, 세포에서 인위적으로 제조된 베시클이다. 또한 상기 세포 유래 베시클은 세포막의 구조인 이중 인지질(phospholipid) 막의 형태를 가지는 것을 특징으로 한다. In an embodiment of the present invention, the cell-derived vesicles are produced by extruding cells, and are artificially prepared vesicles from cells. In addition, the cell-derived vesicle is characterized in that it has the form of a double phospholipid membrane, which is a structure of a cell membrane.
본 발명의 구체예에서, 상기 세포는 세포 유래 베시클의 분리가 가능한 세포라면 제한없이 사용될 수 있으며, 자연계 생물 개체로부터 분리된 세포일 수 있다. 또한, 상기 세포는 원핵세포(prokaryote) 또는 진핵세포(eukaryote)일 수 있다.In an embodiment of the present invention, the cell may be used without limitation as long as it is capable of separating cell-derived vesicles, and may be a cell isolated from a natural biological entity. In addition, the cell may be a prokaryotic cell (prokaryote) or eukaryote (eukaryote).
본 발명의 바람직한 구체예에서, 상기 원핵세포 또는 진핵세포는 적혈구, 단핵구, 대식세포, 수지상세포, T 세포, B 세포, 자연살해세포(natural killer cell, NK cell) 및 줄기세포로 이루어진 군에서 선택된 1종 이상일 수 있다. 상기 줄기세포는 중간엽 줄기세포, 유도만능줄기세포, 배아줄기세포 및 침샘 줄기세포로 이루어진 군에서 선택된 어느 하나 이상일 수 있다. 또한 상기 원핵세포 또는 진핵세포는 선포세포, 근상피세포 및 혈소판으로 이루어진 군에서 선택된 어느 하나 이상일 수 있다.In a preferred embodiment of the present invention, the prokaryotic or eukaryotic cell is selected from the group consisting of red blood cells, monocytes, macrophages, dendritic cells, T cells, B cells, natural killer cells (NK cells) and stem cells. It may be one or more. The stem cells may be any one or more selected from the group consisting of mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, and salivary gland stem cells. In addition, the prokaryotic or eukaryotic cells may be any one or more selected from the group consisting of acinar cells, myoepithelial cells, and platelets.
본 발명의 바람직한 구체예에서, 상기 원핵세포 또는 진핵세포는 HEK293 세포일 수 있고, 바람직하게는 Human embryonic kidney에서 유래한 세포주인 HEK293 부유세포일 수 있다.In a preferred embodiment of the present invention, the prokaryotic or eukaryotic cells may be HEK293 cells, preferably HEK293 floating cells, which are a cell line derived from human embryonic kidney.
본 발명의 구체예에서, 상기 원핵세포 또는 진핵세포는 포유류의 세포일 수 있다.In an embodiment of the present invention, the prokaryotic or eukaryotic cell may be a mammalian cell.
본 발명의 바람직한 구체예에서, 상기 원핵세포 또는 진핵세포는 원생생물(protista), 진균(fungi), 세균(bacteria), 고균(archaea) 및 바이러스(virus)로 이루어진 군에서 선택된 1종 이상의 미생물인 것이 바람직하다.In a preferred embodiment of the present invention, the prokaryotic or eukaryotic cell is one or more microorganisms selected from the group consisting of protista, fungi, bacteria, archaea and viruses. it is desirable
본 발명에 있어서, 원생생물은 단세포성 진핵 생물로, 핵을 가지고 있는 생물 중 가장 단순한 종류로 핵과 세포질 그리고 원형질막이 기본 구조이나 세포벽을 가지고 있는 것도 있다. 운동성을 지난 원생 생물로는 편모충, 아메바, 짚신벌레 등이 있고, 광합성을 하는 조류성 원생 생물은 식물성 플랑크톤이나 유글레나, 그리고 다세포로 이루어진 녹조류, 홍조류, 갈조류 등이 있다.In the present invention, protists are unicellular eukaryotic organisms, which are the simplest kind among organisms having a nucleus, and may have a basic structure or cell wall with a nucleus, cytoplasm, and plasma membrane. Motile protists include flagellates, amoebas, parameciums, etc., and photosynthetic algae protists include phytoplankton, euglena, and multicellular green algae, red algae, and brown algae.
본 발명에 있어서, 진균은 곰팡이, 효모, 버섯을 포함한 72,000종 이상의 균종으로 구성하는 미생물군이다. 핵막이 있는 진핵생물에 속하며, 미토콘드리아, 소포체 등의 세포소기관이 발달하고, 키틴, 글루칸 등으로 구성된 세포벽이 있다. 대부분은 세포성인 균사를 형성하여 신장, 발육하고 유성생식 및 무성생식을 하고 번식체로서 포자형성을 하지만, 일부 균종(효모)은 단세포성 증식을 한다. 주로 부생균으로서 자연계의 유기분해에 관여하지만, 일부는 동식물에 기생 또는 공생한다.In the present invention, fungi are a group of microorganisms composed of more than 72,000 species including molds, yeasts, and mushrooms. It belongs to eukaryotic organisms with a nuclear membrane, has organelles such as mitochondria and endoplasmic reticulum, and has a cell wall composed of chitin and glucan. Most of them form cellular hyphae, elongate, develop, reproduce sexually and asexually, and sporulate as propagules, but some fungal species (yeast) grow unicellularly. It is mainly involved in organic decomposition in nature as a by-product, but some parasitic or symbiotic with animals and plants.
본 발명에 있어서, 세균은 단세포로 이루어져서 활동하는 미생물을 총칭한다. 세균의 크기는 0.5μm부터 0.5mm까지 다양하며, 식물세포나 곰팡이 세포와 마찬가지로 세포벽을 가지고 있으나, 셀룰로오즈(cellulose)가 아닌 펩티도글리칸(peptidoglycan)이 주 성분인 점이 다르다.In the present invention, bacteria collectively refer to microorganisms that are active and composed of single cells. Bacteria vary in size from 0.5 μm to 0.5 mm, and have cell walls like plant cells or fungal cells, but differ in that peptidoglycan, not cellulose, is the main component.
본 발명에 있어서, 고균은 핵이 없는 원핵생물이며, 세포 구조 및 에너지 대사의 측면에서는 같은 원핵생물인 세균과 많은 특징을 공유한다. 그러나 인트론, 히스톤단백질, 전사, 단백질 합성 등 유전적인 측면에서는 진핵생물과 많은 특징을 공유하는 반면 다른 분류군에서는 관찰되지 않는 특징도 가지고 있다.In the present invention, Archaea is a prokaryote without a nucleus, and shares many characteristics with the same prokaryotic bacteria in terms of cell structure and energy metabolism. However, while sharing many characteristics with eukaryotes in terms of genetics such as introns, histone proteins, transcription, and protein synthesis, they also have characteristics not observed in other taxa.
본 발명에 있어서, 바이러스는 DNA나 RNA를 유전체(genome)로 가지고 있으며, 단백질로 둘러 싸여 있는 구조를 가지고 있다. 이에, 바이러스는 비세포성 미생물로 분류되며, 이 때 원핵세포로 분류된다.In the present invention, the virus has DNA or RNA as its genome and has a structure surrounded by proteins. Accordingly, viruses are classified as non-cellular microorganisms, and in this case, as prokaryotic cells.
본 발명의 구체예에서, 상기 양이온성 폴리머 및 핵산의 질소/인 비율(N/P ratio)은 1 내지 64인 것이 바람직하고, 더 바람직하게는 8 내지 32일 수 있다.In an embodiment of the present invention, the nitrogen / phosphorus ratio (N / P ratio) of the cationic polymer and nucleic acid is preferably 1 to 64, and may be more preferably 8 to 32.
본 발명의 구체예에서, 상기 핵산 및 세포 유래 베시클의 비율(mRNA copies/CDV particle)은 70 내지 40000일 수 있고, 바람직하게는 300 내지 4000일 수 있다. In an embodiment of the present invention, the ratio of the nucleic acid to the cell-derived vesicle (mRNA copies/CDV particle) may be 70 to 40000, preferably 300 to 4000.
본 발명의 실시예에서, 양이온성 폴리머를 이용하여 세포 유래 베시클에 핵산를 결합시켰을 때 세포 유래 베시클에 의해 핵산 전달 및 단백질 발현이 향상되는 효과를 확인하였다. 나아가, Branched PEI(25 kDa), Linear PEI(2.5 kDa) 및 jetPEI를 사용하였을 때 모두 CDV에 의한 효과가 관찰되어 이러한 방법이 다양한 PEI에 적용됨을 확인하였다. 특히 jetPEI를 이용한 복합체에서는 positive control인 lipofectamine 대비 독성 감소 효과와 lipofectamine과 비슷한 수준의 단백질 발현을 보였다. 상기 결과들은 polyplex와 CDV 복합체라는 효과적인 mRNA 전달이 가능한 새로운 형태의 약물 전달체로서의 가능성을 보여준다.In an embodiment of the present invention, when nucleic acids were bound to cell-derived vesicles using a cationic polymer, the effect of improving nucleic acid delivery and protein expression by the cell-derived vesicles was confirmed. Furthermore, when branched PEI (25 kDa), linear PEI (2.5 kDa), and jetPEI were used, CDV-induced effects were observed, confirming that this method was applicable to various PEIs. In particular, the complex using jetPEI showed a toxicity reduction effect and protein expression similar to that of lipofectamine, which is a positive control. The above results show the potential of polyplex and CDV complex as a new type of drug delivery system capable of effective mRNA delivery.
본 발명의 다른 양태에 따르면, 본 발명은 양이온성 폴리머, 핵산 및 세포 유래 베시클을 함유하는 핵산 전달용 복합체를 포함하는 핵산 전달용 시약 조성물을 제공한다. 또한 본 발명은 양이온성 폴리머, 핵산 및 세포 유래 베시클을 함유하는 핵산 전달용 복합체를 포함하는 약물 전달체를 제공한다.According to another aspect of the present invention, the present invention provides a reagent composition for nucleic acid delivery comprising a complex for nucleic acid delivery containing a cationic polymer, a nucleic acid and a cell-derived vesicle. In addition, the present invention provides a drug delivery system comprising a nucleic acid delivery complex containing a cationic polymer, a nucleic acid, and a cell-derived vesicle.
본 발명에 따른 핵산 전달용 시약 조성물 및 약물 전달체는, 본 발명의 핵산 전달용 복합체 외에 생리활성물질을 더 포함할 수 있다. 상기 용어, "생리활성물질"은 생체 기능을 증진시키거나 억제시키는 물질로서, 생체의 기능을 조절하는 물질이 결핍되거나 과도하게 분비되어 비정상적인 병태를 보일 때 이의 균형을 유지시켜줄 수 있는 물질을 의미한다. 상기 생리활성물질은 핵산 물질일 수 있고, 상기 핵산 물질은 통상의 기술 분야에 알려진 모든 것을 포함할 수 있으며, 경우에 따라 필요한 양으로 사용될 수 있다.The reagent composition and drug delivery system for nucleic acid delivery according to the present invention may further include a physiologically active substance in addition to the complex for nucleic acid delivery of the present invention. As used herein, "physiologically active substance" refers to a substance that enhances or inhibits a biological function, and refers to a substance that can maintain the balance when a substance that regulates a biological function is deficient or excessively secreted to show an abnormal condition. . The physiologically active material may be a nucleic acid material, and the nucleic acid material may include any material known in the art, and may be used in a necessary amount according to circumstances.
본 발명의 또 다른 양태에 따르면, 본 발명은 양이온성 폴리머, 핵산 및 세포 유래 베시클을 함유하는 핵산 전달용 복합체를 개체에 투여하는 단계;를 포함하는 핵산 전달 방법을 제공한다.According to another aspect of the present invention, the present invention provides a nucleic acid delivery method comprising administering to a subject a nucleic acid delivery complex containing a cationic polymer, a nucleic acid and a cell-derived vesicle.
본 발명의 구체예에서, 상기 개체는 핵산을 전달하고자 하는 대상을 의미하며, 보다 상세하게는 세포, 동물 등일 수 있으나, 이에 본 발명이 제한되지 않는다.In an embodiment of the present invention, the subject refers to a target to which nucleic acid is to be delivered, and more specifically, may be a cell, animal, etc., but the present invention is not limited thereto.
중복되는 내용은 본 명세서의 복잡성을 고려하여 생략하며, 본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 기술분야에서 통상적으로 사용되는 의미를 갖는 것이다.Redundant content is omitted in consideration of the complexity of the present specification, and terms not otherwise defined in the present specification have meanings commonly used in the technical field to which the present invention belongs.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 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 illustrating 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.
[실험예][Experimental example]
실험예 1. HEK293 세포 유래 베지클(HEK-CDV) 생산 및 정제Experimental Example 1. Production and purification of HEK293 cell-derived vesicles (HEK-CDV)
Human embryonic kidney에서 유래한 세포주인 HEK293 부유세포는 4 mM Glutamax(Gibco, #35050061)와 0.2% Pluronic F68(Gibco, #24040032)을 포함하는 Freestyle F17(Gibco, #11995065) 배지로 조건(37℃, DO = 50%, pH=7.3±0.1, PV=15.2)에서 STR200 Bioreactor를 이용해 배양하였다. 최종 세포배양액은 VCD = 6.0x106 cells/mL, Viability> 90%에서 수확하였다.HEK293 floating cells, a cell line derived from human embryonic kidney, were conditioned (37°C, DO = 50%, pH = 7.3 ± 0.1, PV = 15.2) using STR200 Bioreactor. The final cell culture was harvested at VCD = 6.0x10 6 cells/mL, Viability> 90%.
배양한 세포의 압출을 위하여 필터 어셈블리에 총 4개의 카트리지 깊이 필터(Sartopure PP3 Cartridge, 총 막면적: 1.8 m2, retentionrates:0.65㎛)를 장착하였다. 구체적으로, 2개의 필터 어셈블리에 구비되는 각 하단부에 카트리지 깊이 필터(cartridges depth filter)를 2개씩 장착하고, 유휴 장착홀은 캡으로 폐쇄하였다. 이후 각 필터 어셈블리에는 질소(N2)가스 공급을 위한 가스 공급라인을 연결하였다. HEK293 세포 배양액 농도 6.0x106 cells/mL 12.5 L를 2개의 필터 어셈블리에 절반씩 투입하였다. 가스 공급라인의 양쪽 밸브를 열고 0.4 bar로 가압하여 사용된 배지를 수확라인으로 배출하였다. 연동 펌프를 이용해 37℃ PBS 16 L을 수확라인으로 백-플러싱 하였다. 압출을 위하여 가스 공급라인의 한쪽 밸브는 잠그고 다른 한쪽은 열어 한방향으로 재부유된 세포 용액이 필터 어셈블리 내부의 카트리지 필터를 통과하도록 하였다. 좌우 밸브를 반대로 열고 닫기를 반복하여 총 70번 통과하였다. 필터 어셈블리에 공급되는 질소가스 압력은 첫 번째는 1.0 bar, 그리고 2~70번째는 2.5 bar로 수행되었다. For extrusion of the cultured cells, a total of 4 cartridge depth filters (Sartopure PP3 Cartridge, total membrane area: 1.8 m 2 , retention rates: 0.65 μm) were mounted on the filter assembly. Specifically, two cartridge depth filters were mounted on each lower end of the two filter assemblies, and the idle mounting hole was closed with a cap. Thereafter, a gas supply line for supplying nitrogen (N 2 ) gas was connected to each filter assembly. 12.5 L of HEK293 cell culture medium at a concentration of 6.0x10 6 cells/mL was added to each of the two filter assemblies. Both valves of the gas supply line were opened and pressurized at 0.4 bar to discharge the used medium to the harvest line. 16 L of 37 °C PBS was back-flushed into the harvest line using a peristaltic pump. For extrusion, one valve of the gas supply line was closed and the other valve was opened so that the cell solution resuspended in one direction passed through the cartridge filter inside the filter assembly. Opening and closing the left and right valves in opposite directions was repeated a total of 70 times. The nitrogen gas pressure supplied to the filter assembly was 1.0 bar for the first and 2.5 bar for the 2nd to 70th.
압출 용액 내 존재하는 핵산 및 단백질과 같은 불순물을 제거하기 위하여, 십자류 여과 tangential flow filtration(TFF, Repligen CA, US) 장치를 이용하여 정제하였다. 정제를 위하여 750 kDa의 molecular weight cut off(MWCO)를 갖는 직경 0.5 mm, 길이 20 cm의 modified polyethersulfone(mPES) 재질의 hollow fiber membrane(Repligen, D02-E750-05-N)을 사용하였다. TFF 정제된 CDV를 사이즈 배제 크로마토그래피인 qEV10(IZON, #SP3)을 이용하여 제조사의 프로토콜에 따라 추가 정제하였고, 고순도의 균일한 나노 사이즈의 HEK293 세포 유래 베지클(HEK-CDV)을 수득하였다. In order to remove impurities such as nucleic acids and proteins present in the extrusion solution, purification was performed using a tangential flow filtration (TFF, Repligen CA, US) device. For purification, a hollow fiber membrane (Repligen, D02-E750-05-N) made of modified polyethersulfone (mPES) with a diameter of 0.5 mm and a length of 20 cm with a molecular weight cut off (MWCO) of 750 kDa was used. The TFF-purified CDV was further purified according to the manufacturer's protocol using size exclusion chromatography, qEV10 (IZON, #SP3), and high-purity, uniform nano-sized HEK293 cell-derived vesicles (HEK-CDV) were obtained.
실험예 2. bPEI/mRNA/CDV 복합체 및 lPEI/mRNA/CDV 복합체 제조 Experimental Example 2. Preparation of bPEI/mRNA/CDV complex and lPEI/mRNA/CDV complex
EGFP mRNA(TriLink, L-7601)와 다양한 N/P ratio에 따라 필요한 PEI(branched PEI(bPEI) 25 kDa 또는 linear PEI(lPEI) 2.5kDa을 각각 최종 볼륨 10μL가 되도록 PBS에 희석하였다. N/P ratio는 도 1과 같이 계산하였다. mRNA 용액과 PEI 용액(bPEI 또는 lPEI)을 1:1 부피비로 파이펫팅하여 섞은 후 상온에서 20분 동안 반응시켰다. 반응 후 20 μL의 HEK-CDV를 PEI/mRNA 복합체에 첨가하고 파이펫팅하였다. 대조군인 PEI/mRNA에서는 HEK-CDV 대신 20 μL의 PBS를 혼합하였다. 그 후 혼합 용액을 4℃에서 10분 동안 반응시켜, bPEI/mRNA/CDV 또는 lPEI/mRNA/CDV를 얻었다.EGFP mRNA (TriLink, L-7601) and PEI (branched PEI (bPEI) 25 kDa or linear PEI (lPEI) 2.5 kDa required according to various N/P ratios were diluted in PBS to a final volume of 10 μL, respectively. N/P The ratio was calculated as shown in Figure 1. The mRNA solution and the PEI solution (bPEI or lPEI) were mixed by pipetting at a volume ratio of 1: 1, and then reacted at room temperature for 20 minutes. The mixture was added to the complex and pipetted. For control PEI/mRNA, instead of HEK-CDV, 20 μL of PBS was mixed After that, the mixed solution was reacted at 4° C. for 10 minutes and bPEI/mRNA/CDV or lPEI/mRNA/ got CDV.
실험예 3. jetPEI/mRNA/CDV 복합체 제조Experimental Example 3. Preparation of jetPEI/mRNA/CDV Complex
Renilla luciferase mRNA(TriLink, L-7204) 0.2 μg과 N/P ratio에 따라 필요한 jetPEI를 각각 최종 부피 10 μL가 되도록 증류수에 희석하였다. N/P ratio는 도 2의 계산식으로 계산하였다. 희석된 jetPEI 용액을 희석된 mRNA 용액에 첨가하여 빠르게 40-50회 빠르게 파이펫팅하여 혼합하였다. 그 후, 혼합 용액을 실온에서 20분 동안 반응시켜 jetPEI/mRNA 복합체 형성을 유도하였다. 5 μL의 PBS 또는 HEK-CDV를 jetPEI/mRNA 복합체에 첨가하고 40~50회 위아래로 빠르게 파이펫팅하여 혼합하였다. 대조군인 jetPEI/mRNA에서는 HEK-CDV 대신 5 μL의 PBS를 혼합하였다. 그 후 혼합 용액을 4℃에서 10분 동안 반응시켜, jetPEI/mRNA/CDV를 수득하였다.Renilla luciferase mRNA (TriLink, L-7204) 0.2 μg and jetPEI required according to the N/P ratio were each diluted in distilled water to a final volume of 10 μL. The N/P ratio was calculated using the formula shown in FIG. 2. The diluted jetPEI solution was added to the diluted mRNA solution and mixed by pipetting rapidly 40-50 times. Then, the mixed solution was reacted at room temperature for 20 minutes to induce the formation of jetPEI/mRNA complex. 5 μL of PBS or HEK-CDV was added to the jetPEI/mRNA complex and mixed by rapidly pipetting up and down 40-50 times. In the control, jetPEI/mRNA, 5 μL of PBS was mixed instead of HEK-CDV. Thereafter, the mixed solution was reacted at 4° C. for 10 minutes to obtain jetPEI/mRNA/CDV.
상기 실험예 2 및 3에 사용한 bPEI, lPEI 및 jetPEI의 정보는 표 1과 같다.Information on bPEI, lPEI and jetPEI used in Experimental Examples 2 and 3 is shown in Table 1.
실험예 4. PEI/mRNA/CDV 복합체의 유전자 발현 및 세포 독성 평가 Experimental Example 4. Evaluation of gene expression and cytotoxicity of the PEI/mRNA/CDV complex
4-1. PEI/mRNA/CDV 복합체의 유전자 발현4-1. Gene expression of the PEI/mRNA/CDV complex
HEK293 세포(ATCC)를 96 well plate에 2 x 104 cells/well씩 시딩한 후 10% FBS 및 1% Penicillin/Streptomycin을 첨가한 α-MEM(Gibco, 12571063) 배지를 사용하여 37°C, 5% CO2 조건에서 배양하였다. 17시간 후 mRNA 형질감염시키기 위하여, 배지를 제거하였다. total volume이 100μl가 되도록 상기와 동일한 세포 배양 배지와 상기 실험예 2 및 3의 PEI/mRNA/CDV 복합체를 처리하였다(mRNA 0.2μg 기준). 24 시간 후 GFP 단백질 발현 정도를 형광 현미경(Nikon, ECLIPSE Ti2) 및 FACS(Sony, 3800)를 사용하여 분석하였다.After seeding HEK293 cells (ATCC) in a 96-well plate at 2 x 10 4 cells/well, they were cultured in α-MEM (Gibco, 12571063) medium supplemented with 10% FBS and 1% Penicillin/Streptomycin at 37°C and 5 It was cultured under % CO 2 conditions. For mRNA transfection after 17 hours, the medium was removed. The same cell culture medium as above and the PEI/mRNA/CDV complex of Experimental Examples 2 and 3 were treated so that the total volume was 100 μl (based on 0.2 μg of mRNA). After 24 hours, the level of GFP protein expression was analyzed using a fluorescence microscope (Nikon, ECLIPSE Ti2) and FACS (Sony, 3800).
luciferase mRNA의 경우 manufacturer instruction에 따라 Renilla luciferase assay를 진행하여 단백질 발현 정도를 평가하였으며, 발광의 세기는 luminometer(Molecular device, SpectraMax L)를 이용하여 정량하였다.In the case of luciferase mRNA, Renilla luciferase assay was performed according to the manufacturer's instructions to evaluate the level of protein expression, and the intensity of luminescence was quantified using a luminometer (Molecular device, SpectraMax L).
4-2. 세포 독성 평가4-2. Cytotoxicity assessment
실험예 4-1과 동일한 방법으로, HEK293 세포에 PEI/mRNA/CDV 복합체를 처리한 후 24시간 뒤에 새로운 배지 100 μL(CCK-8 용액(Dojindo) 10 μL를 포함)로 교체하였다. 37°C에서 2시간 동안 차광 상태로 배양한 뒤 microplate reader(Molecular device, M2e)를 이용하여 450 nm에서 흡광도를 측정하였다. 세포 독성은 시료를 처리하지 않은 그룹의 흡광도를 기준으로 시료를 처리한 그룹의 흡광도의 비율을 계산하여 평가하였다.In the same manner as in Experimental Example 4-1, HEK293 cells were treated with the PEI/mRNA/CDV complex, and then replaced with 100 μL of a new medium (including 10 μL of CCK-8 solution (Dojindo)) 24 hours later. After incubation at 37°C for 2 hours in a shaded state, absorbance was measured at 450 nm using a microplate reader (Molecular device, M2e). Cytotoxicity was evaluated by calculating the ratio of the absorbance of the group treated with the sample to the absorbance of the group not treated with the sample.
Positive control로 Lipofectamine MessengerMAX™ Transfection Reagent을 사용하였으며 manufacturer instruction에 따라 mRNA(μg) : lipofectamine(μL)=1:3 비율로 처리하였다.Lipofectamine MessengerMAX™ Transfection Reagent was used as a positive control, and mRNA (μg): lipofectamine (μL) = 1:3 ratio was treated according to the manufacturer's instructions.
실험예 5. CDV의 입도 분석Experimental Example 5. Particle size analysis of CDV
PEI/mRNA 복합체가 함유된 CDV의 size 및 zeta-potential은 Dynamic Light Scattering(DLS) 입도분석기(Zetasizer Nano, Malvern)를 이용하여 측정하였다.The size and zeta-potential of CDV containing the PEI/mRNA complex were measured using a Dynamic Light Scattering (DLS) particle size analyzer (Zetasizer Nano, Malvern).
실험예 6. 세포 내 mRNA uptake level 확인Experimental Example 6. Intracellular mRNA uptake level confirmation
세포에 uptake된 mRNA level을 측정하기 위해서 HEK293 세포(ATCC)를 48 well plate에 8 x 104 cells/well씩 시딩한 후 10% FBS 및 1% Penicillin/Streptomycin을 첨가한 α-MEM(Gibco, 12571063) 배지를 사용하여 37°C, 5% CO2 조건에서 배양하였다. 17시간 후 배지를 제거한 후, 상기와 동일한 세포 배양 배지; 및 N/P ratio 10 조건에서 EGFP mRNA 0.25μg를 포함하는 bPEI/mRNA 및 bPEI/mRNA/CDV 1x108 복합체;를 total volume이 200μl가 되도록 처리하였다. 시간별로 배양 후 miRNeasy kit(Qiagen)를 이용하여 세포의 RNA를 추출하였다. 추출한 RNA는 nano-drop으로 정량한 후 동량의 RNA로부터 cDNA를 합성하였다. 합성된 cDNA와 EGFP, GAPDH primer를 이용하여 qPCR을 수행하였다. qPCR로 얻은 Ct 값을 이용하여 세포 내 EGFP mRNA uptake level을 상대정량으로 분석하였다.In order to measure the mRNA level uptaken to the cells, HEK293 cells (ATCC) were seeded in a 48 well plate at 8 x 10 4 cells/well, and then α-MEM (Gibco, 12571063) supplemented with 10% FBS and 1% Penicillin/Streptomycin. ) culture medium at 37°C and 5% CO 2 conditions. After removing the medium after 17 hours, the same cell culture medium as above; and bPEI/mRNA and bPEI/mRNA/CDV 1x10 8 complexes containing 0.25 μg of EGFP mRNA under N/
[실시예][Example]
실시예 1. CDV에 의한 단백질 발현 향상 효과 확인Example 1. Confirmation of protein expression enhancement effect by CDV
CDV에 의한 mRNA 전달 및 단백질 발현 향상 효과를 확인하기 위하여, 다양한 N/P ratio 및 CDV particle를 갖는 bPEI/mRNA/CDV 복합체를 HEK293 세포에 처리하여 transfection 실험을 수행하였다. 모든 복합체는 EGFP mRNA 0.2 μg를 포함한다. 24시간 동안 배양한 후 형광 현미경과 FACS를 통해 세포의 EGFP 발현을 관찰하고 형광의 세기를 측정하였다. 형광 현미경으로 EGFP 발현을 관찰한 결과는 도 3A에 나타내었고, 형광 세기를 정량하여 도 3B에 나타내었다.In order to confirm the mRNA delivery and protein expression enhancement effects by CDV, transfection experiments were performed by treating HEK293 cells with bPEI/mRNA/CDV complexes having various N/P ratios and CDV particles. All complexes contain 0.2 μg of EGFP mRNA. After culturing for 24 hours, EGFP expression of the cells was observed using a fluorescence microscope and FACS, and fluorescence intensity was measured. The result of observing EGFP expression under a fluorescence microscope is shown in FIG. 3A, and the fluorescence intensity is quantified and shown in FIG. 3B.
도 3A 및 B에 나타낸 바와 같이, N/P ratio 16과 32에서 CDV에 의한 mRNA 전달 향상 효과가 관찰되었다. 또한 CDV의 양이 증가함에 따라 transfection 효율이 증가되는 것을 확인하였다. 특히 N/P ratio 16, CDV 1x108 particles 조건에서 대조군(N/P ratio 16, bPEI/mRNA 처리군)보다 EGFP 단백질의 발현이 약 10배 증가된 것을 확인하였다.As shown in FIGS. 3A and B, an effect of enhancing mRNA delivery by CDV was observed at N/P ratios of 16 and 32. In addition, it was confirmed that the transfection efficiency increased as the amount of CDV increased. In particular, it was confirmed that the expression of EGFP protein increased about 10 times compared to the control group (N/
실시예 2. CDV 용량에 따른 bPEI/mRNA/CDV 복합체의 transfection 효율 평가Example 2. Evaluation of transfection efficiency of bPEI/mRNA/CDV complexes according to CDV capacity
CDV에 의한 단백질 발현 향상 효과가 가장 큰 N/P ratio 16 조건에서 제형의 최적화를 위하여, CDV의 양에 따른 bPEI/mRNA/CDV 복합체의 transfection 효율을 확인하였다. 0 부터 1x109 particles까지 총 8가지 조건의 CDV 양으로 bPEI/mRNA/CDV 복합체를 제조하여 HEK293 세포에 처리하였다. 모든 복합체는 EGFP mRNA 0.2 μg를 포함한다. 구체적인 실험 조건은 표 2에 나타내었다.In order to optimize the formulation under the condition of N/
(mRNA copies/CDV particle)Loading ratio
(mRNA copies/CDV particles)
(=0.2 μg)3.64x10 11
(=0.2 μg)
24시간 후 형광 현미경과 FACS를 통해 세포의 EGFP 발현을 관찰 및 정량하였다. 또한 bPEI/mRNA/CDV 복합체의 제타 전위를 측정하였다. 형광 현미경 이미지는 도 4A에 나타내었고, EGFP 정량 결과 및 제타전위 측정 결과는 도 4B에 함께 나타내었다.After 24 hours, EGFP expression of the cells was observed and quantified by fluorescence microscopy and FACS. We also measured the zeta potential of the bPEI/mRNA/CDV complex. The fluorescence microscope image is shown in FIG. 4A, and the EGFP quantification results and zeta potential measurement results are shown together in FIG. 4B.
도 4A 및 B에 나타낸 바와 같이, CDV 1x108, 2.5x108, 5x108 particles를 추가한 bPEI/mRNA/CDV 복합체에서 bPEI/mRNA 대비 EGFP 단백질 발현이 약 10배 증가된 것을 확인하였다. 반면에 CDV 5x108 particles 초과 조건에서는 EGFP 발현 정도가 현저히 감소하였고 이는 CDV 비율의 증가가 단백질 발현 정도의 증가와 비례하지는 않음을 의미한다.As shown in FIGS. 4A and B, it was confirmed that the EGFP protein expression increased about 10-fold compared to bPEI/mRNA in the bPEI/mRNA/CDV complex to which CDV 1x10 8 , 2.5x10 8 , and 5x10 8 particles were added. On the other hand, in the condition of exceeding CDV 5x10 8 particles, the level of EGFP expression decreased significantly, which means that the increase in CDV ratio was not proportional to the increase in protein expression level.
제타 전위 측정 결과, CDV 입자 수가 증가할수록 bPEI/mRNA/CDV의 surface charge가 감소하는 것을 확인하였다. 이는 과량의 CDV로 인한 복합체의 음전하가 세포 내 흡수에 영향을 미친 것으로 사료된다.As a result of zeta potential measurement, it was confirmed that the surface charge of bPEI/mRNA/CDV decreased as the number of CDV particles increased. It is believed that the negative charge of the complex due to the excess CDV affected the uptake into cells.
실시예 3. bPEI 및 bPEI/mRNA/CDV의 세포 독성 평가Example 3. Cytotoxicity evaluation of bPEI and bPEI/mRNA/CDV
bPEI 및 bPEI/mRNA/CDV의 세포 독성을 평가하기 위하여, bPEI 및 bPEI/mRNA/CDV가 처리된 HEK293 세포에서 CCK-8 assay를 수행하였다. 모든 복합체는 EGFP mRNA 0.2μg를 포함한다. CCK-8 assay 결과는 도 5A에 나타내었다. To evaluate the cytotoxicity of bPEI and bPEI/mRNA/CDV, CCK-8 assay was performed on HEK293 cells treated with bPEI and bPEI/mRNA/CDV. All complexes contain 0.2 μg of EGFP mRNA. CCK-8 assay results are shown in Figure 5A.
도 5A에 나타낸 바와 같이, bPEI를 N/P ratio 별로 세포에 처리하였을 때, N/P ratio 32(PEI 농도 = 8 μg/mL) 이상에서 PEI의 양전하에 의한 세포 독성 효과로 cell viability가 감소하는 것을 확인하였다.As shown in Figure 5A, when bPEI was treated with cells by N / P ratio, cell viability was reduced due to the cytotoxic effect caused by the positive charge of PEI at N / P ratio 32 (PEI concentration = 8 μg / mL) or higher confirmed that
상기 도 5A의 결과에 기초하여, 단백질 발현이 가장 높은 N/P ratio 16, CDV 1x108 조건의 bPEI/mRNA/CDV 처리군의 세포 독성을 재확인하였다. 세포 독성 확인 결과는 도 5B에 나타내었다.Based on the results of FIG. 5A, the cytotoxicity of the bPEI/mRNA/CDV treatment group under the N/
도 5B에 나타낸 바와 같이, N/P 16, CDV 1x108 조건의 bPEI/mRNA/CDV는 세포 독성을 보이지 않는 것을 확인하였다. 상기 결과는 CDV 추가에 따른 세포 독성이 없음을 의미한다.As shown in FIG. 5B, it was confirmed that bPEI/mRNA/CDV under N/
실시예 4. CDV에 의한 세포 내 mRNA 전달 향상 효과 확인Example 4. Confirmation of the effect of improving intracellular mRNA delivery by CDV
N/P ratio 16 조건에서 (i) bPEI/mRNA; 또는(ii) bPEI/mRNA/CDV 1x108 complex; 처리에 따른 세포 내 mRNA level를 qPCR을 통해 비교하였다. qPCR 결과는 도 6에 나타내었다. In the N/
도 6에 나타낸 바와 같이, 세포 내 uptake된 mRNA는 bPEI/mRNA 대비 bPEI/mRNA/CDV complex에서 48시간 이후까지 약 2-3배 높은 수준을 유지하였다.As shown in FIG. 6 , uptake of mRNA in cells was maintained at a level about 2-3 times higher in the bPEI/mRNA/CDV complex compared to bPEI/mRNA until 48 hours later.
실시예 5. 다양한 PEI 종류 적용 가능성 조사Example 5. Investigation of the applicability of various types of PEI
bPEI 이외에 다양한 종류의 PEI에서 적용 가능성을 조사하였다. In addition to bPEI, the applicability of various types of PEI was investigated.
5-1. linear PEI(lPEI) 2.5kDa5-1. linear PEI (lPEI) 2.5 kDa
linear PEI(lPEI) 2.5kDa를 이용한 lPEI/mRNA/CDV의 transfection efficiency를 확인하였다. bPEI와 동일한 방법으로, 다양한 N/P ratio에서 mRNA 0.25 μg를 포함하는 lPEI/mRNA/CDV를 제조한 후 HEK293 세포에 처리하였다. lPEI/mRNA/CDV 처리 24시간 후 유세포 분석을 수행하였다. 유세포 분석 결과는 도 7에 나타내었다.The transfection efficiency of lPEI/mRNA/CDV using linear PEI (lPEI) 2.5 kDa was confirmed. In the same manner as in bPEI, lPEI/mRNA/CDV containing 0.25 μg of mRNA was prepared at various N/P ratios and treated with HEK293 cells. Flow cytometric analysis was performed 24 hours after lPEI/mRNA/CDV treatment. The flow cytometry results are shown in FIG. 7 .
도 7에 나타낸 바와 같이, lPEI/mRNA/CDV 처리군은 N/P ratio 64 이상에서 CDV를 추가함에 따라 GFP 단백질 발현이 증가함을 확인하였다. 그러나 동일 mRNA 양에 대하여 Branched PEI 25kDa를 이용한 bPEI/mRNA/CDV와 MFI를 비교하였을 때, lPEI/mRNA/CDV 처리군은 단백질 발현이 낮은 것을 확인하였다.As shown in Figure 7, it was confirmed that the lPEI/mRNA/CDV treatment group increased GFP protein expression as CDV was added at an N/P ratio of 64 or higher. However, when comparing bPEI/mRNA/CDV and MFI using Branched PEI 25kDa for the same amount of mRNA, it was confirmed that the lPEI/mRNA/CDV treatment group showed low protein expression.
5-2. jetPEI5-2. jetPEI
polymer 계열 중 임상적용이 가능한 jetPEI를 이용한 jetPEI/mRNA/CDV의 transfection efficiency를 확인하였다. mRNA는 Renilla luciferase mRNA를 사용하였다. N/P ratio, mRNA/CDV ratio screening을 기반으로 jetPEI/mRNA/CDV Formula #1 및 #2를 선정하였으며, 구체적인 내용은 표 3에 나타내었다.The transfection efficiency of jetPEI/mRNA/CDV using jetPEI, which can be applied clinically among polymers, was confirmed. As mRNA, Renilla luciferase mRNA was used. JetPEI/mRNA/
(mRNA copies/CDV particle)Loading ratio
(mRNA copies/CDV particles)
jetPEI/mRNA/CDV Formula #1 및 #2의 mRNA 용량(0.01~0.2μg)에 따른 효능 및 독성을 확인하였다. 세포 독성 확인 결과는 도 8A에 나타내었고, in vitro transfection efficacy를 분석한 결과는 도 8B에 나타내었다.The efficacy and toxicity of jetPEI/mRNA/
도 8A에 나타낸 바와 같이, 독성 비교 결과 lipofectamine의 경우 용량이 증가할수록 독성이 뚜렷하게 증가한 반면, jetPEI/mRNA/CDV Formula #1 및 #2는 control 대비 80% 이상의 생존율을 보였다.As shown in FIG. 8A, as a result of toxicity comparison, the toxicity of lipofectamine increased markedly as the dose increased, whereas jetPEI/mRNA/
도 8B에 나타낸 바와 같이, 낮은 용량에서부터 CDV에 의해 단백질 발현이 2 배에서 많게는 100 배 이상 향상된 것을 확인하였다. 특히 mRNA 0.1μg부터 jetPEI/mRNA/CDV formula #1 및 #2에 의해 발현된 단백질의 발광세기는 positive control인 lipofectamine과 유사한 수준임을 확인하였다. As shown in FIG. 8B , it was confirmed that protein expression was improved from 2 to 100 times or more by CDV from a low dose. In particular, it was confirmed that the luminescence intensity of proteins expressed by jetPEI/mRNA/
상기 결과는 CDV를 사용함에 따라, 독성을 나타내지 않는 범위의 적은 양의 jetPEI로도 단백질 발현을 증가시킬 수 있음을 의미한다.The above result means that protein expression can be increased even with a small amount of jetPEI in a non-toxic range by using CDV.
종합적으로, 양이온성 폴리머를 이용하여 CDV에 mRNA를 결합시켰을 때 CDV에 의해 mRNA 전달 및 단백질 발현이 향상되는 효과를 확인하였다. Branched PEI(25 kDa), Linear PEI(2.5 kDa) 및 jetPEI를 사용하였을 때 모두 CDV에 의한 효과가 관찰되어 이러한 방법이 다양한 PEI에 적용됨을 확인하였다. 특히 jetPEI를 이용한 복합체에서는 positive control인 lipofectamine 대비 독성 감소 효과와 lipofectamine과 비슷한 수준의 단백질 발현을 보였다. 이러한 결과들은 polyplex와 CDV 복합체라는 효과적인 mRNA 전달이 가능한 새로운 형태의 약물 전달체로서의 가능성을 보여준다.Overall, when mRNA was bound to CDV using a cationic polymer, the effect of enhancing mRNA delivery and protein expression by CDV was confirmed. When branched PEI (25 kDa), linear PEI (2.5 kDa), and jetPEI were used, CDV-induced effects were observed, confirming that this method was applicable to various PEIs. In particular, the complex using jetPEI showed a toxicity reduction effect and protein expression similar to that of lipofectamine, which is a positive control. These results show the potential of polyplex and CDV complex as a new type of drug delivery system capable of effective mRNA delivery.
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다. In the above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (13)
A complex for nucleic acid delivery containing a cationic polymer, nucleic acid and cell-driven vesicles.
상기 양이온성 폴리머는 폴리에틸렌이민(PEI), 폴리프로필렌이민(PPI), 펜타에틸렌헥사민, N,N'-비스(2-아미노에틸)-1,3-프로판디아민, N-(2-아미노에틸)-1,3-프로판디아민, N-(2-아미노프로필)-1,3-프로판디아민, 스퍼민(spermine), 스퍼미딘(spermidine), 1,4-비스(3-아미노프로필)피페라진, 1-(2-아미노에틸)피페라진, 트리(2-아미노에틸)아민, 폴리아미도아민(PAMAM), 폴리(L-라이신)(PLL) 및 키토산으로 이루어진 군에서 선택된 1종 이상인, 핵산 전달용 복합체.
According to claim 1,
The cationic polymer is polyethyleneimine (PEI), polypropyleneimine (PPI), pentaethylene hexamine, N, N'-bis (2-aminoethyl) -1,3-propanediamine, N- (2-aminoethyl )-1,3-propanediamine, N-(2-aminopropyl)-1,3-propanediamine, spermine, spermidine, 1,4-bis(3-aminopropyl)piperazine, At least one selected from the group consisting of 1-(2-aminoethyl)piperazine, tri(2-aminoethyl)amine, polyamidoamine (PAMAM), poly(L-lysine) (PLL), and chitosan, for nucleic acid delivery complex.
상기 양이온성 폴리머는 branched-폴리에틸렌이민(bPEI), linear-폴리에틸렌이민(lPEI) 또는 jet-폴리에틸렌이민(jetPEI)인, 핵산 전달용 복합체.
According to claim 1,
The cationic polymer is a branched-polyethyleneimine (bPEI), linear-polyethyleneimine (lPEI) or jet-polyethyleneimine (jetPEI) complex for nucleic acid delivery.
상기 핵산은 플라스미드 유전자, 안티센스 올리고뉴클레오티드, siRNA, rRNA, RNA, DNA, cDNA, mRNA, shRNA, lncRNA, miRNA 및 tRNA로 이루어진 군에서 선택되는 1종 이상인, 핵산 전달용 복합체.
According to claim 1,
The nucleic acid is at least one selected from the group consisting of plasmid genes, antisense oligonucleotides, siRNA, rRNA, RNA, DNA, cDNA, mRNA, shRNA, lncRNA, miRNA and tRNA, complex for nucleic acid delivery.
상기 세포 유래 베시클은 세포를 압출하여 제조된 것인, 핵산 전달용 복합체.
According to claim 1,
The cell-derived vesicle is prepared by extruding cells, a complex for nucleic acid delivery.
상기 세포는 원핵세포(prokaryote) 또는 진핵세포(eukaryote)인, 핵산 전달용 복합체.
According to claim 1,
The cell is a prokaryotic cell (prokaryote) or eukaryote (eukaryote), the complex for nucleic acid delivery.
상기 원핵세포 또는 진핵세포는 적혈구, 단핵구, 대식세포, 수지상세포, T 세포, B 세포, 자연살해세포(natural killer cell, NK cell) 및 줄기세포로 이루어진 군에서 선택된 1종 이상인, 핵산 전달용 복합체.
According to claim 6,
The prokaryotic or eukaryotic cell is at least one selected from the group consisting of red blood cells, monocytes, macrophages, dendritic cells, T cells, B cells, natural killer cells (NK cells), and stem cells. Complex for nucleic acid delivery .
상기 원핵세포 또는 진핵세포는 HEK293 세포인, 핵산 전달용 복합체.
According to claim 6,
The prokaryotic or eukaryotic cell is a HEK293 cell, a nucleic acid delivery complex.
상기 원핵세포 또는 진핵세포는 원생생물(protista), 진균(fungi), 세균(bacteria), 고균(archaea) 및 바이러스(virus)로 이루어진 군에서 선택된 1종 이상의 미생물인, 핵산 전달용 복합체.
According to claim 6,
The prokaryotic or eukaryotic cell is one or more microorganisms selected from the group consisting of protista, fungi, bacteria, archaea and viruses, complex for nucleic acid delivery.
상기 양이온성 폴리머 및 핵산의 질소/인 비율(N/P ratio)은 1 내지 64인, 핵산 전달용 복합체.
According to claim 1,
The nitrogen / phosphorus ratio (N / P ratio) of the cationic polymer and nucleic acid is 1 to 64, the nucleic acid delivery complex.
상기 핵산 및 세포 유래 베시클의 비율(mRNA copies/CDV particle)은 70 내지 40000인, 핵산 전달용 복합체.
According to claim 1,
The ratio of the nucleic acid and the cell-derived vesicle (mRNA copies / CDV particle) is 70 to 40000, a nucleic acid delivery complex.
A reagent composition for nucleic acid delivery comprising a complex for nucleic acid delivery containing a cationic polymer, a nucleic acid and a cell-derived vesicle.
A drug delivery system comprising a nucleic acid delivery complex containing a cationic polymer, a nucleic acid and a cell-derived vesicle.
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