KR102224580B1 - Nucleic acid construct for simultaneous gene expression and suppression - Google Patents

Nucleic acid construct for simultaneous gene expression and suppression Download PDF

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KR102224580B1
KR102224580B1 KR1020190122794A KR20190122794A KR102224580B1 KR 102224580 B1 KR102224580 B1 KR 102224580B1 KR 1020190122794 A KR1020190122794 A KR 1020190122794A KR 20190122794 A KR20190122794 A KR 20190122794A KR 102224580 B1 KR102224580 B1 KR 102224580B1
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이혁진
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이화여자대학교 산학협력단
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Abstract

The present invention relates to a nucleic acid construct for simultaneous gene expression and suppression. A nucleic acid construct capable of simultaneously expressing and inhibiting gene expression according to the present invention includes both mRNA and siRNA in a single construct, and thus, mRNA and siRNA are simultaneously introduced into the same cell, thereby normally expressing the protein to be expressed, while specifically inhibiting the expression of a certain gene. The mRNA-siRNA nucleic acid construct can express a target protein in antigen-presenting cells and block the activation of an immune checkpoint protein at the same time, so as to activate an immune system and overcome an interference with immune evasion, and thus, is expected to be very useful in the field of immunotherapy, especially in the field of anticancer immunotherapy.

Description

유전자 발현 및 억제가 동시에 가능한 핵산 구조체{Nucleic acid construct for simultaneous gene expression and suppression} Nucleic acid construct for simultaneous gene expression and suppression}

본 발명은 유전자 발현 및 억제가 동시에 가능한 핵산 구조체에 관한 것이다.The present invention relates to a nucleic acid construct capable of simultaneously expressing and suppressing genes.

암은 인류의 건강을 위협하는 최대의 질병 중의 하나로서 세포가 일련의 돌연변이 과정을 거쳐 무제한적이고, 비 조절적인 방식으로 증식 및 불사화되어 발생하는 질병이다. 암과 관련된 다양한 생화학적 기전이 규명되어 그에 따른 치료제가 개발되어 오고 있으나, 아직까지 근본적인 치료방법은 제시되지 않고 있다. 이러한 암치료의 새로운 패러다임으로 자리매김하고 있는 것이 바로 면역항암요법(암면역치료, cancer immunotherapy)이다.Cancer is one of the greatest diseases that threaten human health, and it is a disease that occurs when cells proliferate and immortalize in an unlimited and unregulated manner through a series of mutations. Various biochemical mechanisms related to cancer have been identified, and therapeutic agents have been developed accordingly, but a fundamental treatment method has not yet been suggested. Immuno-chemotherapy (cancer immunotherapy) is positioning itself as a new paradigm for cancer treatment.

상기 면역항암요법은 수동면역치료와 능동면역치료로 구분할 수 있다. 수동면역치료에는 면역관문 억제제(immune checkpoint inhibitor), 면역세포치료제(immune cell therapy), 치료용 항체(therapeutic antibody) 등이 사용되며, 이 중 면역관문억제제는 T세포 억제에 관여하는 면역관문 단백질(immune checkpoint protein)의 활성화를 차단하여 T세포를 활성화시켜 암세포를 공격하는 치료제로서 CTLA-4, PD-1, PD-L1 억제제 등이 있다.The immunotherapy can be divided into passive immunotherapy and active immunotherapy. In passive immunotherapy, immune checkpoint inhibitors, immune cell therapy, therapeutic antibodies, etc. are used. Among these, immune checkpoint inhibitors are immune checkpoint proteins that are involved in T cell suppression. immune checkpoint protein) and activates T cells to attack cancer cells, such as CTLA-4, PD-1, and PD-L1 inhibitors.

능동면역치료에는 암치료 백신, 면역조절제 등이 사용되며, 구체적으로 체내 면역세포를 수집하여 강화시키거나 유전공학적으로 변형시켜 다시 넣어주는 세포치료 방식으로 종양침윤림프구(tumor infiltrating lymphocytes, TIL), T세포 수용체(T cell receptor, TCR), 키메릭항원수용체(chimeric antigen receptor, CAR) 세포치료제 등이 있고, 암세포가 갖고 있는 종양특이적 항원(tumor-specific antigen)을 암환자에게 투여하여 면역체계를 활성화시킴으로써 체내 면역기능을 활발하게 만들어 암세포를 공격하도록 하는 암백신 등이 있다.For active immunotherapy, cancer treatment vaccines and immunomodulators are used. Specifically, tumor infiltrating lymphocytes (TIL) and T There are T cell receptor (TCR), chimeric antigen receptor (CAR) cell therapy, etc., and the immune system is administered by administering tumor-specific antigens of cancer cells to cancer patients. There are cancer vaccines that activate the body's immune function to attack cancer cells.

그 중에서도 수지상세포(dendritic cell, DC)는 면역계의 가장 핵심적인 항원제시세포로서 수지상세포 암백신은 암 항원을 제시하여 항암 면역 반응 활성화를 통해 종양을 효과적으로 제거할 수 있는 이상적인 치료제로 여겨지고 있다. 그러나, 종양 및 종양미세환경(tumor microenvironment, TME)은 직접적으로 수지상세포의 기능장애를 유도하거나 종양 항원을 감추고 면역억제 사이토카인을 다량 분비하여 항암 면역 활성을 억제한다. 특히, 암세포와 이를 공격하려는 면역세포의 힘의 균형이 무너지게 되면 암세포가 본격적인 증식을 개시하며 체내 면역시스템을 교란하는데, 이 때 일부 암세포는 면역세포의 면역관문을 활용하여 면역을 회피하게 된다.Among them, dendritic cells (DC) are the most important antigen-presenting cells of the immune system, and dendritic cell cancer vaccines present cancer antigens and are considered an ideal therapeutic agent that can effectively remove tumors through activation of anti-cancer immune responses. However, tumors and tumor microenvironment (TME) directly induce dysfunction of dendritic cells or hide tumor antigens and secrete large amounts of immunosuppressive cytokines to suppress anti-cancer immune activity. In particular, when the balance between the power of cancer cells and immune cells to attack them is broken, cancer cells start to proliferate in earnest and disturb the body's immune system, in which some cancer cells use the immune gates of immune cells to evade immunity.

따라서, 면역체계를 활성화시켜 능동적으로 암세포에 대한 면역반응을 증가시키면서도, 수동적으로 면역관문 단백질의 활성화를 차단하여 면역회피 방해를 받지 않도록 하는 새로운 면역항암제에 대한 필요성이 대두된다.Accordingly, there is a need for a new anticancer drug that activates the immune system and actively increases the immune response to cancer cells, while passively blocking the activation of the immune checkpoint protein so as not to interfere with immune evasion.

또한, 유전체 시스템의 복잡한 환경, 특히 암 환경에서 특정 유전자들의 상향 또는 하향 조절이 원활하게 이루어질 필요가 있는데, 하나의 물질의 처리로써 이러한 유전자 조절을 이루는 것이 매우 어렵다. 즉, 단일의 분자를 이용하여 생체 내에서 유전자들의 정확하고 빠른 조절을 목적하는 많은 연구들이 있었으나, 충분한 결과를 확보하고 있지 못한 실정이다. In addition, there is a need to smoothly regulate specific genes up or down in a complex environment of a genomic system, particularly in a cancer environment, and it is very difficult to achieve such gene regulation with the treatment of a single substance. In other words, there have been many studies aimed at accurately and rapidly regulating genes in vivo using a single molecule, but sufficient results have not been obtained.

본 발명자들은 mRNA 및 siRNA를 포함하는 핵산 구조체를 제조하고, 상기 구조체가 특정 유전자들을 mRNA 또는 siRNA 각각을 통해 동시에 발현 또는 억제함을 확인하였다. 특히, 항원 mRNA를 발현시켜 면역체계를 활성화시킴과 동시에 면역관문 단백질의 활성화를 억제하여 면역관문 단백질에 의한 면역 회피 기전을 극복할 수 있음을 확인하고, 본 발명을 완성하게 되었다.The present inventors prepared a nucleic acid construct including mRNA and siRNA, and confirmed that the construct simultaneously expressed or suppressed specific genes through mRNA or siRNA, respectively. In particular, it was confirmed that the immune system was activated by expressing the antigen mRNA and at the same time suppressed the activation of the immune checkpoint protein, thereby overcoming the immune evasion mechanism by the immune checkpoint protein, and the present invention was completed.

따라서, 본 발명의 목적은 유전자 발현 및 억제가 동시에 가능한 mRNA 및 siRNA를 포함하는 핵산 구조체를 제공하는 데에 있다.Accordingly, an object of the present invention is to provide a nucleic acid construct including mRNA and siRNA capable of simultaneously expressing and suppressing genes.

상기 목적을 달성하기 위하여, 본 발명은 mRNA 및 siRNA를 포함하는 핵산 구조체를 제공한다.In order to achieve the above object, the present invention provides a nucleic acid construct comprising mRNA and siRNA.

또한, 본 발명은 상기 핵산 구조체 및 핵산 전달체를 포함하는 조성물을 제공한다.In addition, the present invention provides a composition comprising the nucleic acid construct and a nucleic acid delivery system.

또한, 본 발명은 상기 핵산 구조체를 포함하는 면역치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for immunotherapy comprising the nucleic acid construct.

또한, 본 발명은 상기 핵산 구조체를 포함하는 암 백신을 제공한다.In addition, the present invention provides a cancer vaccine comprising the nucleic acid construct.

또한, 본 발명은 상기 핵산 구조체의 제조방법을 제공한다.In addition, the present invention provides a method of manufacturing the nucleic acid construct.

본 발명에 따른 유전자 발현 및 억제가 동시에 가능한 핵산 구조체는 단일 구조체 내에 mRNA와 siRNA를 모두 포함함에 따라 동일한 세포에 mRNA 및 siRNA를 동시에 도입시키고, 이를 통해 발현하고자 하는 단백질을 정상적으로 발현시키면서도, 특정 유전자의 발현은 특이적으로 억제할 수 있다. 상기 mRNA-siRNA 핵산 구조체는 항원 제시 세포에 타겟 단백질을 발현시켜 면역체계를 활성화시킴과 동시에 면역관문 단백질의 활성화를 차단하여 면역회피 방해를 극복할 수 있어, 면역치료 분야, 특히 항암면역치료 분야에서 매우 유용하게 사용될 수 있을 것으로 기대된다.The nucleic acid construct capable of simultaneously expressing and suppressing genes according to the present invention includes both mRNA and siRNA in a single construct, so that mRNA and siRNA are simultaneously introduced into the same cell, and through this, the protein to be expressed is normally expressed, while the Expression can be specifically inhibited. The mRNA-siRNA nucleic acid construct is capable of overcoming immune evasion interference by blocking the activation of immune checkpoint proteins while activating the immune system by expressing a target protein in antigen-presenting cells. It is expected to be very useful.

도 1은 본 발명에 따른 mRNA-siRNA 핵산 구조체의 제조방법을 순서대로 도식화한 것이다.
도 2는 본 발명에 따른 mRNA-siRNA 핵산 구조체의 제조방법을 순서대로 도식화한 것이다. DNA 주형, 상기 주형 DNA를 사용하여 시험관 내 전사된 mRNA(“mRNA-siRNA(SS or AS)”) 및 상기 mRNA에 siRNA를 어닐링(annealing)시켜 제조된 mRNA-siRNA 핵산 구조체를 siRNA의 센스(SS) 또는 안티센스 서열(AS) 표시와 함께 도식화하였다.
도 3은 본 발명에 따른 mRNA-siRNA 핵산 구조체가 세포 내 환경에서 RNase H에 의해 mRNA 및 siRNA로 분리되는 과정을 도식화한 것이다(붉은색 글씨는 DNA 서열에 해당).
도 4는 siRNA의 센스 또는 안티센스 서열, 길이를 달리하는 Oligo(dT)를 다양한 구성으로 조합한 siRNA-Oligo(dT)를 mRNA에 어닐링(annealing)시켜 제조한 mRNA-siRNA 실시예들을 도식화한 것이다(붉은색 글씨는 DNA 서열에 해당).
도 5는 DNA 주형(a) 및 시험관 내 전사 산물(b)에 대하여 전기영동한 결과를 나타낸 도이다.
도 6은 mRNA-siRNA에 대하여 전기영동한 결과를 나타낸 도이다.
도 7은 서로 다른 mRNA-siRNA 실시예들의 단백질(빨간 형광 단백질, GFP) 발현 능력을 평가한 결과를 나타낸 도이다.
도 8은 형질주입 24시간 후, 서로 다른 mRNA-siRNA 실시예들의 단백질(파란 형광 단백질, BFP) 발현 능력 및 유전자(녹색 형광 단백질, GFP) 발현 억제 능력을 평가한 결과를 나타낸 도이다. (a) mBFP 단백질 발현 효율, (b) GFP 단백질 발현 효율.
도 9는 형질주입 48시간 후, 서로 다른 mRNA-siRNA 실시예들의 단백질(파란 형광 단백질, BFP) 발현 능력 및 유전자(녹색 형광 단백질, GFP) 발현 억제 능력을 평가한 결과를 나타낸 도이다. (a) mBFP 단백질 발현 효율, (b) GFP 단백질 발현 효율.
도 10은 본 발명에 따른 핵산 구조체에 오브알부민(Ovalbumin, OVA)을 발현하는 mRNA 및 STAT3 유전자의 발현을 억제하는 siRNA를 도입하고, 상기 mOVA-siSTAT3의 STAT3 단백질 발현 억제 능력을 평가한 결과를 나타낸 도이다.
도 11은 mOVA-siSTAT3의 수지상세포 성숙 촉진 효과를 CD40, CD80 발현량을 통해 확인한 결과를 나타낸 도이다.1 is a schematic diagram of a method for preparing an mRNA-siRNA nucleic acid construct according to the present invention in sequence.
2 is a schematic diagram of a method for preparing an mRNA-siRNA nucleic acid construct according to the present invention in sequence. The DNA template, mRNA (“mRNA-siRNA (SS or AS)”) transcribed in vitro using the template DNA, and the mRNA-siRNA nucleic acid construct prepared by annealing the siRNA to the mRNA are used as the sense of siRNA (SS ) Or antisense sequence (AS).
3 is a schematic diagram of a process in which the mRNA-siRNA nucleic acid construct according to the present invention is separated into mRNA and siRNA by RNase H in the intracellular environment (red letters correspond to DNA sequences).
Figure 4 is a schematic diagram of mRNA-siRNA examples prepared by annealing siRNA-Oligo (dT), which is a combination of oligos (dT) having different lengths and sense or antisense sequences of siRNA, in various configurations ( The red text corresponds to the DNA sequence).
5 is a diagram showing the results of electrophoresis on a DNA template (a) and an in vitro transcription product (b).
6 is a diagram showing the results of electrophoresis for mRNA-siRNA.
7 is a diagram showing the results of evaluating the protein (red fluorescent protein, GFP) expression ability of different mRNA-siRNA examples.
8 is a diagram showing the results of evaluating the protein (blue fluorescent protein, BFP) expression ability and gene (green fluorescent protein, GFP) expression inhibition ability of different mRNA-siRNA examples 24 hours after transfection. (a) mBFP protein expression efficiency, (b) GFP protein expression efficiency.
9 is a diagram showing the results of evaluating the protein (blue fluorescent protein, BFP) expression ability and gene (green fluorescent protein, GFP) expression inhibition ability of different mRNA-siRNA examples 48 hours after transfection. (a) mBFP protein expression efficiency, (b) GFP protein expression efficiency.
FIG. 10 shows the results of introducing mRNA expressing Ovalbumin (OVA) and siRNA that suppresses the expression of STAT3 gene into the nucleic acid construct according to the present invention, and evaluating the ability of mOVA-siSTAT3 to inhibit STAT3 protein expression It is a degree.
11 is a diagram showing the results of confirming the effect of mOVA-siSTAT3 on promoting dendritic cell maturation through the expression levels of CD40 and CD80.

이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.

다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

본 발명에서 용어, “벡터”는 적절한 숙주 세포에서 목적하는 단백질을 발현할 수 있도록 하는 작동 가능하게 연결된 필수적인 조절 요소를 포함하는 핵산 제작물을 의미한다.In the present invention, the term "vector" refers to a nucleic acid construct comprising essential regulatory elements operably linked to enable expression of a desired protein in an appropriate host cell.

본 발명에 있어서, 상기 벡터는 본 발명에 따른 핵산 구조체를 생산하는데 적합한 것이면 제한 없이 사용될 수 있으며, 프로모터 등 전사를 위해 필요한 요소를 포함한다. 상기 벡터는 RNA 벡터 또는 DNA 벡터일 수 있으며, 바람직하게는 DNA 벡터일 수 있다. 상기 벡터는 바이러스 벡터 또는 플라스미드 벡터와 같이 통상의 기술자에게 알려진 벡터이면 제한 없이 사용될 수 있으며, 바람직하게는 DNA 플라스미드 벡터를 사용할 수 있다. 또한, 상기 벡터는 원형 분자인 것을 특징으로 할 수 있다. 상기 벡터, 바람직하게 원형 벡터는, 예를 들어 제한 효소에 의해 선형화(linearization)될 수 있다.In the present invention, the vector may be used without limitation as long as it is suitable for producing the nucleic acid construct according to the present invention, and includes elements necessary for transcription such as a promoter. The vector may be an RNA vector or a DNA vector, preferably a DNA vector. The vector may be used without limitation as long as it is a vector known to a person skilled in the art, such as a viral vector or a plasmid vector, and preferably, a DNA plasmid vector may be used. In addition, the vector may be characterized in that it is a circular molecule. The vector, preferably a circular vector, may be linearized, for example by a restriction enzyme.

본 발명에서 용어, "프로모터"는 전사조절인자들이 결합하는 DNA 염기서열 부위를 의미하며, 본 발명의 목적상 유전자 발현율을 높이기 위하여 강력하고 안정적인 유전자 발현을 유도할 수 있는 프로모터를 사용할 수 있다. 본 발명에 있어서, 상기 프로모터는 바람직하게는 RNA 중합효소 프로모터일 수 있고, 보다 바람직하게는 T7 프로모터 또는 SP6 프로모터일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the term "promoter" refers to a DNA nucleotide sequence site to which transcriptional regulatory factors bind, and for the purposes of the present invention, a promoter capable of inducing strong and stable gene expression may be used to increase the gene expression rate. In the present invention, the promoter may preferably be an RNA polymerase promoter, more preferably a T7 promoter or an SP6 promoter, but is not limited thereto.

본 발명에서 용어, "UTR(untranslation region, 비해독 부분)"은 mRNA의 비해독 부분을 이르는 단어로 일반적으로 코딩 영역의 양 말단을 의미한다. 특히, 5' 말단 부분을 5' UTR로 3' 말단 부분을 3' UTR로 지칭한다.In the present invention, the term "UTR (untranslation region, untranslated portion)" refers to an untranslated portion of an mRNA and generally means both ends of the coding region. In particular, the 5'end portion is referred to as 5'UTR and the 3'end portion is referred to as 3'UTR.

본 발명에서 용어, "폴리(A) 테일(poly(A) tail)"은 폴리아데닐산 또는 폴리아데닐산 절편으로도 불리며, 진핵생물의 mRNA 3' 말단에 보편적으로 존재하는 아데닐산의 연속한 배열을 의미한다. 그 길이는 10 내지 200 뉴클레오티드(nt) 정도이며, 발현벡터 백본(backbone)의 허용 크기에 따라 다양하게 조절될 수 있다. 폴리(A)는 mRNA의 안정화, 해독, 핵에서 세포질로의 수송 등에 관여하는 것으로 알려져 있다.In the present invention, the term "poly(A) tail" is also referred to as a polyadenylic acid or polyadenylic acid fragment, and is a continuous sequence of adenylic acids commonly present at the 3'end of mRNA in eukaryotes. Means. The length is about 10 to 200 nucleotides (nt), and can be variously adjusted according to the allowable size of the expression vector backbone. Poly(A) is known to be involved in mRNA stabilization, translation, and transport from the nucleus to the cytoplasm.

본 발명에서 용어, "siRNA(small interfering RNA)"는 이중가닥 RNA가 다이서(Dicer) 효소에 의해 절단되어 생성되는 18~23 뉴클레오티드(nt) 크기의 작은 RNA 조각으로, 상보적인 서열을 갖는 mRNA에 특이적으로 결합하여 타겟 단백질, mRNA의 발현을 억제하는 데 사용할 수 있다. 상기 siRNA는 센스 RNA와 안티센스 RNA가 이중가닥 RNA 분자를 형성하고, 이때 센스 RNA가 mRNA 중 일부의 연속 뉴클레오티드의 표적 서열과 동일한 핵산 서열을 포함하는 siRNA 분자인 것이 바람직하다. 상기 안티센스 서열은 센스 서열과 상보적인 서열을 가지는 것이 가장 바람직하다.In the present invention, the term "siRNA (small interfering RNA)" refers to a small RNA fragment having a size of 18 to 23 nucleotides (nt) that is produced by cleaving double-stranded RNA by Dicer enzyme, and has a complementary sequence. It can be used to specifically bind to and suppress the expression of a target protein or mRNA. The siRNA is preferably an siRNA molecule comprising a nucleic acid sequence identical to a target sequence of a contiguous nucleotide of some of the mRNA, wherein the sense RNA and the antisense RNA form a double-stranded RNA molecule. Most preferably, the antisense sequence has a sequence complementary to the sense sequence.

본 발명에서, 용어 "상보적인 서열"은 서로 상보적 결합을 이루는 특징을 유지할 수 있다면 100% 상보적 서열 외에 60 내지 100%의 상보적 서열, 바람직하게는 80% 내지 100% 상보적 서열, 더욱 바람직하게는 90 내지 100% 상보적 서열, 더더욱 바람직하게는 95% 내지 100%의 상보적 서열로 이루어지는 것을 포함할 수 있다.In the present invention, the term "complementary sequence" refers to a complementary sequence of 60 to 100% in addition to the 100% complementary sequence, preferably 80% to 100% complementary sequence, and more It may include those consisting of preferably 90 to 100% complementary sequence, even more preferably 95% to 100% of the complementary sequence.

본 발명에서 용어, "표적 유전자"란 상기 siRNA에 의해 발현이 선택적으로 억제되거나 불활성화되는 유전자이다. 이러한 불활성화는, siRNA가 표적 유전자의 mRNA를 절단함에 의해 달성된다.In the present invention, the term "target gene" refers to a gene whose expression is selectively suppressed or inactivated by the siRNA. This inactivation is achieved by siRNA cleaving the mRNA of the target gene.

본 발명에서 용어, “리보뉴클레아제(ribonuclease, RNase)”는 리보핵산을 분해하여 올리고뉴클레오티드 혹은 단일 핵산으로 만드는 반응을 촉매하는 효소로, 바람직하게는 엔도 리보핵산 분해효소(endo ribonuclease)이며, 상기 엔도 리보핵산 분해효소는 RNase A, RNase H, RNase I, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase V1 및 RNase V를 포함한다.In the present invention, the term "ribonuclease (RNase)" is an enzyme that catalyzes the reaction to decompose ribonucleic acid into oligonucleotides or single nucleic acids, preferably endo ribonuclease, The endo ribonucleic acid degrading enzyme includes RNase A, RNase H, RNase I, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase V1 and RNase V.

본 발명에 있어서, 상기 리보핵산은 전령 리보핵산(messenger RNA)일 수 있으며, 상기 리보뉴클레아제로는 바람직하게는 RNase H를 사용할 수 있다. 본 발명의 일 실시예에서는, RNase H를 사용하여 본 발명에 따른 핵산 구조체인 mRNA-siRNA의 폴리(A)-폴리(T) 혼성화 부분의 인산다이에스터 결합(phosphodiester bond)을 가수분해시켜 mRNA에 따른 유전자 발현 및 siRNA에 따른 단백질 발현 억제를 동시에 달성할 수 있음을 확인하고 있다.In the present invention, the ribonucleic acid may be a messenger ribonucleic acid (messenger RNA), and RNase H may be preferably used as the ribonuclease. In an embodiment of the present invention, RNase H is used to hydrolyze the phosphate diester bond of the poly(A)-poly(T) hybridization portion of the mRNA-siRNA of the nucleic acid structure according to the present invention to It has been confirmed that it is possible to simultaneously achieve suppression of protein expression according to gene expression and siRNA.

본 발명에서 용어, “mRNA-siRNA(SS or AS)”는 3' 말단에 siRNA의 센스 또는 안티센스 서열을 포함하는 mRNA를 의미한다. 본 발명의 일 실시예에서는, 폴리(A) 서열 및/또는 siRNA(센스/안티센스) 서열을 포함하는 역방향 프라이머(Reverse Primer, RP)를 사용하여 3’ UTR 바로 옆 3’에 폴리(T) 테일(poly(T) tail) 및 siRNA(안티센스/센스) 서열이 도입된 DNA 주형을 제조하고, 제조한 DNA 주형을 이용하여 시험관 내(in vitro) 전사(in vitro transcription, IVT)를 통해 3' 말단에 siRNA 센스 또는 안티센스 서열을 포함하는 mRNA(“mRNA-siRNA(SS or AS)”)를 제조하였다.In the present invention, the term "mRNA-siRNA (SS or AS)" refers to an mRNA including a sense or antisense sequence of siRNA at the 3'end. In one embodiment of the present invention, using a reverse primer (RP) comprising a poly (A) sequence and / or siRNA (sense / antisense) sequence, a poly (T) tail 3'immediately next to the 3'UTR. (poly(T) tail) and siRNA (antisense/sense) sequences were introduced into a DNA template, and the 3'end through in vitro transcription (IVT) using the prepared DNA template MRNA (“mRNA-siRNA (SS or AS)”) containing siRNA sense or antisense sequence was prepared.

본 발명에서 용어, “mRNA-siRNA”는 상기 제조한 mRNA-siRNA(SS or AS)의 3' 말단에 도입된 siRNA의 센스 또는 안티센스 서열에 상보적으로 결합할 수 있는 “혼성화 서열”을 어닐링한 결과, mRNA-siRNA 3' 말단의 폴리(A) 테일 및 siRNA 서열(센스/안티센스)이 각각 이와 상보적인 혼성화 서열(안티센스/센스) 및 oligo(dT)와 결합하여 이중가닥을 형성하는 핵산 구조체를 의미한다. In the present invention, the term "mRNA-siRNA" is annealing a "hybridization sequence" capable of complementarily binding to the sense or antisense sequence of the siRNA introduced at the 3'end of the prepared mRNA-siRNA (SS or AS). As a result, the poly(A) tail and siRNA sequence (sense/antisense) at the 3′ end of the mRNA-siRNA bind to a hybridization sequence (antisense/sense) and oligo (dT) complementary thereto to form a double-stranded nucleic acid construct. it means.

본 발명에 있어서, 상기 “혼성화 서열”은 siRNA 또는 DNA일 수 있으며, 따라서 “mRNA-siRNA”은 본 명세서 내에서 “mRNA-siRNA/siRNA”또는 “mRNA-siRNA/DNA”로도 표기된다.In the present invention, the “hybridization sequence” may be siRNA or DNA, and thus “mRNA-siRNA” is also referred to as “mRNA-siRNA/siRNA” or “mRNA-siRNA/DNA” in the present specification.

본 발명은 유전자 발현 및 억제가 동시에 가능한 핵산 구조체를 제공한다.The present invention provides a nucleic acid construct capable of simultaneously expressing and suppressing genes.

보다 구체적으로, 본 발명은 mRNA 및 siRNA를 포함하는 핵산 구조체로, (i) 3’ 말단에 5’ 방향에서 3’ 방향으로 순차적으로 위치하는 폴리(A) 테일(poly(A) tail); 및 제1 siRNA 센스 또는 안티센스 서열을 포함하는 mRNA; 및 (ii) 상기 제1 siRNA 서열에 상보적으로 결합하는 혼성화 서열을 포함하는 핵산 구조체를 제공한다.More specifically, the present invention relates to a nucleic acid construct comprising mRNA and siRNA, comprising: (i) a poly(A) tail sequentially positioned in a 3'direction from a 5'direction to a 3'end; And an mRNA comprising a first siRNA sense or antisense sequence; And (ii) a hybridization sequence that complementarily binds to the first siRNA sequence.

본 발명에 있어서, 상기 혼성화 서열은 제1 siRNA 서열에 상보적으로 결합하는 제2 siRNA 안티센스 또는 센스 서열; 또는 제1 siRNA 서열에 상보적으로 결합하는 DNA 안티센스 또는 센스 서열인 것을 특징으로 한다.In the present invention, the hybridization sequence is a second siRNA antisense or sense sequence that complementarily binds to the first siRNA sequence; Or it is characterized in that the DNA antisense or sense sequence complementarily binds to the first siRNA sequence.

본 발명에 있어서, 상기 혼성화 서열은 3’ 말단에 상기 mRNA의 폴리(A) 테일과 상보적으로 결합하는 폴리(T) 테일(poly(T) tail)을 더 포함할 수 있다. 상기 폴리(T) 테일은 2 내지 6 nt 길이일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the hybridization sequence may further include a poly (T) tail complementarily binding to the poly (A) tail of the mRNA at the 3'end. The poly (T) tail may be 2 to 6 nt in length, but is not limited thereto.

본 발명에 있어서, 상기 핵산 구조체는 제1 siRNA 센스 또는 안티센스 서열의 3’ 말단에 추가의 siRNA 센스 또는 안티센스 서열을 위한 제1 링커 서열을 더 포함할 수 있고, 상기 제1 링커 서열은 링커 서열의 3’ 말단에 추가의 siRNA 센스 또는 안티센스 서열을 더 포함할 수 있다.In the present invention, the nucleic acid construct may further include a first linker sequence for an additional siRNA sense or antisense sequence at the 3′ end of the first siRNA sense or antisense sequence, and the first linker sequence An additional siRNA sense or antisense sequence may be further included at the 3'end.

또한, 본 발명에 있어서, 상기 핵산 구조체는 상기 제1 링커 서열; 및 추가의 siRNA 센스 또는 안티센스 서열에 상보적으로 결합하는 혼성화 서열; 및 제2 링커 서열을 더 포함할 수 있다. 상기 혼성화 서열은 상기 제1 링커 서열; 및 추가의 siRNA 센스 또는 안티센스 서열에 상보적으로 결합하는 것을 특징으로 한다.In addition, in the present invention, the nucleic acid construct comprises the first linker sequence; And a hybridization sequence that complementarily binds to an additional siRNA sense or antisense sequence; And a second linker sequence. The hybridization sequence comprises the first linker sequence; And complementary binding to an additional siRNA sense or antisense sequence.

본 발명에 있어서, 링커(linker) 또는 어댑터(adapor) 서열은 통상의 방법에 따른 합성 올리고뉴클레오타이드 서열을 제한 없이 사용할 수 있고, 바람직하게는 상기 제1 링커 서열은 폴리(U) 테일일 수 있고, 상기 제2 링커 서열은 제1 링커 서열과 상보적으로 결합하는 폴리(A) 테일일 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the linker (linker) or adapter (adapor) sequence may be used without limitation a synthetic oligonucleotide sequence according to a conventional method, preferably the first linker sequence may be a poly (U) tail, The second linker sequence may be a poly(A) tail complementary to the first linker sequence, but is not limited thereto.

상기 링커 또는 어댑터 서열은 DNA와 혼성화되지 않는 부위를 포함할 수 있다.The linker or adapter sequence may include a site that does not hybridize with DNA.

본 발명에 있어서, 상기 핵산 구조체는 하기 그림에서 붉은 점선으로 표시한 반복단위를 복수 개 더 포함할 수 있으며, 바람직하게는 1개 내지 5개 더 포함할 수 있다. 추가되는 반복단위의 개수는 당업자가 목적에 따라 적절히 조절할 수 있다.In the present invention, the nucleic acid construct may further include a plurality of repeating units indicated by a red dotted line in the following figure, preferably 1 to 5 more. The number of added repeating units can be appropriately adjusted by a person skilled in the art according to the purpose.

Figure 112019101328436-pat00001
Figure 112019101328436-pat00001

구체적으로, 상기 핵산 구조체는 제1 siRNA 센스 또는 안티센스 서열의 3’ 말단에 (a) 및 (b)를 포함하는 반복단위를 복수 개 더 포함할 수 있고, 상기 (a) 및 (b)는, (a) 추가의 siRNA 센스 또는 안티센스 서열을 위한 제1 링커 서열; 및 추가의 siRNA 센스 또는 안티센스 서열; 및 (b) 혼성화 서열; 및 제2 링커 서열인 것을 특징으로 하며, 이 때 상기 혼성화 서열은 상기 제1 링커 서열; 및 추가의 siRNA 센스 또는 안티센스 서열에 상보적으로 결합하는 것을 특징으로 한다. Specifically, the nucleic acid construct may further include a plurality of repeating units including (a) and (b) at the 3'end of the first siRNA sense or antisense sequence, and (a) and (b), (a) a first linker sequence for an additional siRNA sense or antisense sequence; And additional siRNA sense or antisense sequences; And (b) a hybridization sequence; And a second linker sequence, wherein the hybridization sequence includes the first linker sequence; And complementary binding to an additional siRNA sense or antisense sequence.

반복단위의 일부로 추가되는 siRNA는 타겟으로 하는 유전자가 서로 같거나 다를 수 있으며, 상기 핵산 구조체는 세포 내로 주입되면 RNase H에 의해 mRNA-siRNA의 혼성화 부위가 절단되어 독립된 이중가닥 siRNA로 분리되어 효과적으로 표적 유전자의 발현을 억제할 수 있다. 서로 상이한 2종 이상의 siRNA를 포함하는 경우, 2 이상의 타겟 유전자를 동시에 발현 억제할 수 있다.The siRNA added as a part of the repeat unit may have the same or different target genes, and when the nucleic acid construct is injected into the cell, the hybridization site of mRNA-siRNA is cleaved by RNase H and separated into an independent double-stranded siRNA to effectively target. It can suppress the expression of the gene. When two or more siRNAs that are different from each other are included, two or more target genes can be simultaneously suppressed.

또한, 본 발명은 본 발명에 따른 핵산 구조체 및 핵산 전달체를 포함하는 조성물을 제공한다. In addition, the present invention provides a composition comprising a nucleic acid construct and a nucleic acid delivery system according to the present invention.

본 발명에서, 용어 “핵산 전달체(nucleic acid delivery system)”는 본 발명에 따른 핵산 구조체의 체내로의 전달효율을 높이기 위한 것으로, 체내에서의 안정성이 우수할 뿐 아니라 약물로서의 제조 공정이 간단하다는 장점이 있다.In the present invention, the term “nucleic acid delivery system” is for increasing the delivery efficiency of the nucleic acid construct according to the present invention into the body, and has the advantage of excellent stability in the body as well as a simple manufacturing process as a drug. There is this.

상기 핵산 전달체는, 이에 제한되는 것은 아니나, 바이러스성 벡터, 비바이러스성 벡터, 리포좀, 양이온성 고분자, 미셀(micelle), 에멀젼 및 나노입자(nanoparticles)를 포함할 수 있고, 바람직하게는 나노입자일 수 있다. 상기 양이온성 고분자에는 키토산, 아텔로콜라겐(atelocollagen), 양이온성 폴리펩타이드(cationic polypeptide) 등과 같은 천연고분자와 poly(L-lysin), 선형 또는 분지형 PEI(polyethylene imine), 사이클로덱스트린계열 다가양이온(cyclodextrin-based polycation), 덴드리머(dendrimer) 등과 같은 합성고분자를 포함한다.The nucleic acid delivery system may include, but is not limited to, viral vectors, non-viral vectors, liposomes, cationic polymers, micelles, emulsions, and nanoparticles, preferably nanoparticles. I can. The cationic polymer includes natural polymers such as chitosan, atelocollagen, cationic polypeptide, etc., poly(L-lysin), linear or branched polyethylene imine (PEI), cyclodextrin-based polycation ( and synthetic polymers such as cyclodextrin-based polycation) and dendrimers.

본 발명에 있어서, 상기 나노입자는 이에 제한되는 것은 아니나, 자성입자(magnetic bead), 금(Au) 나노입자, 은(Ag) 나노입자, 백금(Pt) 나노입자, 양자점(Quantum dot), 상방전환 나노입자(upconversion nanoparticle, UCNP), 그래핀(graphene)-나노입자 복합체, 색 염색 나노입자(color dyed particles) 및 라텍스(latex) 나노입자, 실리카 나노입자(silica nanoparticle), 탄소유도체 나노입자(carbon derivative nanopartilces; 예컨대, 탄소나노튜브(carbon nanotube) 및 고형리피드 나노입자(solid lipid nanoparticles)로 이루어지는 군에서 선택되는 어느 하나일 수 있다.In the present invention, the nanoparticles are not limited thereto, but magnetic beads, gold (Au) nanoparticles, silver (Ag) nanoparticles, platinum (Pt) nanoparticles, quantum dots, upper side Upconversion nanoparticles (UCNP), graphene-nanoparticle complexes, color dyed particles and latex nanoparticles, silica nanoparticles, carbon derivative nanoparticles ( carbon derivative nanopartilces; For example, it may be any one selected from the group consisting of carbon nanotubes and solid lipid nanoparticles.

본 발명에 따른 핵산 구조체는 상기와 같은 나노입자에 도입되는 경우 상기 핵산 구조체의 타겟 세포로의 전달이 촉진되어, 비교적 낮은 농도의 투여량으로도 타겟 세포로 전달되어 높은 타겟 단백질 발현 유도 및 표적 유전자 발현 억제 기능을 나타낼 수 있다. 또한, 타겟 이외의 타 장기 및 세포로의 비 특이적인 mRNA-siRNA의 전달을 방지할 수 있다.When the nucleic acid construct according to the present invention is introduced into the above-described nanoparticles, the delivery of the nucleic acid construct to the target cell is promoted, and the nucleic acid construct is delivered to the target cell even at a relatively low concentration to induce high target protein expression and target genes. It can exhibit an expression inhibitory function. In addition, it is possible to prevent the delivery of non-specific mRNA-siRNA to other organs and cells other than the target.

또한, 본 발명은 본 발명에 따른 핵산 구조체를 포함하는 면역치료용 약학적 조성물을 제공한다. 본 발명에 따른 약학적 조성물은 면역 반응을 증가시키거나, 특정 질병, 감염 또는 질환의 치료 또는 예방에 바람직한 면역 반응의 일부를 선택적으로 상승시킬 수 있다.In addition, the present invention provides a pharmaceutical composition for immunotherapy comprising the nucleic acid construct according to the present invention. The pharmaceutical composition according to the present invention may increase the immune response, or may selectively increase some of the immune responses that are desirable for the treatment or prevention of certain diseases, infections or diseases.

본 발명에 있어서, 상기 면역치료는 바람직하게는 암 면역치료일 수 있다.In the present invention, the immunotherapy may preferably be cancer immunotherapy.

본 발명에 있어서, 상기 암은 뇌종양, 양성성상세포종, 악성성상세포종, 뇌하수체 선종, 뇌수막종, 뇌림프종, 핍지교종, 두개내인종, 상의세포종, 뇌간종양, 두경부 종양, 후두암, 구인두암, 비강/부비동암, 비인두암, 침샘암, 하인두암, 갑상선암, 흉부종양, 소세포성 폐암, 비소세포성 폐암, 흉선암, 종격동 종양, 식도암, 유방암, 남성유방암, 복부종양, 위암, 간암, 담낭암, 담도암, 췌장암, 소장암, 대장암, 항문암, 방광암, 신장암, 남성생식기종양, 음경암, 요도암, 전립선암, 여성생식기종양, 자궁경부암, 자궁내막암, 난소암, 자궁육종, 질암, 여성외부생식기암, 여성요도암, 피부암, 골수종, 백혈병 및 악성 림프종으로 이루어진 군으로부터 선택되는 어느 하나일 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the cancer is a brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendroglioma, intracranial tumor, epistemoma, brain stem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal/sinus cancer , Nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, gastric cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, Small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, urethral cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer , Female urethral cancer, skin cancer, myeloma, leukemia, and malignant lymphoma may be any one selected from the group consisting of, but is not limited thereto.

본 발명에 있어서, 상기 약학적 조성물은 본 발명에 따른 핵산 구조체를 단독으로 포함하거나 하나 이상의 약학적으로 허용되는 담체, 부형제 또는 희석제를 포함하여 제공될 수 있다.In the present invention, the pharmaceutical composition may be provided by including the nucleic acid construct according to the present invention alone or including one or more pharmaceutically acceptable carriers, excipients, or diluents.

상기 "약학적으로 허용되는"이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 의미하며, 상기 담체, 부형제 및 희석제의 예로는 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 폴리비닐피롤리톤, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있으나 이에 제한되는 것은 아니다. The term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause an allergic reaction such as gastrointestinal disorders, dizziness or similar reactions when administered to a human, and examples of the carrier, excipient and diluent Lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolitone, water, methyl Hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils, but are not limited thereto.

본 발명에 따른 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 세프 테졸에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calciumcarbonate), 수크로스(sucrose) 또는 락토오스(lactose), 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 단순희석제인 물, 리퀴드 파라핀 이외에 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(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.The pharmaceutical composition according to 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. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient, such as starch, calcium carbonate, and sucrose. ) Or lactose (lactose), gelatin, etc. are mixed to prepare. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

상기 약학적 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나, 바람직한 효과를 위해서, 1일 0.0001 내지 100mg/kg으로, 바람직하게는 0.001 내지 100mg/kg으로 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The preferred dosage of the pharmaceutical composition varies depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, it is good to administer at 0.0001 to 100 mg/kg per day, preferably at 0.001 to 100 mg/kg. Administration may be administered once a day, or may be divided several times. The above dosage does not limit the scope of the present invention in any way.

상기 약학적 조성물은 다양한 경로로 투여될 수 있으며, 예를 들어 경구, 직장 또는 정맥, 근육, 피하, 복강, 자궁내 경막 또는 뇌혈관내 주사에 의해 투여될 수 있다. The pharmaceutical composition may be administered by various routes, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intraperitoneal, intrauterine dural or cerebrovascular injection.

또한, 본 발명은 본 발명에 따른 핵산 구조체를 포함하는 암 백신을 제공한다. 본 발명에 따른 핵산 구조체는 항원제시세포에 항원을 표지하여 면역반응을 유도시킬 수 있으며, 동시에 면역 관문 단백질의 발현을 억제할 수 있다. 상기 핵산 구조체는 나노입자와 결합한 상태로 암 백신에 포함될 수 있다.In addition, the present invention provides a cancer vaccine comprising the nucleic acid construct according to the present invention. The nucleic acid construct according to the present invention can induce an immune response by labeling the antigen presenting cell with an antigen, and at the same time inhibit the expression of an immune checkpoint protein. The nucleic acid construct may be included in a cancer vaccine in a state in which the nanoparticles are bound.

본 발명에 있어서, 상기 암 백신은 수지상세포를 더 포함하는 것일 수 있다. 예를 들어, 상기 암 백신은 생체 외(ex vivo)에서 본 발명에 따른 핵산 구조체를 수지상세포에 전달한 다음, 형질감염된 수지상세포를 전달하는 형태로 제공될 수 있다. 상기 수지상세포는 대상체의 면역원성을 증가시킬 수 있어, 대상체 내 종양의 증식 및/또는 전이를 효과적으로 예방 또는 억제할 수 있다.In the present invention, the cancer vaccine may further include dendritic cells. For example, the cancer vaccine may be provided in the form of delivering the nucleic acid construct according to the present invention to dendritic cells ex vivo, and then delivering the transfected dendritic cells. The dendritic cells can increase the immunogenicity of the subject, and thus can effectively prevent or inhibit the proliferation and/or metastasis of a tumor in the subject.

본 명세서에서 용어, "수지상세포"는 항원을 세포 내부로 흡수하고, 이를 처리하여 항원 또는 항원으로부터 유래된 펩타이드를 MHC 클라스 I 복합체 또는 MHC 클래스 복합체와 함께 제시하는 항원제시세포(antigen presenting cell)를 의미한다. 수지상세포는 성숙도에 따라 미성숙 수지상세포와 성숙 수지상세포로 분류한다.As used herein, the term "dendritic cell" refers to an antigen presenting cell that absorbs an antigen into a cell and processes it to present an antigen or a peptide derived from the antigen together with the MHC class I complex or the MHC class complex. it means. Dendritic cells are classified into immature dendritic cells and mature dendritic cells according to their maturity.

상기 "미성숙 수지상세포"는 성숙 초기 단계에서 발견되는 것으로서, 성숙 수지상세포와 마찬가지로 CD14와 같은 세포 표면 마커를 발현하지 않으며, HLA-DR, CD86, CD80, CD83 또는 CD40을 낮은 수준으로 발현하고, CD1a 및 CCR1, CCR2, CCR5 및 CXCR1을 통상적 수준으로 발현하는 수지상세포를 말한다.The "immature dendritic cells" are found in the early stages of maturation, do not express cell surface markers such as CD14 like mature dendritic cells, and express HLA-DR, CD86, CD80, CD83 or CD40 at low levels, and CD1a And dendritic cells expressing CCR1, CCR2, CCR5 and CXCR1 at normal levels.

상기 "성숙 수지상세포"는 미성숙 수지상세포가 성숙화되어 형성된 세포를 의미하며, B 세포 및 T 세포 활성에 관여하는 세포 표면 마커, 예를 들어 MHC 클라스 I 또는 MHC 클래스 (HLA-DR), 세포 부착인자(CD54, CD18, CD11), 공동 자극 인자(예를 들어, CD86, CD80, CD83 또는 CD40)이 미성숙 수지상세포에 비하여 높은 수준 또는 상대적으로 증가된 수준으로 발현하는 세포를 의미한다.The "mature dendritic cells" refers to cells formed by maturation of immature dendritic cells, and cell surface markers involved in B cell and T cell activity, for example, MHC class I or MHC class (HLA-DR), cell adhesion factor (CD54, CD18, CD11), co-stimulatory factor (eg, CD86, CD80, CD83 or CD40) refers to a cell that expresses at a higher or relatively increased level compared to immature dendritic cells.

예를 들어, 본 발명의 일 실시예에서는 본 발명에 따른 핵산 구조체에 오브알부민(Ovalbumin, OVA)을 발현하는 mRNA 및 STAT3 유전자의 발현을 억제하는 siRNA를 도입한 mRNA-siSTAT3을 형질주입한 결과, CD40 및 CD80 마커의 발현량이 현저히 증가하여 분리된 mRNA 및 siRNA를 혼합하여 투여하는 것보다 수지상세포 성숙 촉진 효과가 훨씬 우수함을 확인하였다.For example, in one embodiment of the present invention, as a result of transfecting the nucleic acid construct according to the present invention with mRNA-siSTAT3 into which mRNA expressing Ovalbumin (OVA) and siRNA that suppresses the expression of STAT3 gene were introduced, It was confirmed that the expression levels of CD40 and CD80 markers were significantly increased, and the effect of promoting dendritic cell maturation was much better than that of administering the isolated mRNA and siRNA mixed.

또한, 본 발명은In addition, the present invention

1) 3’ 말단에 5' 방향에서 3' 방향으로 순차적으로 위치하는 폴리(T) 테일(poly(T) tail) 및 siRNA 안티센스 또는 센스 서열이 도입된 DNA 주형을 제조하는 단계; 1) preparing a DNA template into which a poly(T) tail and siRNA antisense or sense sequence are sequentially positioned in the 3'direction from the 5'direction to the 3'end;

2) DNA 주형에 RNA 중합효소를 포함하는 반응용액을 처리하여 3’ 말단에 siRNA 서열을 포함하는 mRNA를 얻는 단계; 및 2) treating a reaction solution containing RNA polymerase in a DNA template to obtain an mRNA containing an siRNA sequence at the 3'end; And

3) 3' 말단에 Oligo(dT)가 연결된 siRNA를 상기 mRNA 산물에 어닐링하는 단계; 를 포함하는 mRNA 및 siRNA를 포함하는 핵산 구조체의 제조방법을 제공한다.3) annealing the siRNA to which the Oligo (dT) is linked to the 3'end to the mRNA product; It provides a method for producing a nucleic acid construct comprising mRNA and siRNA comprising a.

상기 1) 단계에 있어서, DNA 주형은 mRNA 합성을 위한 전사주형으로서, T7 프로모터(promoter), 5’ UTR, 오픈 리딩 프레임(open reading frame, ORF) 및 3’ UTR을 포함하는 DNA 벡터(pUCIDT-Amp vector)를 제조하였고, 상기 DNA를 주형으로 PCR을 수행하여 벡터를 선형화한 후 증폭시킨 것일 수 있다.In the step 1), the DNA template is a transcription template for mRNA synthesis, a DNA vector comprising a T7 promoter, 5'UTR, open reading frame (ORF), and 3'UTR (pUCIDT- Amp vector) was prepared, and PCR was performed using the DNA as a template to linearize the vector and then amplify it.

PCR은 DNA 벡터를 1차 주형으로 하여 PCR 반응 혼합액, 정방향 프라이머(forward primer)로서 5’-TTGGACCCTCGTACAGAAGCTAATACG-3’(서열번호 4)으로 이루어진 올리고뉴클레오티드, 역방향 프라이머(reverse primer)로서 “5’ - siRNA 서열 - 폴리(A) - DNA 벡터와 특이적으로 결합하는 프라이머 서열 - 3’ (역방향 프라이머)” 구성에 기반한 것을 사용하여 수행하였다. 구체적으로, 상기 역방향 프라이머는 폴리(A) 서열 및/또는 siRNA(센스/안티센스) 서열을 포함하여, 제조된 DNA 주형의 3' UTR 바로 옆 3’에 폴리(T) 테일(poly(T) tail) 및 siRNA(안티센스/센스) 서열을 도입시켰다. 본 발명의 일 실시예에 있어서, 상기 PCR 반응 혼합액은 2X TOPsimpleTM DryMIX-HOT(enzynomics, 한국)을 사용하였으나, 이에 제한되는 것은 아니다.PCR is a PCR reaction mixture using a DNA vector as the primary template, an oligonucleotide consisting of 5'-TTGGACCCTCGTACAGAAGCTAATACG-3' (SEQ ID NO: 4) as a forward primer, and "5'-siRNA as a reverse primer. Sequence-poly(A)-primer sequence that specifically binds to the DNA vector-3'(reverse primer)" was carried out using a configuration based one. Specifically, the reverse primer includes a poly (A) sequence and/or siRNA (sense/antisense) sequence, and a poly (T) tail 3'immediately next to the 3'UTR of the prepared DNA template. ) And siRNA (antisense/sense) sequences were introduced. In one embodiment of the present invention, the PCR reaction mixture was 2X TOPsimple TM DryMIX-HOT (enzynomics, Korea), but is not limited thereto.

DNA 주형을 제조하기 위한 PCR의 조건은 PCR 수행을 위한 3단계, 즉 변성(Denaturation) 단계, 혼성(Annealing) 단계, 연장(Elongation) 단계를 수행하기 위한 온도 및 시간을 각각 유지할 수 있고, 상기 변성 단계를 수행하기 위한 조건은 85℃ 내지 105℃ 5분 내지 15분, 바람직하게는 95℃ 10분, 상기 혼성 단계를 수행하기 위한 조건은 40℃ 내지 65℃ 20초 내지 40초, 바람직하게는 59℃ 30초, 상기 연장 단계를 수행하기 위한 조건은 50℃ 내지 80℃ 30초 내지 2분, 바람직하게는 72℃ 1분일 수 있다. 상기 PCR 수행을 위한 특정된 온도 및 시간 범위는 PCR을 수행함에 있어서 실현 가능한 범위 내에서 조절 가능하다.The conditions of PCR for preparing the DNA template can be maintained at the temperature and time for performing the three steps for performing PCR, that is, the denaturation step, the annealing step, and the elongation step, and the denaturation Conditions for performing the step are 85 ℃ to 105 ℃ 5 minutes to 15 minutes, preferably 95 ℃ 10 minutes, conditions for performing the hybrid step is 40 ℃ to 65 ℃ 20 seconds to 40 seconds, preferably 59 ℃ 30 seconds, conditions for performing the extending step may be 50 ℃ to 80 ℃ 30 seconds to 2 minutes, preferably 72 ℃ 1 minute. The specified temperature and time range for performing the PCR can be adjusted within a range that is feasible in performing the PCR.

본 발명의 일 실시예에서, 상기 DNA 주형을 제조하기 위한 PCR의 조건은 초기변성(Initial denaturation) 95℃ 10분 후, 변성 95℃ 3초, 혼성 59℃ 30초, 연장 72℃ 1분을 1회전으로 총 30회전을 진행하고, 추가 연장(Final elongation) 72℃ 5분을 진행하였다. 또한, 증폭된 PCR 반응산물은 AccuPrep®PCR Purification Kit(Bioneer, South Korea)를 사용하여 정제하였다.In one embodiment of the present invention, the conditions of PCR for preparing the DNA template are after initial denaturation 95°C for 10 minutes, denaturation 95°C for 3 seconds, hybridization 59°C for 30 seconds, and extension 72°C for 1 minute. A total of 30 rotations were performed by rotation, and a final elongation was performed at 72° C. for 5 minutes. In addition, the amplified PCR reaction product was purified using AccuPrep® PCR Purification Kit (Bioneer, South Korea).

상기 2) 단계는 시험관 내 전사(in vitro transcription, IVT) 단계로, 1) 단계에서 제조한 DNA 주형에 RNA 중합효소를 포함하는 반응용액을 처리하여 3’ 말단에 siRNA 서열을 포함하는 mRNA(“mRNA-siRNA”)를 제조하는 것을 특징으로 할 수 있다. 본 발명의 일 실시예에서, 상기 시험관 내 전사는 제조한 DNA 주형에 반응용액을 처리하여 37℃에서 16시간 동안 배양한 뒤, Turbo DNase I를 1μl 추가하여 37℃에서 15분간 반응시켜 수행하였다. 다만, 상기 시험관 내 전사를 위한 구체적인 반응조건은 실현 가능한 범위 내에서 조절 가능하다.The step 2) is an in vitro transcription (IVT) step, and the DNA template prepared in step 1) is treated with a reaction solution containing RNA polymerase, and the mRNA containing the siRNA sequence at the 3'end (“ mRNA-siRNA”) may be characterized by production. In one embodiment of the present invention, the in vitro transcription was performed by treating the prepared DNA template with a reaction solution and incubating for 16 hours at 37°C, and then adding 1 μl of Turbo DNase I to react at 37°C for 15 minutes. However, the specific reaction conditions for in vitro transfer can be adjusted within a realizable range.

상기 3) 단계에 있어서, 어닐링(annealing)을 위한 조건은 서열-의존적이며 환경적 변수에 따라 다양하나, 40 내지 80℃ 5분 내지 15분, 바람직하게는 75℃ 10분일 수 있다. 본 발명의 일 실시예에서, 상기 어닐링은 75℃ 10분 수행 후 4℃까지 패스트 쿨링(급냉)하여 수행하였다.In step 3), the conditions for annealing are sequence-dependent and vary depending on environmental variables, but may be 40 to 80°C for 5 minutes to 15 minutes, preferably 75°C for 10 minutes. In an embodiment of the present invention, the annealing was performed by fast cooling (quick cooling) to 4°C after 10 minutes at 75°C.

이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐 당해 기술분야에서 통상의 지식을 가진 자라면 이로부터 다양한 변형 및 균등한 다른 실시예가 가능하다는 점을 이해할 것이다. 따라서 본 발명의 진정한 기술적 보호 범위는 첨부된 특허청구 범위의 기술적 사상에 의하여 정해져야 할 것이다.Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and those of ordinary skill in the art will understand that various modifications and equivalent other examples are possible therefrom. Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.

실시예Example . 본 발명에 따른 핵산 구조체의 제조. Preparation of nucleic acid construct according to the present invention

DNA 주형 제조DNA template preparation

유전자 발현 및 억제가 동시에 가능한 핵산 구조체를 제조하기 위해, 다음과 같이 실험을 수행하였다. 먼저, mRNA 합성을 위한 전사주형으로서 T7 프로모터(promoter), 5' UTR, 오픈 리딩 프레임(open reading frame, ORF) 및 3' UTR을 포함하는 DNA 벡터(pUCIDT-Amp vector)를 제조하였다. 상기 DNA를 주형으로 PCR을 수행하여 벡터를 선형화하고 T7 프로모터부터 3' UTR까지 증폭시켰다. PCR은 TOPsimple™ DryMIX-HOT(enzynomics, 한국)을 사용하여 수행하였고, 구체적인 PCR 조건은 하기 표 1과 같다. 증폭된 PCR 반응산물은 AccuPrep® PCR Purification Kit(Bioneer, South Korea)를 사용하여 정제하였다.In order to prepare a nucleic acid construct capable of simultaneous gene expression and suppression, an experiment was performed as follows. First, as a transcription template for mRNA synthesis, a DNA vector (pUCIDT-Amp vector) including a T7 promoter, 5'UTR, open reading frame (ORF), and 3'UTR was prepared. The DNA was subjected to PCR as a template to linearize the vector and amplify it from the T7 promoter to the 3'UTR. PCR was performed using TOPsimple™ DryMIX-HOT (enzynomics, Korea), and specific PCR conditions are shown in Table 1 below. The amplified PCR reaction product was purified using AccuPrep® PCR Purification Kit (Bioneer, South Korea).

[표 1][Table 1]

Figure 112019101328436-pat00002
Figure 112019101328436-pat00002

이 때, 폴리(A) 서열 및/또는 siRNA(센스/안티센스) 서열을 포함하는 역방향 프라이머(Reverse Primer, RP)를 사용하여 3’ UTR 바로 옆 3’에 폴리(T) 테일(poly(T) tail) 및 siRNA(안티센스/센스) 서열이 도입된 DNA 주형을 제조하였다(도 1의 a). 즉, 상기 역방향 프라이머는 다음 구성에 기반한 것을 사용하였다.In this case, using a reverse primer (RP) containing a poly (A) sequence and/or an siRNA (sense/antisense) sequence, a poly (T) tail (poly(T)) immediately next to the 3'UTR 3' tail) and siRNA (antisense/sense) sequences were introduced into a DNA template (Fig. 1 a). That is, the reverse primer was used based on the following configuration.

5' - 5'- siRNAsiRNA 서열 - Sequence- 폴리Poly (A) - DNA 벡터와 특이적으로 결합하는 (A)-specifically binding to the DNA vector 프라이머primer 서열 - 3' (역방향 Sequence-3'(reverse 프라이머primer ))

본 실시예에 있어서, 본 발명자들은 상기 역방향 프라이머로 120 nt 폴리(A) 서열만으로 이루어진 프라이머, 120 nt 폴리(A) 서열과 siRNA(센스/안티센스) 서열을 포함하는 프라이머, 녹색 형광 단백질(Green Fluorescent Protein, GFP) mRNA를 표적하는 siRNA(이하, siGFP), 빨간 형광 단백질(Red fluorescent Protein, RFP) 및 파랑 형광 단백질(Blue fluorescent Protein, BFP)을 코딩하는 mRNA를 사용하였다. In this example, the present inventors used a primer comprising only a 120 nt poly (A) sequence as the reverse primer, a primer comprising a 120 nt poly (A) sequence and an siRNA (sense/antisense) sequence, and a green fluorescent protein. Protein, GFP) mRNA targeting siRNA (hereinafter, siGFP), red fluorescent protein (RFP), and mRNA encoding blue fluorescent protein (BFP) were used.

mRNAmRNA -- siRNAsiRNA 합성 synthesis

상기 실시예에서 제조한 DNA 주형을 사용하여 시험관 내(in vitro) 전사(in vitro transcription, IVT)를 수행하여 말단에 siRNA 서열을 포함하는 mRNA(이하, “mRNA-siRNA(SS or AS)”라 한다)를 합성하였다. T7 RNA 중합효소, rATP, rCTP, rGTP, rUTP 및 캡 유사체(cap analogue)를 포함하는 반응 용액(MEGAscript™T7 Transcription Kit (Ambion, USA)을 사용하여 시험관 내 전사를 수행하고, DNA 분해효소(DNase) 처리로 주형 DNA를 제거하고 mRNA만을 회수하였다. Using a DNA template prepared in Example vitro (in vitro) transcription (in vitro transcription, IVT) mRNA ( hereinafter, "SS or AS) mRNA- siRNA (" containing siRNA sequences at the terminal by performing the LA To) was synthesized. Transcription was performed in vitro using a reaction solution containing T7 RNA polymerase, rATP, rCTP, rGTP, rUTP, and cap analogue (MEGAscript™ T7 Transcription Kit (Ambion, USA), and DNA degrading enzyme (DNase)). ), the template DNA was removed and only the mRNA was recovered.

구체적으로, 반응용액을 튜브에 넣고 37℃에서 16시간 동안 배양한 뒤, Turbo DNase I를 1μl 추가하여 37℃에서 15분간 반응시키고, 반응산물을 MEGAclear™ Kit(Ambion, USA)를 사용하여 정제하였다. 반응용액을 구성하는 모든 성분 및 이의 용량, 농도는 하기 표 2와 같다. 이를 통해 합성된 mRNA는 캡 유사체를 전사 반응에 첨가함으로써 생산된 5'-캡(5'-cap)을 5' 말단에 포함하였다(도 1의 b).Specifically, the reaction solution was put into a tube and incubated at 37°C for 16 hours, then 1 μl of Turbo DNase I was added to react at 37°C for 15 minutes, and the reaction product was purified using MEGAclear™ Kit (Ambion, USA). . All components constituting the reaction solution, and their capacity and concentration are shown in Table 2 below. The mRNA synthesized through this included a 5'-cap (5'-cap) produced by adding a cap analog to the transcriptional reaction at the 5'end (Fig. 1b).

[표 2][Table 2]

Figure 112019101328436-pat00003
Figure 112019101328436-pat00003

본 발명에 따른 핵산 구조체 제조Preparation of nucleic acid construct according to the present invention

다음으로, dTdTdTdTdTdT와 연결시킨 siRNA(siRNA-Oligo(dT))를 포함하는 역방향 프라이머를 상기 실시예에서 제조한 mRNA-siRNA(SS or AS)에 어닐링(annealing)하여, mRNA-siRNA(SS or AS)의 일부가 혼성화 서열과 상보적으로 결합하여 이중가닥을 형성하는, 본 발명에 따른 핵산 구조체(“mRNA-siRNA”를 제조하였다. 이 때, 상기 혼성화 서열은 폴리(T)를 포함하는 siRNA 서열 또는 폴리(T)를 포함하는 DNA 서열에 해당한다. Next, a reverse primer containing siRNA (siRNA-Oligo (dT)) linked with dTdTdTdTdTdT was annealed to the mRNA-siRNA (SS or AS) prepared in the above example, and mRNA-siRNA (SS or AS A nucleic acid construct according to the present invention (“mRNA-siRNA”) was prepared in which a part of) was complementarily combined with a hybridization sequence to form a double strand. In this case, the hybridization sequence was a siRNA sequence including poly(T). Or it corresponds to a DNA sequence containing poly (T).

구체적으로, 어닐링은 mRNA-siRNA(SS or AS)와 siRNA-Oligo(dT)를 동일한 튜브에 넣어 혼합하고, 75℃에서 10분간 반응시킨 뒤, 4℃까지 패스트 쿨링(급냉)하였다. 이 때, dTdTdTdTdTdT 부위는 mRNA-siRNA(SS or AS)의 폴리(A) 테일(poly(A) tail)에 어닐링되고, siRNA 서열(안티센스/센스)은 mRNA-siRNA(SS or AS)의 3' 말단에 도입된 siRNA에 상보적으로 어닐링되었고(도 1의 c), 그 결과 mRNA-siRNA(SS or AS) 3’ 말단의 폴리(A) 테일 및 siRNA 서열(센스/안티센스)이 각각 이와 상보적인 siRNA(안티센스/센스) 및 oligo(dT)와 결합하여 이중가닥을 형성하는 본 발명에 따른 핵산 구조체가 형성되었다(이하, “(또는 mRNA-siRNA/siRNA 또는 mRNA-siRNA/DNA)”라 한다). 상기 mRNA-siRNA의 제조과정을 도식화하여 도 2에 나타내었다. Specifically, for annealing, mRNA-siRNA (SS or AS) and siRNA-Oligo (dT) were put in the same tube and mixed, reacted at 75° C. for 10 minutes, and then fast cooled to 4° C. (quick cooling). At this time, the dTdTdTdTdTdT site is annealed to the poly(A) tail of the mRNA-siRNA (SS or AS), and the siRNA sequence (antisense/sense) is 3'of the mRNA-siRNA (SS or AS). It was annealed complementarily to the siRNA introduced at the end (Fig. 1 c), and as a result, the poly(A) tail and siRNA sequence (sense/antisense) at the 3'end of the mRNA-siRNA (SS or AS) were complementary to this, respectively. A nucleic acid construct according to the present invention that forms a double strand by binding with siRNA (antisense/sense) and oligo (dT) was formed (hereinafter referred to as “(or mRNA-siRNA/siRNA or mRNA-siRNA/DNA”)) . A schematic diagram of the preparation process of the mRNA-siRNA is shown in FIG. 2.

본 실시예 및 실험예에서 사용한 DNA 벡터, 모든 프라이머 서열, 모든 siRNA 서열을 하기 표 3에 나타내었다. 모든 서열은 IDT(유전자 합성서비스)로부터 제공받아 사용하였다.The DNA vectors, all primer sequences, and all siRNA sequences used in this Example and Experimental Example are shown in Table 3 below. All sequences were provided and used from IDT (gene synthesis service).

유전자 이름Gene name 서열 정보Sequence information 서열번호Sequence number pUCIDT-AMP_mTagBFP2pUCIDT-AMP_mTagBFP2 TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAAATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCATCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCAACGCGATGACGATGGATAGCGATTCATCGATGAGCTGACCCGATCGCCGCCGCCGGAGGGTTGCGTTTGAGACGGGCGACAGATGAGGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTAGAGGATCGAACCCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGTGTCTAAGGGCGAAGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACCGTGGACAACCATCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAGGGCACCCAGACCATGAGAATCAAGGTGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCGACATCCTGGCTACTAGCTTCCTCTACGGCAGCAAGACCTTCATCAACCACACCCAGGGCATCCCCGACTTCTTCAAGCAGTCCTTCCCTGAGGGCTTCACATGGGAGAGAGTCACCACATACGAAGACGGGGGCGTGCTGACCGCTACCCAGGACACCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCAAGATCAGAGGGGTGAACTTCACATCCAACGGCCCTGTGATGCAGAAGAAAACACTCGGCTGGGAGGCCTTCACCGAGACGCTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAAACGACATGGCCCTGAAGCTCGTGGGCGGGAGCCATCTGATCGCAAACGCCAAGACCACATATAGATCCAAGAAACCCGCTAAGAACCTCAAGATGCCTGGCGTCTACTATGTGGACTACAGACTGGAAAGAATCAAGGAGGCCAACAACGAGACCTACGTCGAGCAGCACGAGGTGGCAGTGGCCAGATACTGCGACCTCCCTAGCAAACTGGGGCACAAGCTTAATGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTGAGGGTCTAGAACTAGTGTCGACGCAAATCAGTTCTGGACCAGCGAGCTGTGCTGCGACTCGTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGTCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTACCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAAATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCATCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCAACGCGATGACGATGGATAGCGATTCATCGATGAGCTGACCCGATCGCCGCCGCCGGAGGGTTGCGTTTGAGACGGGCGACAGATGAGGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTAGAGGATCGAACCCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGTGTCTAAGGGCGAAGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACCGTGGACAACCATCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAGGGCACCCAGACCATGAGAATCAAGGTGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCGACATCCTGGCTACTAGCTTCCTCTACGGCAGCAAGACCTTCATCAACCACACCCAGGGCATCCCCGACTTCTTCAAGCAGTCCTTCCCTGAGGGCTTCACATGGGAGAGAGTCACCACATACGAAGACGGGGGCGTGCTGAC CGCTACCCAGGACACCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCAAGATCAGAGGGGTGAACTTCACATCCAACGGCCCTGTGATGCAGAAGAAAACACTCGGCTGGGAGGCCTTCACCGAGACGCTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAAACGACATGGCCCTGAAGCTCGTGGGCGGGAGCCATCTGATCGCAAACGCCAAGACCACATATAGATCCAAGAAACCCGCTAAGAACCTCAAGATGCCTGGCGTCTACTATGTGGACTACAGACTGGAAAGAATCAAGGAGGCCAACAACGAGACCTACGTCGAGCAGCACGAGGTGGCAGTGGCCAGATACTGCGACCTCCCTAGCAAACTGGGGCACAAGCTTAATGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTGAGGGTCTAGAACTAGTGTCGACGCAAATCAGTTCTGGACCAGCGAGCTGTGCTGCGACTCGTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAA ATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGTCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTC GCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTACCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC 1One pUCIDT-AMP_DsRed Express2 RFPpUCIDT-AMP_DsRed Express2 RFP TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAAATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCATCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCAACGCGATGACGATGGATAGCGATTCATCGATGAGCTGACCCGATCGCCGCCGCCGGAGGGTTGCGTTTGAGACGGGCGACAGATGAGGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTAGAGGATCGAACCCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGATAGCACTGAGAACGTCATCAAGCCCTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCAAGCCCTACGAGGGCACCCAGACCGCCAAGCTGCAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCCAGTACGGCTCCAAGGTGTACGTGAAGCACCCCGCCGACATCCCCGACTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCACCTTCATCTACCACGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACTCTGGGCTGGGAGCCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCCACAAGGCGCTGAAGCTGAAGGGCGGCGGCCACTACCTGGTGGAGTTCAAGTCAATCTACATGGCCAAGAAGCCCGTGAAGCTGCCCGGCTACTACTACGTGGACTCCAAGCTGGACATCACCTCCCACAACGAGGACTACACCGTGGTGGAGCAGTACGAGCGCGCCGAGGCCCGCCACCACCTGTTCCAGTAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTGAGGGTCTAGAACTAGTGTCGACGCAAATCAGTTCTGGACCAGCGAGCTGTGCTGCGACTCGTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGTCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTACCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAAATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCATCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCAACGCGATGACGATGGATAGCGATTCATCGATGAGCTGACCCGATCGCCGCCGCCGGAGGGTTGCGTTTGAGACGGGCGACAGATGAGGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTAGAGGATCGAACCCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGATAGCACTGAGAACGTCATCAAGCCCTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCAAGCCCTACGAGGGCACCCAGACCGCCAAGCTGCAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCCAGTACGGCTCCAAGGTGTACGTGAAGCACCCCGCCGACATCCCCGACTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGT GACCCAGGACTCCTCCCTGCAGGACGGCACCTTCATCTACCACGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACTCTGGGCTGGGAGCCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCCACAAGGCGCTGAAGCTGAAGGGCGGCGGCCACTACCTGGTGGAGTTCAAGTCAATCTACATGGCCAAGAAGCCCGTGAAGCTGCCCGGCTACTACTACGTGGACTCCAAGCTGGACATCACCTCCCACAACGAGGACTACACCGTGGTGGAGCAGTACGAGCGCGCCGAGGCCCGCCACCACCTGTTCCAGTAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTGAGGGTCTAGAACTAGTGTCGACGCAAATCAGTTCTGGACCAGCGAGCTGTGCTGCGACTCGTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGTCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCAT TGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTACCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC 22 pOVApOVA gatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattcctcgaaagacaactcagagttcaccatgggctccatcggtgcagcaagcatggaattttgttttgatgtattcaaggagctcaaagtccaccatgccaatgagaacatcttctactgccccattgccatcatgtcagctctagccatggtatacctgggtgcaaaagacagcaccaggacacaaataaataaggttgttcgctttgataaacttccaggattcggagacagtattgaagctcagtgtggcacatctgtaaacgttcactcttcacttagagacatcctcaaccaaatcaccaaaccaaatgatgtttattcgttcagccttgccagtagactttatgctgaagagagatacccaatcctgccagaatacttgcagtgtgtgaaggaactgtatagaggaggcttggaacctatcaactttcaaacagctgcagatcaagccagagagctcatcaattcctgggtagaaagtcagacaaatggaattatcagaaatgtccttcagccaagctccgtggattctcaaactgcaatggttctggttaatgccattgtcttcaaaggactgtgggagaaagcatttaaggatgaagacacacaagcaatgcctttcagagtgactgagcaagaaagcaaacctgtgcagatgatgtaccagattggtttatttagagtggcatcaatggcttctgagaaaatgaagatcctggagcttccatttgccagtgggacaatgagcatgttggtgctgttgcctgatgaagtctcaggccttgagcagcttgagagtataatcaactttgaaaaactgactgaatggaccagttctaatgttatggaagagaggaagatcaaagtgtacttacctcgcatgaagatggaggaaaaatacaacctcacatctgtcttaatggctatgggcattactgacgtgtttagctcttcagccaatctgtctggcatctcctcagcagagagcctgaagatatctcaagctgtccatgcagcacatgcagaaatcaatgaagcaggcagagaggtggtagggtcagcagaggctggagtggatgctgcaagcgtctctgaagaatttagggctgaccatccattcctcttctgtatcaagcacatcgcaaccaacgccgttctcttctttggcagatgtgtttccccttaaaaagaagaaagctgaaaaactctgtcccttccaacaagacccagagcactgtagtatcaggggtaaaatgaaaagtatgttatctgctgcatccagacttcataaaagctggagcttaatctagagggccctattctatagtgtcacctaaatgctagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgc caggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaacc gtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcc tgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacggg ccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattcctcgaaagacaactcagagttcaccatgggctccatcggtgcagcaagcatggaattttgttttgatgtattcaaggagctcaaagtccaccatgccaatgagaacatcttctactgccccattgccatcatgtcagctctagccatggtatacctgggtgcaaaagacagcaccaggacacaaataaataaggttgttcgctttgataaacttccaggattcggagacagtattgaagctcagtgtggcacatctgtaaacgttcactcttcact tagagacatcctcaaccaaatcaccaaaccaaatgatgtttattcgttcagccttgccagtagactttatgctgaagagagatacccaatcctgccagaatacttgcagtgtgtgaaggaactgtatagaggaggcttggaacctatcaactttcaaacagctgcagatcaagccagagagctcatcaattcctgggtagaaagtcagacaaatggaattatcagaaatgtccttcagccaagctccgtggattctcaaactgcaatggttctggttaatgccattgtcttcaaaggactgtgggagaaagcatttaaggatgaagacacacaagcaatgcctttcagagtgactgagcaagaaagcaaacctgtgcagatgatgtaccagattggtttatttagagtggcatcaatggcttctgagaaaatgaagatcctggagcttccatttgccagtgggacaatgagcatgttggtgctgttgcctgatgaagtctcaggccttgagcagcttgagagtataatcaactttgaaaaactgactgaatggaccagttctaatgttatggaagagaggaagatcaaagtgtacttacctcgcatgaagatggaggaaaaatacaacctcacatctgtcttaatggctatgggcattactgacgtgtttagctcttcagccaatctgtctggcatctcctcagcagagagcctgaagatatctcaagctgtccatgcagcacatgcagaaatcaatgaagcaggcagagaggtggtagggtcagcagaggctggagtggatgctgcaagcgtctctgaagaatttagggctgaccatccattcctcttctgtatcaagcacatcgcaaccaacgccgttctcttctttggcagatgtgtttccccttaaaaagaagaaagctgaaaaactctgtcccttccaacaagacccagagcactgtagtatcaggggtaaaatgaaaagtatgttatctgctgc atccagacttcataaaagctggagcttaatctagagggccctattctatagtgtcacctaaatgctagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattctttt 33 Forward primer- pUCIDT-AMP_mTagBFP2Forward primer- pUCIDT-AMP_mTagBFP2 5'-TTGGACCCTCGTACAGAAGCTAATACG-3'
5'-TTGGACCCTCGTACAGAAGCTAATACG-3'
 44 Reverse primer- pUCIDT-AMP_mTagBFP2-siGFP (AS)Reverse primer- pUCIDT-AMP_mTagBFP2-siGFP (AS) 5'-AAG CAA GCT GAC CCT GAA GTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTT CCT ACT CAG GCT TTA TTC AAA GAC CA-3' (Italic: sequence for antisense strand of siGFP ) 5'- AAG CAA GCT GAC CCT GAA GTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTT CCT ACT CAG GCT TTA TTA CA-3' (Italic: sequence for antisense strand of siGFP ) 55 SS siGFPSS siGFP 5′-GCA AGC UGA CCC UGA AGU U (dT)2 or 6-3′5′-GCA AGC UGA CCC UGA AGU U (dT) 2 or 6 -3′ 6, 76, 7 Reverse primer- pUCIDT-AMP_mTagBFP2-siGFP (SS)Reverse primer- pUCIDT-AMP_mTagBFP2-siGFP (SS) 5'-AAA ACT TCA GGG TCA GCT TGC TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTT CCT ACT CAG GCT TTA TTC AAA GAC CA-3'
(Italic: sequence for sense strand of siGFP ) 5'- AAA ACT TCA GGG TCA GCT TGC TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTT CCT ACT CAG GCT TTA TTA CA-3'
(Italic: sequence for sense strand of siGFP ) 88 AS siGFPAS siGFP 5′-AAC UUC AGG GUC AGC UUG C (dT) 2 or 6-3′5′-AAC UUC AGG GUC AGC UUG C (dT) 2 or 6 -3′ 9, 109, 10 Forward primer- pUCIDT-AMP_DsRed Express2 RFPForward primer- pUCIDT-AMP_DsRed Express2 RFP 5'-TTGGACCCTCGTACAGAAGCTAATACG-3'5'-TTGGACCCTCGTACAGAAGCTAATACG-3' 44 Reverse primer- pUCIDT-AMP_DsRed Express2 RFP-siGFP (AS)Reverse primer- pUCIDT-AMP_DsRed Express2 RFP-siGFP (AS) 5'-AAG CAA GCT GAC CCT GAA GTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTT CCT ACT CAG GCT TTA TTC AAA GAC CA-3' (Italic: sequence for antisense strand of siGFP ) 5'- AAG CAA GCT GAC CCT GAA GTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTT CCT ACT CAG GCT TTA TTA CA-3' (Italic: sequence for antisense strand of siGFP ) 55 SS siGFPSS siGFP 5′-GCA AGC UGA CCC UGA AGU U (dT)2 or 6-3′5′-GCA AGC UGA CCC UGA AGU U (dT) 2 or 6 -3′ 6, 76, 7 Reverse primer- pUCIDT-AMP_DsRed Express2 RFP-siGFP (SS)Reverse primer- pUCIDT-AMP_DsRed Express2 RFP-siGFP (SS) 5'-AAA ACT TCA GGG TCA GCT TGC TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTT CCT ACT CAG GCT TTA TTC AAA GAC CA-3'
(Italic: sequence for sense strand of siGFP ) 5'- AAA ACT TCA GGG TCA GCT TGC TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTT CCT ACT CAG GCT TTA TTA CA-3'
(Italic: sequence for sense strand of siGFP ) 88 AS siGFPAS siGFP 5′-AAC UUC AGG GUC AGC UUG C (dT) 2 or 6-3′5′-AAC UUC AGG GUC AGC UUG C (dT) 2 or 6 -3′ 9, 109, 10 Forward primer- pOVAForward primer- pOVA 5'-GAGAACCCACTGCTTACTGG-3'5'-GAGAACCCACTGCTTACTGG-3' 1111 Reverse primer- pOVA-siSTAT3 (AS)Reverse primer- pOVA-siSTAT3 (AS) 5'-AAA GTA GGT GCA GGA ACT GTG TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TCA GCG AGC TCT AGC ATT TAG G-3'
(Italic: sequence for antisense strand of siSTAT3 ) 5'- AAA GTA GGT GCA GGA ACT GTG TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TCA GCG TAG AGC TCT G-3'
(Italic: sequence for antisense strand of siSTAT3 ) 1212 SS siSTAT3SS siSTAT3 5′-AGU AGG UGC AGG AAC UGU G (dT) 2 or 6-3′5′-AGU AGG UGC AGG AAC UGU G (dT) 2 or 6 -3′ 13, 1413, 14 Reverse primer- pOVA-siSTAT3 (SS)Reverse primer- pOVA-siSTAT3 (SS) 5'-AAC ACA GTT CCT GCA CCT ACT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TCA GCG AGC TCT AGC ATT TAG G-3'
(Italic: sequence for sense strand of siSTAT3 ) 5'- AAC ACA GTT CCT GCA CCT ACT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TCA GCG AGC TCT AGC ATT -3'
(Italic: sequence for sense strand of siSTAT3 ) 1515 AS siSTAT3AS siSTAT3 5′-CAC AGU UCC UGC ACC UAC U(dT) 2 or 6-3′5′-CAC AGU UCC UGC ACC UAC U(dT) 2 or 6 -3′ 16, 1716, 17

본 발명에 따른 mRNA-siRNA는 세포 내 환경에서 Rnase H에 의해 mRNA의 폴리(A)와 siRNA의 폴리(T) 간에 형성된 이중가닥 혼성체가 절단되어, 타겟 단백질을 발현시킬 수 있는 mRNA와 특정 유전자의 발현을 억제시킬 수 있는 siRNA로 분리되고, 이를 통해 대상체에서 단백질의 발현과 억제를 동시에 가능하게 한다(도 3). The mRNA-siRNA according to the present invention is a double-stranded hybrid formed between poly(A) of mRNA and poly(T) of siRNA by Rnase H in the intracellular environment. It is separated into siRNA capable of inhibiting expression, thereby enabling simultaneous expression and suppression of the protein in the subject (Fig. 3).

한편, 도 3에 나타낸 바와 같이, 본 발명에 따른 mRNA-siRNA는 mRNA의 3' 말단에 siRNA가 하나 이상 연결될 수 있는 것을 특징으로 한다. 이 때, 첫번째 siRNA의 도입 이후 두번째 siRNA부터는 상기 어닐링과 동일한 조건으로 실험을 수행하되, 역방향 프라이머에 siRNA 서열을 추가하여 진행한다. 추가되는 siRNA의 경우, 역방향 프라이머에 대하여 도입하고자 하는 개수만큼 siRNA를 도입하여 추가할 수 있음을 특징으로 한다. 예를 들어, 두번째 siRNA를 도입하기 위해 사용한 모든 프라이머를 하기 표 4에 나타내었다.On the other hand, as shown in Figure 3, the mRNA-siRNA according to the present invention is characterized in that one or more siRNA can be linked to the 3'end of the mRNA. At this time, from the second siRNA after the introduction of the first siRNA, the experiment is performed under the same conditions as the annealing, but the siRNA sequence is added to the reverse primer. In the case of an added siRNA, it is characterized in that it can be added by introducing as many siRNAs as the number to be introduced with respect to the reverse primer. For example, all primers used to introduce the second siRNA are shown in Table 4 below.

유전자 이름Gene name 서열 정보Sequence information 서열번호Sequence number Forward primer- pUCIDT-AMP_mTagBFP2Forward primer- pUCIDT-AMP_mTagBFP2 TTGGACCCTCGTACAGAAGCTAATACGTTGGACCCTCGTACAGAAGCTAATACG 44 Reverse primer- pUCIDT-AMP_mTagBFP2-siGFP (2개) (AS)Reverse primer- pUCIDT-AMP_mTagBFP2-siGFP (2EA) (AS) AAG CAA GCT GAC CCT GAA GTT AAAA AAG CAA GCT GAC CCT GAA GTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTT CCT ACT CAG GCT TTA TTC AAA GAC CA(Italic: sequence for antisense strand of siGFP 2개) AAG CAA GCT GAC CCT GAA GTT AAAA AAG CAA GCT GAC CCT GAA GTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTT CCT ACT CAG GAC TTA TTC AAA(Italic: sequence for antisense strand of siGFP 2) 1818 Reverse primer- pUCIDT-AMP_mTagBFP2-siGFP (2개) (SS)Reverse primer- pUCIDT-AMP_mTagBFP2-siGFP (2EA) (SS) AAA ACT TCA GGG TCA GCT TGC AAAA AAA ACT TCA GGG TCA GCT TGC TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTT CCT ACT CAG GCT TTA TTC AAA GAC CA(Italic: sequence for sense strand of siGFP 2개) AAA ACT TCA GGG TCA GCT TGC AAAA AAA ACT TCA GGG TCA GCT TGC TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTC TTT TTT TTT TTT TTT TTT CTT CCT ACT CAG GCT T GAC CA (Italic: sequence for sense strand of siGFP 2ea ) 1919 AS siGFPAS siGFP 5′-AAC UUC AGG GUC AGC UUG C (dT) 2 or 6-3′5′-AAC UUC AGG GUC AGC UUG C (dT) 2 or 6 -3′ 9, 109, 10 SS siGFPSS siGFP 5′-GCA AGC UGA CCC UGA AGU U (dT)2 or 6-3′5′-GCA AGC UGA CCC UGA AGU U (dT) 2 or 6 -3′ 6, 76, 7

본 실시예에 있어서, 본 발명자들은 하기와 같이 siRNA의 센스 또는 안티센스 서열, 길이를 달리하는 Oligo(dT)를 다양한 구성으로 조합한 siRNA-Oligo(dT)를 사용하여 mRNA의 단백질 발현 효율 및 siRNA의 유전자 발현 억제능을 평가하고, 유전자 발현 및 억제 효과가 우수한 핵산 구조체의 최적 구조를 도출하고자 하였다(도 4).In this example, the present inventors use siRNA-Oligo (dT), which combines the sense or antisense sequence of siRNA and Oligo (dT) of different lengths in various configurations as follows, The ability to inhibit gene expression was evaluated, and an optimal structure of a nucleic acid construct having excellent gene expression and inhibitory effects was attempted (FIG. 4).

5' - 5'- siGFP와siGFP and 상보적으로 결합하는 Complementary siRNAsiRNA 서열( order( 안티센스Antisense /센스) - /sense) - dTdTdTdT - 3' (T2) -3'(T2)

5’ - 5’- siGFP와siGFP and 상보적으로 결합하는 Complementary siRNAsiRNA 서열( order( 안티센스Antisense /센스) - dTdTdTdTdTdT - 3’ (T6) /Sense)-dTdTdTdTdTdT-3’ (T6)

5’ - 5’- siGFP와siGFP and 상보적으로 결합하는 DNA 서열( DNA sequences that bind complementarily ( 안티센스Antisense /센스) - /sense) - dTdTdTdTdTdTdTdT - 3’ (DNA) -3’ (DNA)

실험예Experimental example 1. 각 1. Each 반응산물의Reaction product 확인 Confirm

상기 실시예의 각 반응이 끝난 후, 반응 산물을 1% 아가로스 겔에 100V 전압으로 30분간 전기영동하여 PCR 반응 산물, 시험관 내 전사 산물을 확인한 결과를 도 5에 나타내었다.After each reaction in the above example was completed, the reaction product was electrophoresed on a 1% agarose gel at a voltage of 100 V for 30 minutes to confirm the PCR reaction product and the in vitro transcription product, and the results are shown in FIG. 5.

도 5의 a에 나타낸 바와 같이, 역방향 프라이머를 사용하여 증폭된 DNA 단편의 크기는 각각 971 bp(poly(A)120, 레인 4) 및 992 bp(poly(A)120-AS siGFP, 레인 5)임을 확인하였다. 상기 결과를 통해, PCR을 수행함에 따라 3’ UTR 바로 옆 3’에 폴리(T) tail 및 siRNA 서열이 도입되도록 디자인된 역방향 프라이머를 통해 DNA 주형이 효과적으로 증폭되었음을 확인하였다.As shown in FIG. 5A, the sizes of the DNA fragments amplified using reverse primers were 971 bp (poly(A) 120, lane 4) and 992 bp (poly(A) 120-AS siGFP, lane 5), respectively. Was confirmed. Through the above results, it was confirmed that the DNA template was effectively amplified through reverse primers designed to introduce poly(T) tail and siRNA sequences into 3′ immediately next to the 3′ UTR as PCR was performed.

또한, 도 5의 b에 나타낸 바와 같이, 상기 DNA 주형을 사용하여 시험관 내 전사를 수행한 결과, 각각 3' 말단에 poly(A)120 서열이 도입된 mRFP(레인 2) 및 3' 말단에 poly(A)120-AS siGFP 서열이 도입된 mRFP(레인 3)가 합성되었음을 확인하였다.In addition, as shown in b of FIG. 5, as a result of performing in vitro transcription using the DNA template, mRFP (lane 2) and poly(Lane 2) in which poly(A)120 sequences were introduced at the 3'ends, respectively, (A) It was confirmed that mRFP (lane 3) into which the 120-AS siGFP sequence was introduced was synthesized.

마찬가지로, 상기 실시예에 기재한 방법으로 제조한 mRNA-siRNA 핵산 구조체를 확인한 결과를 도 6에 나타내었다.Similarly, the result of confirming the mRNA-siRNA nucleic acid construct prepared by the method described in the above Example is shown in FIG. 6.

도 6의 a에 나타낸 바와 같이, mRNA의 3' 말단에 poly(A)120 서열만 도입된 것과 poly(A)120과 안티센스 siGFP 서열이 함께 도입된 형태의 mRFP를 센스 siGFP 가닥과 패스트 쿨링을 시킨 결과, poly(A)120과 안티센스 siGFP 서열이 함께 도입된 형태의 mRFP에 주입한 센스 siGFP의 밴드가 옅어지는 것을 확인함으로써 센스 siGFP가 성공적으로 poly(A)120과 안티센스 siGFP 서열이 도입된 mRFP에 어닐링되었음을 확인하였다.As shown in FIG. 6A, the poly(A)120 sequence was introduced at the 3′ end of the mRNA, and the mRFP in the form of the poly(A)120 and antisense siGFP sequence introduced together was fast cooled with the sense siGFP strand. As a result, it was confirmed that the band of the sense siGFP injected into the mRFP in which the poly(A)120 and antisense siGFP sequences were introduced together was confirmed to be lightened, so that the sense siGFP was successfully introduced into the mRFP into which the poly(A)120 and antisense siGFP sequences were introduced. It was confirmed that it was annealed.

또한, 도 6의 b에 나타낸 바와 같이, FITC 표지된 센스 siGFP(FITC-SS siGFP, 레인 1)보다, 3' 말단에 안티센스 siGFP가 도입된 mRFP(mRFP-AS siGFP)에 대하여 FITC 표지된 센스 siGFP가 어닐링된 경우(mRFP-AS siGFP + FITC-SS siGFP, 레인 2) 밴드가 더 위쪽에서 나타남을 확인하였다. 상기 결과를 통해, 3' 말단에 siRNA 서열을 포함하는 mRNA와 상기 siRNA 서열에 상보적으로 결합하는 siRNA를 어닐링시키면, 상호 간에 부분적으로 혼성화된 mRNA-siRNA 핵산 구조체가 형성됨을 확인하였다.In addition, as shown in b of Figure 6, FITC-labeled sense siGFP for mRFP (mRFP-AS siGFP) with antisense siGFP introduced at the 3'end than FITC-labeled sense siGFP (FITC-SS siGFP, lane 1) When was annealed (mRFP-AS siGFP + FITC-SS siGFP, lane 2), it was confirmed that the band appeared from the upper side. Through the above results, it was confirmed that the mRNA-siRNA nucleic acid structure partially hybridized to each other was formed when the mRNA containing the siRNA sequence at the 3'end and the siRNA complementarily bound to the siRNA sequence were annealed.

실험예Experimental example 2. 각 구조체의 단백질 발현 능력 평가 2. Evaluation of protein expression ability of each construct

상기 실시예에서 제조한 서로 다른 mRNA-siRNA 핵산 구조체의 단백질 발현 효과를 확인하였다. 구체적으로, 세포를 96-웰 플레이트에 웰당 1 x 104개로 시딩하고, 형질주입 시약을 사용하여 세포를 10 pg mRNA/cell의 용량의 각 구조체로 형질주입(transfection)시켰다. 형질주입 24시간 후, 유세포 분석기 FACS Calibur(BD Biosciences, CA)를 사용하여 각 실시예에서 발현되는 빨간 형광 단백질의 형광 강도를 측정하여 각 구조체의 번역 효율(translation efficiency)을 확인하고 그 결과를 도 7에 나타내었다. 실험에 사용한 모든 실시예를 하기 표 5에 나타내었다. 하기 표 5에 있어서, mRNA-SS/SS 또는 mRNA-SS/AS는 siGFP-T2 서열과 어닐링 된 것을 의미하며, mRNA-AS/SS(DNA) 또는 mRNA-AS/AS(DNA)는 siGFP 서열을 가지고 있는 DNA에 추가적으로 dT 서열이 4개 도입된 서열과 어닐링 된 것을 의미한다.Protein expression effects of different mRNA-siRNA nucleic acid constructs prepared in the above example were confirmed. Specifically, cells were seeded at 1 x 10 4 per well in a 96-well plate, and cells were transfected with each construct at a dose of 10 pg mRNA/cell using a transfection reagent. 24 hours after transfection, the fluorescence intensity of the red fluorescent protein expressed in each example was measured using a flow cytometer FACS Calibur (BD Biosciences, CA) to confirm the translation efficiency of each construct and the result was plotted. It is shown in 7. All examples used in the experiment are shown in Table 5 below. In Table 5 below, mRNA-SS/SS or mRNA-SS/AS means that the siGFP-T2 sequence is annealed, and mRNA-AS/SS (DNA) or mRNA-AS/AS (DNA) represents the siGFP sequence. It means that the dT sequence was annealed with the introduced sequence of 4 additional dT sequences.

본 발명의 실시예Examples of the present invention mRNA-siRNA(SS or AS)
(5'-3')mRNA-siRNA (SS or AS)
(5'-3') siRNA 및/또는 DNA (5'-3')siRNA and/or DNA (5'-3') 실시예Example mRNAmRNA siRNAsiRNA siRNAsiRNA Oligo(dT)Oligo(dT) -- -- -- -- BlankBlank mRFPmRFP -- -- -- mRNAmRNA siGFP(SS)siGFP(SS) -- -- mRNA-SSmRNA-SS siGFP(SS)siGFP(SS) TT(T2)TT(T2) mRNA-SS/SSmRNA-SS/SS siGFP(AS)siGFP(AS) TT(T2)TT(T2) mRNA-SS/ASmRNA-SS/AS siGFP(SS) (DNA)siGFP(SS) (DNA) TTTT(DNA)TTTT (DNA) mRNA-SS/SS(DNA)mRNA-SS/SS (DNA) siGFP(AS) (DNA)siGFP(AS) (DNA) TTTT(DNA)TTTT (DNA) mRNA-SS/AS(DNA)mRNA-SS/AS (DNA) siGFP(AS)siGFP(AS) -- -- mRNA-ASmRNA-AS siGFP(SS)siGFP(SS) TT(T2)TT(T2) mRNA-AS/SSmRNA-AS/SS siGFP(AS)siGFP(AS) TT(T2)TT(T2) mRNA-AS/ASmRNA-AS/AS siGFP(SS) (DNA)siGFP(SS) (DNA) TTTT(DNA)TTTT (DNA) mRNA-AS/SS(DNA)mRNA-AS/SS (DNA) siGFP(AS) (DNA)siGFP(AS) (DNA) TTTT(DNA)TTTT (DNA) mRNA-AS/AS(DNA)mRNA-AS/AS (DNA)

도 7에 나타낸 바와 같이, 3' 말단에 siRNA 서열을 포함하는 mRNA를 형질주입한 실시예에서 공통적으로 번역 효율이 낮아진 것을 확인하였다. 반면, mRNA(SS)에 siGFP(AS)-TTTT(DNA)를 어닐링시킨 실시예와 mRNA(AS)에 siGFP(SS)-TTTT(DNA)를 어닐링시킨 실시예의 경우 감소된 번역 효율이 현저히 증가하며, mRFP을 형질주입한 경우보다도 형광 강도가 강하여 mRNA의 단백질 발현 능력이 완전히 회복되었음을 확인하였다.As shown in FIG. 7, it was confirmed that the translation efficiency was lowered in common in the examples in which mRNA containing an siRNA sequence was transfected at the 3'end. On the other hand, in the example in which siGFP (AS)-TTTT (DNA) was annealed to mRNA (SS) and the example in which siGFP (SS)-TTTT (DNA) was annealed to mRNA (AS), the reduced translation efficiency was significantly increased. , It was confirmed that the fluorescence intensity was stronger than that of the case where mRFP was transfected, and the protein expression ability of the mRNA was completely recovered.

상기 결과를 통해, 3' 말단에 siRNA(센스/안티센스) 서열을 포함하는 mRNA와, 상기 siRNA에 상보적으로 결합하는 siRNA(안티센스/센스) 서열 및 mRNA에 상보적으로 결합하는 Oligo(dT)를 포함하는 siRNA를 혼성화시키면, siRNA 서열의 결합에 의해 저해된 mRNA의 번역 효율이, mRNA-siRNA(SS or AS)의 siRNA 서열에 어닐링(annealing)된 DNA 서열에 의해 회복될 수 있음을 확인하였다. 이는 본 발명에 따른 mRNA-siRNA/DNA 하이브리드 구조체가 세포 내 RNase H에 의해 잘려나가는 기전에 의한 것으로 보인다. 또한, mRNA-siRNA(SS or AS)에 어닐링시킨 siRNA-dTdT(T2)의 경우 단백질 번역 효율이 저해되는 것을 확인함으로써 mRNA-siRNA(SS or AS)와 어닐링된 DNA의 시퀀스의 길이가 적어도 2개 이상은 되어야 세포 내 RNase H에 의해 추가적으로 도입된 siRNA 시퀀스가 잘려나가 mRNA 단백 번역 효율이 회복될 수 있음을 알 수 있었다. 따라서, 이후 실험에서는 mRNA-siRNA(SS or AS)에 siRNA-dTdTdTdTdTdT(T6)를 어닐링시켜 세포 내 도입 시 RNase H에 의해 mRNA와 siRNA로 분리되어 mRNA의 단백질 번역 효율 및 siRNA의 단백질 억제 효율이 유지될 수 있도록 디자인하였다.Through the above results, an mRNA containing an siRNA (sense/antisense) sequence at the 3'end, an siRNA (antisense/sense) sequence complementarily binding to the siRNA, and an Oligo (dT) complementarily binding to the mRNA When the included siRNA is hybridized, it was confirmed that the translation efficiency of the mRNA inhibited by the binding of the siRNA sequence can be recovered by the DNA sequence annealed to the siRNA sequence of the mRNA-siRNA (SS or AS). This seems to be due to the mechanism by which the mRNA-siRNA/DNA hybrid construct according to the present invention is cut off by intracellular RNase H. In addition, in the case of siRNA-dTdT (T2) annealed to mRNA-siRNA (SS or AS), it was confirmed that protein translation efficiency was inhibited, so that the length of the sequence of mRNA-siRNA (SS or AS) and annealed DNA was at least two. It was found that the efficiency of mRNA protein translation could be restored after the siRNA sequence additionally introduced by RNase H in the cell was cut off. Therefore, in subsequent experiments, siRNA-dTdTdTdTdTdT(T6) was annealed to mRNA-siRNA (SS or AS), and when introduced into cells, it was separated into mRNA and siRNA by RNase H, maintaining the efficiency of mRNA protein translation and siRNA protein suppression efficiency. It was designed to be.

실험예Experimental example 3. 각 구조체의 단백질 발현 능력 및 유전자 발현 억제 능력 평가 3. Evaluation of protein expression ability and gene expression inhibition ability of each structure

상기 실시예에서 제조한 서로 다른 mRNA-siRNA 구조체의 단백질 발현 능력 및 유전자 발현 억제 효과를 확인하였다. 구체적으로, 세포를 96-웰 플레이트에 웰당 1 x 104개로 시딩하고, 형질주입 시약을 사용하여 세포를 10 pg mRNA/cell의 용량의 각 구조체로 형질주입(transfection)시켰다. 형질주입 24, 48시간 후, 유세포 분석기 FACS Calibur(BD Biosciences, CA)를 사용하여 각 실시예에서 발현되는 파란 형광 단백질 및 녹색 형광 단백질의 형광 강도를 측정하여 각 구조체의 단백질 발현 효율을 확인하고 그 결과를 도 8 및 도 9에 나타내었다. 실험에 사용한 모든 실시예를 하기 표 6에 나타내었다.The protein expression ability and gene expression inhibition effect of the different mRNA-siRNA constructs prepared in the above example were confirmed. Specifically, cells were seeded at 1 x 10 4 per well in a 96-well plate, and cells were transfected with each construct at a dose of 10 pg mRNA/cell using a transfection reagent. After 24 and 48 hours of transfection, the fluorescence intensity of the blue and green fluorescent proteins expressed in each example was measured using a flow cytometer FACS Calibur (BD Biosciences, CA) to confirm the protein expression efficiency of each construct. The results are shown in FIGS. 8 and 9. All examples used in the experiment are shown in Table 6 below.

mRNA-siRNA(SS or AS)
(5'-3')mRNA-siRNA (SS or AS)
(5'-3') siRNA 및/또는 DNA (5'-3')siRNA and/or DNA (5'-3') 실시예Example mRNAmRNA siRNAsiRNA siRNAsiRNA Oligo(dT)Oligo(dT) -- -- -- -- BlankBlank mBFPmBFP -- -- -- mBFPmBFP -- -- siGFPsiGFP TT(T2)TT(T2) siGFP-T2siGFP-T2 -- -- siGFPsiGFP TTTTTT(T6)TTTTTT(T6) siGFP-T6siGFP-T6 mBFPmBFP siGFP(SS)siGFP(SS) -- -- mBFP-SSmBFP-SS siGFP(AS)siGFP(AS) TT(T2)TT(T2) mBFP-SS/AS-T2mBFP-SS/AS-T2 siGFP(AS)siGFP(AS) TTTTTT(T6)TTTTTT(T6) mBFP-SS/AS-T6mBFP-SS/AS-T6 siGFP(AS)siGFP(AS) TTTT(DNA)TTTT (DNA) mBFP-SS/AS(DNA)mBFP-SS/AS (DNA) siGFP(AS)siGFP(AS) -- -- mBFP-ASmBFP-AS siGFP(SS)siGFP(SS) TT(T2)TT(T2) mBFP-AS/SS-T2mBFP-AS/SS-T2 siGFP(SS)siGFP(SS) TTTTTT(T6)TTTTTT(T6) mBFP-AS/SS-T6mBFP-AS/SS-T6 siGFP(SS)siGFP(SS) TTTT(DNA)TTTT (DNA) mBFP-AS/SS(DNA)mBFP-AS/SS (DNA)

도 8의 a에 나타낸 바와 같이, 3’ 말단에 siRNA 서열을 포함하는 mRNA를 형질주입한 실시예에서 파란 형광 강도가 감소했으며, 이를 통해 siRNA 서열을 연결함에 따라 단백질 발현 효율이 낮아진 것을 확인하였다. 반면, mBFP(SS)에 siGFP(AS)-TTTTTT(T6), siGFP(AS)-TTTT(DNA)를 어닐링시킨 실시예와 mBFP(AS)에 siGFP(SS)-TTTTTT(T6), siGFP(SS)-TTTT(DNA)를 어닐링시킨 실시예의 경우 감소된 번역 효율이 현저히 증가하며, mBFP을 형질주입한 경우보다도 형광 강도가 강하여 mRNA의 단백질 발현 능력이 완전히 회복되었음을 확인하였다. As shown in FIG. 8A, in the example in which the mRNA containing the siRNA sequence at the 3'end was transfected, the blue fluorescence intensity was decreased, and it was confirmed that the protein expression efficiency was lowered by linking the siRNA sequence through this. On the other hand, mBFP (SS) siGFP (AS) -TTTTTT (T6), siGFP (AS)-TTTT (DNA) annealing example and mBFP (AS) siGFP (SS) -TTTTTT (T6), siGFP (SS In the case of )-TTTT (DNA) annealing, the reduced translation efficiency was remarkably increased, and the fluorescence intensity was stronger than that of the mBFP-transfected case, confirming that the protein expression ability of mRNA was completely recovered.

상기 결과를 통해, 3’ 말단에 siRNA(센스/안티센스) 서열을 포함하는 mRNA와, 상기 siRNA에 상보적으로 결합하는 siRNA(안티센스/센스) 서열을 포함하는 siRNA를 혼성화시키는 경우(mRNA-siRNA), mRNA에 siRNA 서열을 연결시킴에 따른 번역 효율의 감소를 극복할 수 있음을 확인하였다. 나아가, 감소된 번역 효율은 어닐링되는 siRNA의 3' 말단에 위치하는 Oligo(dT)가 T2인 경우보다는 T6이, T6인 경우보다는 T4일 때 현저히 증가함을 확인하였다. Through the above results, when hybridizing an mRNA comprising an siRNA (sense/antisense) sequence at the 3'end and an siRNA comprising an siRNA (antisense/sense) sequence complementarily binding to the siRNA (mRNA-siRNA) , It was confirmed that the reduction in translation efficiency can be overcome by linking the siRNA sequence to the mRNA. Furthermore, it was confirmed that the reduced translation efficiency was significantly increased when the oligo (dT) located at the 3'end of the annealed siRNA was T6 rather than T2 and T4 rather than T6.

한편, 도 8의 b에 나타낸 바와 같이, mBFP(SS)에 siGFP(AS)-TTTTTT(T6)을 어닐링시킨 실시예와 mBFP(AS)에 siGFP(SS)-TTTTTT(T6)을 어닐링시킨 실시예에서 녹색 형광 강도가 감소했으며, 특히 측정된 형광 강도가 정상 siRNA를 형질주입한 경우와 유사함을 확인하였다. On the other hand, as shown in b of Figure 8, mBFP (SS) in an embodiment of annealing siGFP (AS)-TTTTTT (T6) to mBFP (AS) and an embodiment of annealing siGFP (SS) -TTTTTT (T6) to mBFP (AS) It was confirmed that the green fluorescence intensity decreased, and in particular, the measured fluorescence intensity was similar to that of the normal siRNA transfected case.

상기 결과를 통해, mRNA의 단백질 발현과는 독립적으로, 3’ 말단에 siRNA(센스/안티센스) 서열을 포함하는 mRNA와, 상기 siRNA에 상보적으로 결합하는 siRNA(안티센스/센스) 서열을 포함하는 siRNA를 혼성화시키는 경우(mRNA-siRNA), 상기 siRNA가 표적하는 유전자의 발현이 현저히 억제됨을 확인하였다. 특히, 단백질 발현 억제 효율은 어닐링되는 siRNA의 3' 말단에 위치하는 Oligo(dT)가 T6인 경우 가장 높음을 확인하였다. Through the above results, independently of the protein expression of the mRNA, an mRNA comprising an siRNA (sense/antisense) sequence at the 3'end, and a siRNA comprising an siRNA (antisense/sense) sequence complementary to the siRNA When hybridizing (mRNA-siRNA), it was confirmed that the expression of the gene targeted by the siRNA was remarkably suppressed. In particular, it was confirmed that the protein expression inhibition efficiency was highest when the oligo (dT) located at the 3'end of the annealed siRNA was T6.

이와 같은 결과는 형질주입 48시간 후에도 유사하게 나타남을 확인하였다. 다만, 도 9의 a에 나타낸 바와 같이, mRNA에 siRNA 서열을 연결시킴에 따른 감소된 번역 효율은 어닐링되는 siRNA의 3’ 말단에 위치하는 Oligo(dT)가 T2 또는 T4일 때 가장 크게 증가하였다. 또한, 도 9의 b에 나타낸 바와 같이, 유전자 발현 억제 효율은 mBFP(AS)에 siGFP(SS)-TTTTTT(T6)을 어닐링시킨 실시예에서 가장 높았다.It was confirmed that these results were similar even after 48 hours of transfection. However, as shown in Fig. 9a, the reduced translation efficiency by linking the siRNA sequence to the mRNA was greatest when the oligo (dT) located at the 3'end of the siRNA to be annealed was T2 or T4. In addition, as shown in b of Fig. 9, the gene expression suppression efficiency was highest in the examples in which siGFP(SS)-TTTTTT(T6) was annealed to mBFP(AS).

상술한 분석 결과, 본 발명에 따른 mRNA-siRNA 핵산 구조체는 고효율로 세포에 동시에 도입되고, 도입된 후 세포 내 RNase에 의해 mRNA와 siRNA간에 형성된 이중가닥 혼성체가 절단되어 타겟 단백질을 발현시킬 수 있는 mRNA와 특정 유전자의 발현을 억제시킬 수 있는 siRNA로 분리됨을 확인하였다. 나아가, 이와 같은 특이적 구조를 가짐으로써, 본 발명에 따른 mRNA-siRNA 핵산 구조체는 하나의 세포에서 mRNA에 의한 타겟 단백질의 발현 및 siRNA에 의한 특정 유전자의 발현 억제를 동시에 성공적으로 달성할 수 있음을 확인하였다.As a result of the above analysis, the mRNA-siRNA nucleic acid construct according to the present invention is simultaneously introduced into the cell with high efficiency, and after the introduction, the double-stranded hybrid formed between the mRNA and the siRNA is cleaved by the intracellular RNase to express the target protein. And siRNA that can inhibit the expression of specific genes. Furthermore, by having such a specific structure, the mRNA-siRNA nucleic acid construct according to the present invention can simultaneously successfully achieve the expression of a target protein by mRNA and suppression of expression of a specific gene by siRNA in one cell. Confirmed.

실험예Experimental example 4. 본 발명에 따른 핵산 구조체의 면역원성 향상 능력 평가 4. Evaluation of the ability to improve immunogenicity of the nucleic acid construct according to the present invention

mRNA는 톨-유사 수용체(toll-like receptor, TLR)를 자극하여 면역반응을 유도하는 효과가 있다고 알려져 있다. 즉, mRNA는 자체적으로 면역반응 유도 효과가 있는 동시에 항원(단백질) 발현이 가능하여 별도의 어쥬번트(adjuvant) 없이도 백신으로 활용 가능하다. 한편, 이와 같은 mRNA 기반 백신은 수지상세포나 T 세포에 특정 항원을 발현시키는데, 이 때 STAT3 단백질은 면역반응 활성화를 억제하여 수지상세포의 성숙(maturation)을 억제한다. It is known that mRNA has the effect of inducing an immune response by stimulating a toll-like receptor (TLR). In other words, mRNA can be used as a vaccine without a separate adjuvant, as it has the effect of inducing an immune response and can express antigen (protein) at the same time. On the other hand, such an mRNA-based vaccine expresses specific antigens on dendritic cells or T cells. At this time, STAT3 protein inhibits maturation of dendritic cells by inhibiting activation of immune responses.

이에, 본 발명자들은 본 발명에 따른 핵산 구조체를 활용하여, 항원을 발현하도록 설계된 mRNA 구조체에 STAT3의 발현을 억제하는 siRNA 서열을 도입하면 면역회피 방해를 받지 않으면서 동시에 수지상세포의 성숙을 효과적으로 촉진할 수 있을 것으로 판단하고 다음과 같이 실험을 수행하였다. 먼저, 상기 실시예에 기재한 것과 동일한 방법으로, 본 발명에 따른 핵산 구조체에 오브알부민(Ovalbumin, OVA)을 발현하는 mRNA 및 STAT3 유전자의 발현을 억제하는 siRNA를 도입하였다. 비교실험군으로는 STAT3을 표적하는 siRNA(siSTAT3), 오브알부민을 발현하는 mRNA와 siSTAT3의 단순 혼합물(mOVA + siSTAT3)을 사용하였다. 실험에 사용한 모든 mRNA와 siRNA 서열은 상기 실시예 내 표 3에 기재하였다.Therefore, the present inventors utilize the nucleic acid construct according to the present invention to effectively promote the maturation of dendritic cells without interfering with immune evasion by introducing an siRNA sequence that inhibits the expression of STAT3 into an mRNA construct designed to express an antigen. It was judged to be possible and the experiment was conducted as follows. First, in the same manner as described in the above examples, mRNA expressing Ovalbumin (OVA) and siRNA inhibiting the expression of STAT3 gene were introduced into the nucleic acid construct according to the present invention. As a comparative experimental group, a simple mixture of STAT3 targeting siRNA (siSTAT3), ovalbumin-expressing mRNA and siSTAT3 (mOVA + siSTAT3) was used. All mRNA and siRNA sequences used in the experiment are listed in Table 3 in the above Examples.

4-1. 4-1. STAT3STAT3 단백질 발현 억제 효과 Protein expression inhibitory effect

골수-유래 수지상세포(bone marrow-derived dendritic cell, BMDC)에 상기 제조한 siSTAT3, mOVA 및 siSTAT3의 단순 혼합물(mOVA 및 siSTAT3의 단순 혼합물) 및 mOVA-siSTAT3/siSTAT3 구조체를 각각 처리하였다. 구체적으로, BMDC(1 × 106 cells/웰)를 10% FBS와 항생제(100 unit/ml 페니실린 및 100 μg/ml 스트렙토마이신)를 함유한 X-vivo 15 media으로 37℃에서 24시간 동안 24-웰 플레이트에서 배양하였다. 배양한 BMDC를 대상으로 siSTAT3, mOVA + siSTAT3 및 mOVA-siSTAT3/siSTAT3을 각각 형질주입하였다. 각 BMDC에서 STAT3 유전자의 발현 정도를 qRT-PCR 방법을 사용하여 분석하고, 그 결과를 도 10에 나타내었다.Bone marrow-derived dendritic cells (BMDC) were treated with a simple mixture of siSTAT3, mOVA, and siSTAT3 (a simple mixture of mOVA and siSTAT3) and mOVA-siSTAT3/siSTAT3 constructs, respectively. Specifically, BMDC (1 × 10 6 cells/well) was mixed with X-vivo 15 media containing 10% FBS and antibiotics (100 unit/ml penicillin and 100 μg/ml streptomycin) at 37°C for 24 hours. Incubated in well plates. The cultured BMDCs were transfected with siSTAT3, mOVA + siSTAT3, and mOVA-siSTAT3/siSTAT3, respectively. The degree of expression of the STAT3 gene in each BMDC was analyzed using the qRT-PCR method, and the results are shown in FIG. 10.

도 10에 나타낸 바와 같이, 본 발명에 따른 핵산 구조체에 mOVA 및 siSTAT3을 도입한 mOVA-siSTAT3/siSTAT3 처리 시, mOVA 및 siSTAT3을 단순 혼합한 mOVA + siSTAT3에 비해 STAT3 발현이 현저히 억제됨을 확인하였다. 상기 결과를 통해, 분리된 mRNA 및 siRNA를 혼합하여 투여하는 것보다 단백질 발현을 목적으로 하는 mRNA와 siRNA가 결합된 형태의 mRNA-siRNA 핵산 구조체를 투여하는 경우에 면역원성 향상 효과가 훨씬 우수함을 확인하였다.As shown in Figure 10, when mOVA-siSTAT3/siSTAT3 treatment with mOVA and siSTAT3 introduced into the nucleic acid construct according to the present invention, it was confirmed that STAT3 expression was significantly suppressed compared to mOVA + siSTAT3 simply mixed with mOVA and siSTAT3. From the above results, it was confirmed that the effect of improving immunogenicity is much better when the mRNA-siRNA nucleic acid construct in which the mRNA and siRNA for protein expression are combined is administered rather than a mixture of isolated mRNA and siRNA I did.

4-2. 4-2. 수지상세포Dendritic cells 성숙 촉진 효과 Maturation promotion effect

상기 실험예 4-1에 기재한 것과 동일한 방법으로 골수-유래 수지상세포(bone marrow-derived dendritic cell, BMDC)에 상기 제조한 siSTAT3, mOVA + siSTAT3 및 mOVA-siSTAT3/siSTAT3 구조체를 각각 처리하여 12시간 내지 48시간 동안 배양하였다. 일부 세포를 취하여 세포 표면에서 발현하는 CD40 및 CD80 마커의 발현양을 유세포분석(Fluorescence-activated cell sorting, FACS)을 통하여 분석하였다. 그 결과를 도 11에 나타내었다.The siSTAT3, mOVA + siSTAT3 and mOVA-siSTAT3/siSTAT3 structures prepared above were treated in bone marrow-derived dendritic cells (BMDC) in the same manner as described in Experimental Example 4-1 for 12 hours. To 48 hours. Some cells were taken and the amount of expression of the CD40 and CD80 markers expressed on the cell surface was analyzed through flow cytometry (Fluorescence-activated cell sorting, FACS). The results are shown in FIG. 11.

도 11에 나타낸 바와 같이, 본 발명에 따른 핵산 구조체에 mOVA 및 siSTAT3을 도입한 mOVA-siSTAT3/siSTAT3 처리 시, mOVA 및 siSTAT3을 단순 혼합한 mOVA + siSTAT3에 비해 성숙한 수지상세포의 표현형인 CD40 및 CD80의 발현이 모두 현저하게 증가함을 확인하였다. 상기 결과를 통해, 분리된 mRNA 및 siRNA를 혼합하여 투여하는 것보다 단백질 발현을 목적으로 하는 mRNA와 siRNA가 결합된 형태의 mRNA-siRNA 핵산 구조체를 투여하는 경우에 수지상세포 성숙 촉진 효과가 훨씬 우수함을 확인하였다.As shown in Figure 11, when mOVA-siSTAT3/siSTAT3 treatment with mOVA and siSTAT3 introduced into the nucleic acid construct according to the present invention, compared to mOVA + siSTAT3, which is a simple mixture of mOVA and siSTAT3, of CD40 and CD80, which are the phenotypes of mature dendritic cells. It was confirmed that all of the expression significantly increased. From the above results, it was found that the effect of promoting dendritic cell maturation was much better when the mRNA-siRNA nucleic acid construct in which the mRNA and siRNA for protein expression were combined is administered rather than a mixture of isolated mRNA and siRNA. Confirmed.

상기와 같은 실험들을 통해, 본 발명에 따른 mRNA-siRNA 핵산 구조체는 mRNA와 siRNA가 동시에 세포에 도입되어, mRNA는 발현하고자 하는 단백질을 정상적으로 발현하고, siRNA는 특정 유전자의 발현을 특이적으로 억제할 수 있음을 확인하였다. 상기 결과를 통해, 본 발명에 따른 핵산 구조체는 항원 제시 세포에 타겟 단백질을 발현시켜 면역체계를 활성화시킴과 동시에, 면역관문 단백질의 활성화를 차단하여 면역회피 방해를 극복할 수 있어, 면역치료 분야, 특히 항암면역치료 분야에서 매우 유용하게 사용될 수 있음을 확인하였다.Through the above experiments, the mRNA-siRNA nucleic acid construct according to the present invention allows mRNA and siRNA to be simultaneously introduced into cells, so that the mRNA normally expresses the protein to be expressed, and siRNA specifically inhibits the expression of a specific gene. It was confirmed that it can be. Through the above results, the nucleic acid construct according to the present invention can overcome the obstacle to immune evasion by blocking the activation of the immune checkpoint protein while at the same time activating the immune system by expressing the target protein in the antigen-presenting cell, In particular, it was confirmed that it can be very useful in the field of anticancer immunotherapy.

<110> Ewha University - Industry Collaboration Foundation <120> Nucleic acid construct for simultaneous gene expression and suppression <130> EWHA1-60P <160> 19 <170> KoPatentIn 3.0 <210> 1 <211> 3782 <212> DNA <213> Artificial Sequence <220> <223> pUCIDT-AMP_mTagBFP2 <400> 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accaaatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcatcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgcaac gcgatgacga tggatagcga 420 ttcatcgatg agctgacccg atcgccgccg ccggagggtt gcgtttgaga cgggcgacag 480 atgaggctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 540 tccatagaag acaccgggac cgatccagcc tccggactct agaggatcga acccttttgg 600 accctcgtac agaagctaat acgactcact atagggaaat aagagagaaa agaagagtaa 660 gaagaaatat aagagccacc atggtgtcta agggcgaaga gctgattaag gagaacatgc 720 acatgaagct gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg 780 gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc 840 ctctcccctt cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca 900 tcaaccacac ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat 960 gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc 1020 tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg 1080 gccctgtgat gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg 1140 ctgacggcgg cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc 1200 tgatcgcaaa cgccaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc 1260 ctggcgtcta ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct 1320 acgtcgagca gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc 1380 acaagcttaa tgctgccttc tgcggggctt gccttctggc catgcccttc ttctctccct 1440 tgcacctgta cctcttggtc tttgaataaa gcctgagtag gaagtgaggg tctagaacta 1500 gtgtcgacgc aaatcagttc tggaccagcg agctgtgctg cgactcgtgg cgtaatcatg 1560 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 1620 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 1680 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 1740 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 1800 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 1860 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 1920 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 1980 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 2040 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 2100 gtcgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 2160 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 2220 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 2280 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 2340 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 2400 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 2460 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 2520 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 2580 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 2640 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 2700 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 2760 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 2820 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 2880 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 2940 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 3000 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 3060 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 3120 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 3180 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 3240 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 3300 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 3360 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 3420 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 3480 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 3540 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactctacc tttttcaata 3600 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 3660 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta 3720 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg 3780 tc 3782 <210> 2 <211> 3749 <212> DNA <213> Artificial Sequence <220> <223> pUCIDT-AMP_DsRed Express2 RFP <400> 2 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accaaatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcatcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgcaac gcgatgacga tggatagcga 420 ttcatcgatg agctgacccg atcgccgccg ccggagggtt gcgtttgaga cgggcgacag 480 atgaggctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 540 tccatagaag acaccgggac cgatccagcc tccggactct agaggatcga acccttttgg 600 accctcgtac agaagctaat acgactcact atagggaaat aagagagaaa agaagagtaa 660 gaagaaatat aagagccacc atggatagca ctgagaacgt catcaagccc ttcatgcgct 720 tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg 780 agggcaagcc ctacgagggc acccagaccg ccaagctgca ggtgaccaag ggcggccccc 840 tgcccttcgc ctgggacatc ctgtcccccc agttccagta cggctccaag gtgtacgtga 900 agcaccccgc cgacatcccc gactacaaga agctgtcctt ccccgagggc ttcaagtggg 960 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc 1020 aggacggcac cttcatctac cacgtgaagt tcatcggcgt gaacttcccc tccgacggcc 1080 ccgtaatgca gaagaagact ctgggctggg agccctccac cgagcgcctg tacccccgcg 1140 acggcgtgct gaagggcgag atccacaagg cgctgaagct gaagggcggc ggccactacc 1200 tggtggagtt caagtcaatc tacatggcca agaagcccgt gaagctgccc ggctactact 1260 acgtggactc caagctggac atcacctccc acaacgagga ctacaccgtg gtggagcagt 1320 acgagcgcgc cgaggcccgc caccacctgt tccagtaggc tgccttctgc ggggcttgcc 1380 ttctggccat gcccttcttc tctcccttgc acctgtacct cttggtcttt gaataaagcc 1440 tgagtaggaa gtgagggtct agaactagtg tcgacgcaaa tcagttctgg accagcgagc 1500 tgtgctgcga ctcgtggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 1560 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 1620 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 1680 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 1740 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 1800 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 1860 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 1920 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 1980 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 2040 cctcgtgcgc tctcctgttc cgaccctgtc gcttaccgga tacctgtccg cctttctccc 2100 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 2160 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 2220 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 2280 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 2340 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 2400 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 2460 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 2520 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 2580 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 2640 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 2700 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 2760 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 2820 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 2880 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 2940 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 3000 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 3060 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 3120 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 3180 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 3240 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 3300 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 3360 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 3420 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 3480 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 3540 aatactcata ctctaccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 3600 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 3660 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa 3720 aaataggcgt atcacgaggc cctttcgtc 3749 <210> 3 <211> 6711 <212> DNA <213> Artificial Sequence <220> <223> pOVA <400> 3 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 60 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag 120 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg 180 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 240 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc 300 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg 360 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa 420 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt 480 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc 540 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc 600 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg 660 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag 720 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg 780 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg 840 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac 900 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg 960 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc 1020 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg 1080 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc 1140 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc 1200 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc 1260 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc 1320 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg 1380 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt 1440 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 1500 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 1560 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc 1620 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 1680 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 1740 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 1800 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 1860 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 1920 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 1980 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 2040 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 2100 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 2160 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 2220 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 2280 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 2340 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 2400 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 2460 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 2520 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 2580 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 2640 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 2700 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 2760 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 2820 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 2880 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 2940 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 3000 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 3060 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 3120 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 3180 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 3240 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 3300 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 3360 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 3420 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 3480 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 3540 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 3600 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 3660 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 3720 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt 3780 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat 3840 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc 3900 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg 3960 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat 4020 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat 4080 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 4140 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc 4200 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 4260 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 4320 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 4380 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 4440 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 4500 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 4560 ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc 4620 actgcttact ggcttatcga aattaatacg actcactata gggagaccca agcttggtac 4680 cgagctcgga tccactagta acggccgcca gtgtgctgga attcctcgaa agacaactca 4740 gagttcacca tgggctccat cggtgcagca agcatggaat tttgttttga tgtattcaag 4800 gagctcaaag tccaccatgc caatgagaac atcttctact gccccattgc catcatgtca 4860 gctctagcca tggtatacct gggtgcaaaa gacagcacca ggacacaaat aaataaggtt 4920 gttcgctttg ataaacttcc aggattcgga gacagtattg aagctcagtg tggcacatct 4980 gtaaacgttc actcttcact tagagacatc ctcaaccaaa tcaccaaacc aaatgatgtt 5040 tattcgttca gccttgccag tagactttat gctgaagaga gatacccaat cctgccagaa 5100 tacttgcagt gtgtgaagga actgtataga ggaggcttgg aacctatcaa ctttcaaaca 5160 gctgcagatc aagccagaga gctcatcaat tcctgggtag aaagtcagac aaatggaatt 5220 atcagaaatg tccttcagcc aagctccgtg gattctcaaa ctgcaatggt tctggttaat 5280 gccattgtct tcaaaggact gtgggagaaa gcatttaagg atgaagacac acaagcaatg 5340 cctttcagag tgactgagca agaaagcaaa cctgtgcaga tgatgtacca gattggttta 5400 tttagagtgg catcaatggc ttctgagaaa atgaagatcc tggagcttcc atttgccagt 5460 gggacaatga gcatgttggt gctgttgcct gatgaagtct caggccttga gcagcttgag 5520 agtataatca actttgaaaa actgactgaa tggaccagtt ctaatgttat ggaagagagg 5580 aagatcaaag tgtacttacc tcgcatgaag atggaggaaa aatacaacct cacatctgtc 5640 ttaatggcta tgggcattac tgacgtgttt agctcttcag ccaatctgtc tggcatctcc 5700 tcagcagaga gcctgaagat atctcaagct gtccatgcag cacatgcaga aatcaatgaa 5760 gcaggcagag aggtggtagg gtcagcagag gctggagtgg atgctgcaag cgtctctgaa 5820 gaatttaggg ctgaccatcc attcctcttc tgtatcaagc acatcgcaac caacgccgtt 5880 ctcttctttg gcagatgtgt ttccccttaa aaagaagaaa gctgaaaaac tctgtccctt 5940 ccaacaagac ccagagcact gtagtatcag gggtaaaatg aaaagtatgt tatctgctgc 6000 atccagactt cataaaagct ggagcttaat ctagagggcc ctattctata gtgtcaccta 6060 aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 6120 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 6180 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 6240 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 6300 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac 6360 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 6420 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 6480 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 6540 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca 6600 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 6660 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt t 6711 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer- pUCIDT-AMP_mTagBFP2 <400> 4 ttggaccctc gtacagaagc taatacg 27 <210> 5 <211> 170 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer- pUCIDT-AMP_mTagBFP2-siGFP (AS) <400> 5 aagcaagctg accctgaagt tttttttttt tttttttttt tttttttttt tttttttttt 60 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt tcttcctact caggctttat tcaaagacca 170 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SS siGFP <400> 6 gcaagcugac ccugaaguut t 21 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SS siGFP <400> 7 gcaagcugac ccugaaguut ttttt 25 <210> 8 <211> 170 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer- pUCIDT-AMP_mTagBFP2-siGFP (SS) <400> 8 aaaacttcag ggtcagcttg cttttttttt tttttttttt tttttttttt tttttttttt 60 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt tcttcctact caggctttat tcaaagacca 170 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS siGFP <400> 9 aacuucaggg ucagcuugct t 21 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> AS siGFP <400> 10 aacuucaggg ucagcuugct ttttt 25 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer- pOVA <400> 11 gagaacccac tgcttactgg 20 <210> 12 <211> 163 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer- pOVA-siSTAT3 (AS) <400> 12 aaagtaggtg caggaactgt gttttttttt tttttttttt tttttttttt tttttttttt 60 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt ttcagcgagc tctagcattt agg 163 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SS siSTAT3 <400> 13 aguaggugca ggaacugugt t 21 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SS siSTAT3 <400> 14 aguaggugca ggaacugugt ttttt 25 <210> 15 <211> 163 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer- pOVA-siSTAT3 (SS) <400> 15 aacacagttc ctgcacctac tttttttttt tttttttttt tttttttttt tttttttttt 60 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt ttcagcgagc tctagcattt agg 163 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS siSTAT3 <400> 16 cacaguuccu gcaccuacut t 21 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> AS siSTAT3 <400> 17 cacaguuccu gcaccuacut ttttt 25 <210> 18 <211> 195 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer- pUCIDT-AMP_mTagBFP2-siGFP (2)(AS) <400> 18 aagcaagctg accctgaagt taaaaaagca agctgaccct gaagtttttt tttttttttt 60 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt tttttttttt tttttttttt ttttttcttc ctactcaggc 180 tttattcaaa gacca 195 <210> 19 <211> 195 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer- pUCIDT-AMP_mTagBFP2-siGFP (2) (SS) <400> 19 aaaacttcag ggtcagcttg caaaaaaaac ttcagggtca gcttgctttt tttttttttt 60 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt tttttttttt tttttttttt ttttttcttc ctactcaggc 180 tttattcaaa gacca 195 <110> Ewha University-Industry Collaboration Foundation <120> Nucleic acid construct for simultaneous gene expression and suppression <130> EWHA1-60P <160> 19 <170> KoPatentIn 3.0 <210> 1 <211> 3782 <212> DNA <213> Artificial Sequence <220> <223> pUCIDT-AMP_mTagBFP2 <400> 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accaaatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcatcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgcaac gcgatgacga tggatagcga 420 ttcatcgatg agctgacccg atcgccgccg ccggagggtt gcgtttgaga cgggcgacag 480 atgaggctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 540 tccatagaag acaccgggac cgatccagcc tccggactct agaggatcga acccttttgg 600 accctcgtac agaagctaat acgactcact atagggaaat aagagagaaa agaagagtaa 660 gaagaaatat aagagccacc atggtgtcta agggcgaaga gctgattaag gagaacatgc 720 acatgaagct gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg 780 gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc 840 ctctcccctt cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca 900 tcaaccacac ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat 960 gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc 1020 tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg 1080 gccctgtgat gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg 1140 ctgacggcgg cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc 1200 tgatcgcaaa cgccaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc 1260 ctggcgtcta ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct 1320 acgtcgagca gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc 1380 acaagcttaa tgctgccttc tgcggggctt gccttctggc catgcccttc ttctctccct 1440 tgcacctgta cctcttggtc tttgaataaa gcctgagtag gaagtgaggg tctagaacta 1500 gtgtcgacgc aaatcagttc tggaccagcg agctgtgctg cgactcgtgg cgtaatcatg 1560 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 1620 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 1680 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 1740 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 1800 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 1860 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 1920 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 1980 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 2040 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 2100 gtcgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 2160 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 2220 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 2280 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 2340 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 2400 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 2460 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 2520 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 2580 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 2640 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 2700 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 2760 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 2820 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 2880 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 2940 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 3000 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 3060 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 3120 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 3180 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 3240 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 3300 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 3360 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 3420 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 3480 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 3540 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactctacc tttttcaata 3600 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 3660 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta 3720 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg 3780 tc 3782 <210> 2 <211> 3749 <212> DNA <213> Artificial Sequence <220> <223> pUCIDT-AMP_DsRed Express2 RFP <400> 2 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accaaatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcatcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgcaac gcgatgacga tggatagcga 420 ttcatcgatg agctgacccg atcgccgccg ccggagggtt gcgtttgaga cgggcgacag 480 atgaggctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 540 tccatagaag acaccgggac cgatccagcc tccggactct agaggatcga acccttttgg 600 accctcgtac agaagctaat acgactcact atagggaaat aagagagaaa agaagagtaa 660 gaagaaatat aagagccacc atggatagca ctgagaacgt catcaagccc ttcatgcgct 720 tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg 780 agggcaagcc ctacgagggc acccagaccg ccaagctgca ggtgaccaag ggcggccccc 840 tgcccttcgc ctgggacatc ctgtcccccc agttccagta cggctccaag gtgtacgtga 900 agcaccccgc cgacatcccc gactacaaga agctgtcctt ccccgagggc ttcaagtggg 960 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc 1020 aggacggcac cttcatctac cacgtgaagt tcatcggcgt gaacttcccc tccgacggcc 1080 ccgtaatgca gaagaagact ctgggctggg agccctccac cgagcgcctg tacccccgcg 1140 acggcgtgct gaagggcgag atccacaagg cgctgaagct gaagggcggc ggccactacc 1200 tggtggagtt caagtcaatc tacatggcca agaagcccgt gaagctgccc ggctactact 1260 acgtggactc caagctggac atcacctccc acaacgagga ctacaccgtg gtggagcagt 1320 acgagcgcgc cgaggcccgc caccacctgt tccagtaggc tgccttctgc ggggcttgcc 1380 ttctggccat gcccttcttc tctcccttgc acctgtacct cttggtcttt gaataaagcc 1440 tgagtaggaa gtgagggtct agaactagtg tcgacgcaaa tcagttctgg accagcgagc 1500 tgtgctgcga ctcgtggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 1560 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 1620 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 1680 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 1740 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 1800 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 1860 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 1920 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 1980 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 2040 cctcgtgcgc tctcctgttc cgaccctgtc gcttaccgga tacctgtccg cctttctccc 2100 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 2160 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 2220 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 2280 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 2340 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 2400 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 2460 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 2520 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 2580 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 2640 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 2700 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 2760 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 2820 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 2880 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 2940 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 3000 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 3060 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 3120 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 3180 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 3240 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 3300 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 3360 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 3420 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 3480 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 3540 aatactcata ctctaccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 3600 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 3660 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa 3720 aaataggcgt atcacgaggc cctttcgtc 3749 <210> 3 <211> 6711 <212> DNA <213> Artificial Sequence <220> <223> pOVA <400> 3 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 60 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag 120 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg 180 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 240 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc 300 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg 360 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa 420 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt 480 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc 540 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc 600 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg 660 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag 720 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg 780 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg 840 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac 900 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg 960 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc 1020 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg 1080 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc 1140 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc 1200 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc 1260 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc 1320 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg 1380 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt 1440 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 1500 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 1560 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc 1620 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 1680 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 1740 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 1800 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 1860 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 1920 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 1980 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 2040 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 2100 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 2160 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 2220 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 2280 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 2340 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 2400 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 2460 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 2520 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 2580 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 2640 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 2700 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 2760 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 2820 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 2880 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 2940 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 3000 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 3060 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 3120 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 3180 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 3240 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 3300 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 3360 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 3420 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 3480 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 3540 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 3600 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 3660 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 3720 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt 3780 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat 3840 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc 3900 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg 3960 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat 4020 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat 4080 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 4140 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc 4200 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 4260 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 4320 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 4380 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 4440 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 4500 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 4560 ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc 4620 actgcttact ggcttatcga aattaatacg actcactata gggagaccca agcttggtac 4680 cgagctcgga tccactagta acggccgcca gtgtgctgga attcctcgaa agacaactca 4740 gagttcacca tgggctccat cggtgcagca agcatggaat tttgttttga tgtattcaag 4800 gagctcaaag tccaccatgc caatgagaac atcttctact gccccattgc catcatgtca 4860 gctctagcca tggtatacct gggtgcaaaa gacagcacca ggacacaaat aaataaggtt 4920 gttcgctttg ataaacttcc aggattcgga gacagtattg aagctcagtg tggcacatct 4980 gtaaacgttc actcttcact tagagacatc ctcaaccaaa tcaccaaacc aaatgatgtt 5040 tattcgttca gccttgccag tagactttat gctgaagaga gatacccaat cctgccagaa 5100 tacttgcagt gtgtgaagga actgtataga ggaggcttgg aacctatcaa ctttcaaaca 5160 gctgcagatc aagccagaga gctcatcaat tcctgggtag aaagtcagac aaatggaatt 5220 atcagaaatg tccttcagcc aagctccgtg gattctcaaa ctgcaatggt tctggttaat 5280 gccattgtct tcaaaggact gtgggagaaa gcatttaagg atgaagacac acaagcaatg 5340 cctttcagag tgactgagca agaaagcaaa cctgtgcaga tgatgtacca gattggttta 5400 tttagagtgg catcaatggc ttctgagaaa atgaagatcc tggagcttcc atttgccagt 5460 gggacaatga gcatgttggt gctgttgcct gatgaagtct caggccttga gcagcttgag 5520 agtataatca actttgaaaa actgactgaa tggaccagtt ctaatgttat ggaagagagg 5580 aagatcaaag tgtacttacc tcgcatgaag atggaggaaa aatacaacct cacatctgtc 5640 ttaatggcta tgggcattac tgacgtgttt agctcttcag ccaatctgtc tggcatctcc 5700 tcagcagaga gcctgaagat atctcaagct gtccatgcag cacatgcaga aatcaatgaa 5760 gcaggcagag aggtggtagg gtcagcagag gctggagtgg atgctgcaag cgtctctgaa 5820 gaatttaggg ctgaccatcc attcctcttc tgtatcaagc acatcgcaac caacgccgtt 5880 ctcttctttg gcagatgtgt ttccccttaa aaagaagaaa gctgaaaaac tctgtccctt 5940 ccaacaagac ccagagcact gtagtatcag gggtaaaatg aaaagtatgt tatctgctgc 6000 atccagactt cataaaagct ggagcttaat ctagagggcc ctattctata gtgtcaccta 6060 aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 6120 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 6180 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 6240 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 6300 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac 6360 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 6420 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 6480 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 6540 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca 6600 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 6660 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt t 6711 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer- pUCIDT-AMP_mTagBFP2 <400> 4 ttggaccctc gtacagaagc taatacg 27 <210> 5 <211> 170 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer- pUCIDT-AMP_mTagBFP2-siGFP (AS) <400> 5 aagcaagctg accctgaagt tttttttttt tttttttttt tttttttttt tttttttttt 60 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt tcttcctact caggctttat tcaaagacca 170 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SS siGFP <400> 6 gcaagcugac ccugaaguut t 21 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SS siGFP <400> 7 gcaagcugac ccugaaguut ttttt 25 <210> 8 <211> 170 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer- pUCIDT-AMP_mTagBFP2-siGFP (SS) <400> 8 aaaacttcag ggtcagcttg cttttttttt tttttttttt tttttttttt tttttttttt 60 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt tcttcctact caggctttat tcaaagacca 170 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS siGFP <400> 9 aacuucaggg ucagcuugct t 21 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> AS siGFP <400> 10 aacuucaggg ucagcuugct ttttt 25 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer- pOVA <400> 11 gagaacccac tgcttactgg 20 <210> 12 <211> 163 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer- pOVA-siSTAT3 (AS) <400> 12 aaagtaggtg caggaactgt gttttttttt tttttttttt tttttttttt tttttttttt 60 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt ttcagcgagc tctagcattt agg 163 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SS siSTAT3 <400> 13 aguaggugca ggaacugugt t 21 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SS siSTAT3 <400> 14 aguaggugca ggaacugugt ttttt 25 <210> 15 <211> 163 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer- pOVA-siSTAT3 (SS) <400> 15 aacacagttc ctgcacctac tttttttttt tttttttttt tttttttttt tttttttttt 60 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt ttcagcgagc tctagcattt agg 163 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AS siSTAT3 <400> 16 cacaguuccu gcaccuacut t 21 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> AS siSTAT3 <400> 17 cacaguuccu gcaccuacut ttttt 25 <210> 18 <211> 195 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer- pUCIDT-AMP_mTagBFP2-siGFP (2)(AS) <400> 18 aagcaagctg accctgaagt taaaaaagca agctgaccct gaagtttttt tttttttttt 60 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt tttttttttt tttttttttt ttttttcttc ctactcaggc 180 tttattcaaa gacca 195 <210> 19 <211> 195 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer- pUCIDT-AMP_mTagBFP2-siGFP (2) (SS) <400> 19 aaaacttcag ggtcagcttg caaaaaaaac ttcagggtca gcttgctttt tttttttttt 60 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt tttttttttt tttttttttt ttttttcttc ctactcaggc 180 tttattcaaa gacca 195

Claims (20)

mRNA 및 siRNA를 포함하는 핵산 구조체로,
(i) 3' 말단에 5' 방향에서 3' 방향으로 순차적으로 위치하는 폴리(A) 테일(poly(A) tail); 및 제1 siRNA 센스 또는 안티센스 서열을 포함하는 mRNA; 및
(ii) 상기 제1 siRNA 서열에 상보적으로 결합하는 혼성화 서열을 포함하는 핵산 구조체로서,
상기 혼성화 서열은 3' 말단에 상기 mRNA의 폴리(A) 테일과 상보적으로 결합하는 폴리(T) 테일(poly(T) tail)을 더 포함하는 것인, 핵산 구조체.
A nucleic acid construct containing mRNA and siRNA,
(i) a poly(A) tail sequentially positioned from the 5'direction to the 3'direction at the 3'end; And an mRNA comprising a first siRNA sense or antisense sequence; And
(ii) a nucleic acid construct comprising a hybridization sequence that complementarily binds to the first siRNA sequence,
The hybridization sequence further comprises a poly (T) tail complementary to the poly (A) tail of the mRNA at the 3'end (poly (T) tail).
 제1항에 있어서, 상기 혼성화 서열은 제1 siRNA 서열에 상보적으로 결합하는 제2 siRNA 안티센스 또는 센스 서열; 또는 제1 siRNA 서열에 상보적으로 결합하는 DNA 안티센스 또는 센스 서열인 것을 특징으로 하는, 핵산 구조체.
The method of claim 1, wherein the hybridization sequence comprises: a second siRNA antisense or sense sequence complementary to the first siRNA sequence; Or a DNA antisense or sense sequence that binds complementarily to the first siRNA sequence, characterized in that the nucleic acid construct.
 삭제delete 제1항에 있어서, 상기 폴리(T) 테일은 2 내지 6 nt 길이인 것을 특징으로 하는, 핵산 구조체.
The nucleic acid construct according to claim 1, wherein the poly(T) tail is 2 to 6 nt in length.
 제1항에 있어서, 상기 핵산 구조체는 상기 mRNA가 코딩하는 타겟 단백질의 발현 및 표적 유전자의 발현 억제를 동시에 유발하는 것을 특징으로 하는, 핵산 구조체.
The nucleic acid construct according to claim 1, wherein the nucleic acid construct simultaneously induces the expression of the target protein encoded by the mRNA and the suppression of the expression of the target gene.
 제1항에 있어서, 제1 siRNA 센스 또는 안티센스 서열의 3' 말단에 추가의 siRNA 센스 또는 안티센스 서열을 위한 제1 링커 서열을 더 포함하는 것을 특징으로 하는, 핵산 구조체.
The nucleic acid construct according to claim 1, further comprising a first linker sequence for an additional siRNA sense or antisense sequence at the 3'end of the first siRNA sense or antisense sequence.
 제6항에 있어서, 상기 제1 링커 서열은 폴리(U) 테일(poly(U) tail)인 것을 특징으로 하는, 핵산 구조체.
The nucleic acid construct according to claim 6, wherein the first linker sequence is a poly(U) tail.
 제6항에 있어서, 상기 제1 링커 서열의 3’ 말단에 추가의 siRNA 센스 또는 안티센스 서열을 더 포함하는 것을 특징으로 하는, 핵산 구조체.
The nucleic acid construct according to claim 6, further comprising an additional siRNA sense or antisense sequence at the 3'end of the first linker sequence.
 제8항에 있어서, 혼성화 서열; 및 제2 링커 서열을 더 포함하는 것을 특징으로 하는 핵산 구조체로서,
상기 혼성화 서열은 상기 제1 링커 서열; 및 추가의 siRNA 센스 또는 안티센스 서열에 상보적으로 결합하는 것을 특징으로 하는, 핵산 구조체.
The method of claim 8, further comprising: a hybridization sequence; And as a nucleic acid construct further comprising a second linker sequence,
The hybridization sequence comprises the first linker sequence; And complementary binding to an additional siRNA sense or antisense sequence.
 제9항에 있어서, 상기 혼성화 서열은 추가의 siRNA 서열에 상보적으로 결합하는 제2 siRNA 안티센스 또는 센스 서열; 또는 추가의 siRNA 서열에 상보적으로 결합하는 DNA 안티센스 또는 센스 서열인 것을 특징으로 하는, 핵산 구조체.
The method of claim 9, wherein the hybridization sequence is a second siRNA antisense or sense sequence that complementarily binds to an additional siRNA sequence; Or a DNA antisense or sense sequence that complementarily binds to an additional siRNA sequence, a nucleic acid construct.
 제9항에 있어서, 상기 제2 링커 서열은 제1 링커 서열과 상보적으로 결합하는 폴리(A) 테일(poly(A) tail)인 것을 특징으로 하는, 핵산 구조체.
The nucleic acid construct according to claim 9, wherein the second linker sequence is a poly(A) tail complementary to the first linker sequence.
 제1항에 있어서, 상기 핵산 구조체는 제1 siRNA 센스 또는 안티센스 서열의 3’ 말단에 다음 (a) 및 (b)를 포함하는 반복단위를 복수 개 더 포함하는 것을 특징으로 하는, 핵산 구조체:
(a) 추가의 siRNA 센스 또는 안티센스 서열을 위한 제1 링커 서열; 및 추가의 siRNA 센스 또는 안티센스 서열; 및
(b) 혼성화 서열; 및 제2 링커 서열로서, 상기 혼성화 서열은 상기 제1 링커 서열; 및 추가의 siRNA 센스 또는 안티센스 서열에 상보적으로 결합하는 것임.
The nucleic acid construct of claim 1, wherein the nucleic acid construct further comprises a plurality of repeat units comprising the following (a) and (b) at the 3'end of the first siRNA sense or antisense sequence:
(a) a first linker sequence for an additional siRNA sense or antisense sequence; And additional siRNA sense or antisense sequences; And
(b) hybridization sequence; And a second linker sequence, wherein the hybridization sequence comprises: the first linker sequence; And complementary binding to an additional siRNA sense or antisense sequence.
 제1항, 제2항 및 제4항 내지 제12항 중 어느 한 항의 핵산 구조체 및 핵산 전달체를 포함하는 조성물.
A composition comprising the nucleic acid construct of any one of claims 1, 2 and 4 to 12 and a nucleic acid delivery system.
 제13항에 있어서, 상기 핵산 전달체는 나노입자인 것을 특징으로 하는, 조성물.
The composition of claim 13, wherein the nucleic acid delivery system is a nanoparticle.
 제1항, 제2항 및 제4항 내지 제12항 중 어느 한 항의 핵산 구조체를 포함하는 면역치료용 약학적 조성물.
A pharmaceutical composition for immunotherapy comprising the nucleic acid construct of any one of claims 1, 2 and 4 to 12.
 제15항에 있어서, 상기 면역치료는 암 면역치료인 것을 특징으로 하는, 약학적 조성물.
The pharmaceutical composition of claim 15, wherein the immunotherapy is cancer immunotherapy.
 제15항에 있어서, 상기 암은 뇌종양, 양성성상세포종, 악성성상세포종, 뇌하수체 선종, 뇌수막종, 뇌림프종, 핍지교종, 두개내인종, 상의세포종, 뇌간종양, 두경부 종양, 후두암, 구인두암, 비강/부비동암, 비인두암, 침샘암, 하인두암, 갑상선암, 흉부종양, 소세포성 폐암, 비소세포성 폐암, 흉선암, 종격동 종양, 식도암, 유방암, 남성유방암, 복부종양, 위암, 간암, 담낭암, 담도암, 췌장암, 소장암, 대장암, 항문암, 방광암, 신장암, 남성생식기종양, 음경암, 요도암, 전립선암, 여성생식기종양, 자궁경부암, 자궁내막암, 난소암, 자궁육종, 질암, 여성외부생식기암, 여성요도암, 피부암, 골수종, 백혈병 및 악성림프종으로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는, 약학적 조성물.
The method of claim 15, wherein the cancer is a brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendroglioma, intracranial tumor, epistemoma, brain stem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal/sinus Cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, gastric cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer , Small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, urethral cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external reproductive system A pharmaceutical composition, characterized in that it is at least one selected from the group consisting of strange cancer, female urethral cancer, skin cancer, myeloma, leukemia, and malignant lymphoma.
 제1항, 제2항 및 제4항 내지 제12항 중 어느 한 항의 핵산 구조체를 포함하는 암 백신.
A cancer vaccine comprising the nucleic acid construct of any one of claims 1, 2 and 4 to 12.
 제18항에 있어서, 상기 암 백신은 수지상세포를 더 포함하는 것을 특징으로 하는, 암 백신.
The cancer vaccine of claim 18, wherein the cancer vaccine further comprises dendritic cells.
 1) 3’ 말단에 5' 방향에서 3' 방향으로 순차적으로 위치하는 폴리(T) 테일(poly(T) tail) 및 siRNA 안티센스 또는 센스 서열이 도입된 DNA 주형을 제조하는 단계;
2) DNA 주형에 RNA 중합효소를 포함하는 반응용액을 처리하여 3’ 말단에 siRNA 서열을 포함하는 mRNA를 얻는 단계; 및
3) 3' 말단에 Oligo(dT)가 연결된 siRNA를 상기 mRNA 산물에 어닐링하는 단계; 를 포함하는 mRNA 및 siRNA를 포함하는 핵산 구조체의 제조방법.1) preparing a DNA template into which a poly(T) tail and siRNA antisense or sense sequences are sequentially positioned in the 3'direction from the 5'direction to the 3'end;
2) treating a reaction solution containing an RNA polymerase in a DNA template to obtain an mRNA containing an siRNA sequence at the 3'end; And
3) annealing the siRNA to which the Oligo (dT) is linked to the 3'end to the mRNA product; A method for producing a nucleic acid construct comprising mRNA and siRNA comprising a.
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KR20150080232A (en) * 2013-12-31 2015-07-09 연세대학교 산학협력단 Magnetic nano-vector for transferring nucleic acids specific to liver cell and magnatic resonance imaging
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