장기이식에서의 RNA간섭 치료법의 전망

김재영 대한이식학회 2015 Korean Journal of Transplantation Vol.29 No.3

RNA간섭(RNA interference, RNAi)은 작은 크기의RNA가 염기서열 특이적으로 유전자 발현을 조절하는 세포 내에 정상적으로 존재하는 현상을 말한다(1-3). RNA 간섭에 관여하는 작은 RNA는 크게 두 가지가 있는데, 하나는 miRNA (microRNA)이고, 다른 하나는 siRNA (small interfering RNA)이다(4,5). miRNA는 원래 세포 내 유전자로부터 발현되는 전사체로써 전사후유전자침묵(posttranscriptional gene silencing)을 매개하며, siRNA는 외부로부터 세포 내로 도입된 이중가닥 RNA (dsRNA)로 자신과상보적인 염기서열을 지닌 mRNA를 절단한다(4,5). RNA 간섭의 작용방식이 표적 mRNA의 염기서열에 고도로 특이적이기 때문에 염기서열만 안다면 이론적으로 원하는어떠한 유전자의 발현도 조절 가능하다. 이러한 이유로, 다양한 의생명 연구에 광범위하게 이용되고 있으며, 최근에는 전통적인 약물치료의 대안으로써 다양한 질환에 RNA 간섭 치료법을 적용하려는 시도가 늘고 있다. 본 논문에서는 RNA간섭의 원리, 적용 및 장기이식에서의 RNA간섭치료연구의 예들을 살펴 봄으로써, 장기이식 분야에서RNA간섭 치료법의 임상적용에 대해 전망해보고자 한다. RNA interference (RNAi) is a normal cellular process in which small RNAs control gene expression. siRNAs introduced into cells suppress gene expression through their recognition and cleavage of cognate mRNAs in a sequence specific manner. Due to its highly specific mode of action, RNAi has recently been tested for treatment or prevention of various diseases including organ transplantation as well as basic biomedical research. However, to achieve clinical success, there are some important issues that should be fully validated. First, siRNAs should be properly designed to avoid off-target effects. Second, siRNAs must be modified so as not to induce innate immune responses. Third, selective delivery of siRNA into desired organs or tissues is required. Despite such prerequisites, siRNAs are thought to be superior to traditional small molecule drug in terms of new drug development. In addition, in case of heart and islet transplantation which probably requires preservation of organs or cultivation of tissues for a while, siRNAs can be added to preserving solution or medium to control target gene expression during this period. In many research studies, mediators of innate immune response, inflammation, and cell death have been tested for alleviation of tissue injury and immune rejection after transplantation as potent targets of RNAi. We suggest that elucidation of exact mechanisms for tissue injury and immune rejection and subsequent selection and validation of target of RNAi in future studies might be helpful in enabling RNAi-based therapy in clinical organ transplantation to become a reality.

RNA-Dependent RNA Polymerase 6 Is Required for Efficient hpRNA-Induced Gene Silencing in Plants

RIKNOHARMOKO,이균오,Wahyu Indra Duwi Fanata,유재용,고기성,임영길,엠다나짐우단,Tri Agus Siswoyo,이승식,김둘이,이상열 한국분자세포생물학회 2013 Molecules and cells Vol.35 No.3

In plants, transgenes with inverted repeats are used to in-duce efficient RNA silencing, which is also frequently induced by highly transcribed sense transgenes. RNA silencing induced by sense transgenes is dependent on RNA-dependent RNA polymerase 6 (RDR6), which con-verts single-stranded (ss) RNA into double-stranded (ds) RNA. By contrast, it has been proposed that RNA silencing induced by self-complementary hairpin RNA (hpRNA) does not require RDR6, because the hpRNA can directly fold back on itself to form dsRNA. However, it is unclear whether RDR6 plays a role in hpRNA-induced RNA silencing by amplifying dsRNA to spread RNA silencing within the plant. To address the efficiency of hpRNA-induced RNA silencing in the presence or absence of RDR6, Wild type (WT, Col-0) and rdr6-11 Arabidopsis thaliana lines expressing green fluorescent protein (GFP) were generated and transformed with a GFP-RNA interference (RNAi) construct. Whereas most GFP-RNAi-transformed WT lines exhibited almost complete silencing of GFP expression in the T1 generation, various levels of GFP expression remained among the GFP-RNAi-transformed rdr6-11 lines. Homozygous expression of GFP-RNAi in the T3 generation was not sufficient to induce complete GFP silencing in several rdr6-11 lines. Our results indicate that RDR6 is required for efficient hpRNA-induced RNA silencing in plants.

RNA 간섭을 통한 Porcine Endogenous Retrovirus의 발현 억제

이현아,구본철,권모선,김태완 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10

최근 돼지의 장기를 사람에게 이식하는 이종간 장기 이식에 관한 연구가 급속히 발전되고 있다. 그러나 돼지의 장기를 이식할 경우 가장 큰 문제점 중의 하나는 돼지 genome 내에 존재하는 내인성 레트로바이러스(porcine endogenous retrovirus; PERV)가 인간에게 그대로 전이될 수 있다는 것이다. 이에 대한 대안으로 최근 활발히 연구되고 있는 RNA 간섭을 통한 PERV RNA의 발현을 최대한 억제하는 방법이 제안되고 있는데, RNA 간섭(RNA interference)은 doublestranded RNA (dsRNA)가 상보적인 표적 mRNA를 분해하여 결과적으로 표적 단백질의 발현을 특이적으로 억제하는 현상을 의미한다. 본 연구에서는 PERV에 대한 RNA 간섭 현상을 일으키는 shRNA 유전자를 레트로바이러스 벡터를 이용하여 돼지세포에 RNA)가 상보적인 표적 mRNA를 분해하여 결과적으로 표적 단백질의 발현을 특이적으로 억제하는 현상을 의미한다. 도입한 후 PERV의 발현율 감소 여부를 조사하였다. 그 결과, gag-pol 유전자와 env유전자 발현은 각각 대조군 세포의 4%와 10% 정도로 억제되었다. 한편, virus 입자의 생산에서 gag-pol 유전자는 대조군 세포에 비해 300배 이상 억제되었으며, env 유전자에서는 20만 배 이상 억제되었다. 이상의 결과를 미루어 볼 때 형질 전환 돼지를 이용한 이종 장기 이식에 있어서 RNA 간섭 현상을 이용한 PERV의 발현을 억제하는 시도는 생물학적 안전성을 크게 증가시킬 수 있을 것으로 사료된다. In recent years the number of patients waiting for organ transplantation has greatly outpaced the supply of human organs available, which leads to a renewed interest in pig-to-human xenotransplantation as an alternative. However, one of the biggest barriers in the xenotransplantation is presence of porcine endogenous retroviruses (PERV) that can infect human cells. In this study, to present a possible solution for this problem, we tried to inhibit expression of PERVs using shRNAs (short hairpin RNA) at the level of RNA synthesis and virus release. The shRNA targeting the sequence of PERV A, B type was cloned into pSIREN-RetroQ vector under the control of polymerase-III U6-RNA gene promoter. Quantitative real-time PCR was performed to detect any alterations in mRNA production of PERV A, B targeted by the shRNA in each clone. Depending on the target sequence of the shRNA, the transaiption of PERV was decreased to as much as 4% and the number of progeny viruses was reduced to less than 1/200,000. Transgenic pigs producing such shRNAs may result in a highly reduced PERV expression in cells and organs/ which is a prerequisite for safe xenotransplantations.

RNA 간섭을 통한 Porcine Endogenous Retrovirus 의 발현 억제

이현아,구본철,권모선,김태완 한국동물생명공학회(구 한국동물번식학회) 2006 Reproductive & developmental biology Vol.30 No.3

최근 돼지의 장기를 사람에게 이식하는 이종간 장기 이식에 관한 연구가 급속히 발전되고 있다. 그러나 돼지의 장기를 이식할 경우 가장 큰 문제점 중의 하나는 돼지 genome 내에 존재하는 내인성 레트로바이러스(porcine endogenous retrovirus; PERV)가 인간에게 그대로 전이될 수 있다는 것이다. 이에 대한 대안으로 최근 활발히 연구되고 있는 RNA 간섭을 통한 PERV RNA의 발현을 최대한 억제하는 방법이 제안되고 있는데, RNA 간섭(RNA interference)은 double- stranded RNA (dsRNA)가 상보적인 표적 mRNA를 분해하여 결과적으로 표적 단백질의 발현을 특이적으로 억제하는 현상을 의미한다. 본 연구에서는 PERV에 대한 RNA 간섭 현상을 일으키는 shRNA 유전자를 레트로바이러스 벡터를 이용하여 돼지세포에 RNA)가 상보적인 표적 mRNA를 분해하여 결과적으로 표적 단백질의 발현을 특이적으로 억제하는 현상을 의미한다. 도입한 후 PERV의 발현율 감소 여부를 조사하였다. 그 결과, gag-pol 유전자와 env 유전자 발현은 각각 대조군 세포의 4%와 10% 정도로 억제되었다. 한편, virus 입자의 생산에서 gag-pol 유전자는 대조군 세포에 비해 300배 이상 억제되었으며, env 유전자에서는 20만 배 이상 억제되었다. 이상의 결과를 미루어 볼 때 형질 전환 돼지를 이용한 이종 장기 이식에 있어서 RNA 간섭 현상을 이용한 PERV의 발현을 억제하는 시도는 생물학적 안전성을 크게 증가시킬 수 있을 것으로 사료된다. In recent years the number of patients waiting for organ transplantation has greatly outpaced the supply of human organs available, which leads to a renewed interest in pig-to-human xenotransplantation as an alternative. However, one of the biggest barriers in the xenotransplantation is presence of porcine endogenous retroviruses (PERV) that can infect human cells. In this study, to present a possible solution for this problem, we tried to inhibit expression of PERVs using shRNAs (short hairpin RNA) at the level of RNA synthesis and virus release. The shRNA targeting the sequence of PERV A, B type was cloned into pSIREN-RetroQ vector under the control of polymerase-Ⅲ U6-RNA gene promoter. Quantitative real-time PCR was performed to detect any alterations in mRNA production of PERV A, B targeted by the shRNA in each clone. Depending on the target sequence of the shRNA, the transcription of PERV was decreased to as much as 4% and the number of progeny viruses was reduced to less than 1/200,000. Transgenic pigs producing such shRNAs may result in a highly reduced PERV expression in cells and organs, which is a prerequisite for safe xenotransplantations.

Targeted gene suppression through double-stranded RNA application using easy-to-use methods in Arabidopsis thaliana

Park Minsu,Um Tae Young,Jang Geupil,Choi Yang Do,Shin Chanseok 한국응용생명화학회 2022 Applied Biological Chemistry (Appl Biol Chem) Vol.65 No.1

RNA interference (RNAi) is an RNA-dependent gene silencing process that is regulated by the interaction between the RNA-induced silencing complex (RISC) and double-stranded RNA (dsRNA). Exogenous dsRNAs are imported directly into the cytoplasm, where they are cleaved by Dicer into short dsRNA fragments of 20–25 base pairs. These short dsRNA fragments, called small interfering RNAs (siRNAs) have sequence-specific interaction with target genes. The guide strand, onto which siRNAs are incorporated in the RISC interacts with the target mRNA sequence, thereby inducing cleavage and degradation of target messenger RNAs (mRNAs) by ribonucleases. Recent studies have shown that plant dsRNA treatments can induce RNAi. However, the dsRNA application methods and delivery systems involved have not been well examined. In this study, dsRNA was introduced to Arabidopsis thaliana by two methods: dipping and spray. We synthesized two dsRNAs designed to target mRNAs encoding enhanced green fluorescent protein (EGFP). After applying dsRNAs that target EGFP, we found an obvious reduction in GFP expression. This was determined using fluorescence microscopy and quantitative reverse transcription PCR to assess the mRNA levels of the auxin-sensitive reporter DR5-EGFP Arabidopsis thaliana. Our data revealed that applying target gene-specific exogenous dsRNAs can induce suppression of target genes of interest whether the dipping or spray method is used. This study therefore provides a foundation for understanding how to apply and deliver dsRNAs in plants. RNA interference (RNAi) is an RNA-dependent gene silencing process that is regulated by the interaction between the RNA-induced silencing complex (RISC) and double-stranded RNA (dsRNA). Exogenous dsRNAs are imported directly into the cytoplasm, where they are cleaved by Dicer into short dsRNA fragments of 20–25 base pairs. These short dsRNA fragments, called small interfering RNAs (siRNAs) have sequence-specific interaction with target genes. The guide strand, onto which siRNAs are incorporated in the RISC interacts with the target mRNA sequence, thereby inducing cleavage and degradation of target messenger RNAs (mRNAs) by ribonucleases. Recent studies have shown that plant dsRNA treatments can induce RNAi. However, the dsRNA application methods and delivery systems involved have not been well examined. In this study, dsRNA was introduced to Arabidopsis thaliana by two methods: dipping and spray. We synthesized two dsRNAs designed to target mRNAs encoding enhanced green fluorescent protein ( EGFP ). After applying dsRNAs that target EGFP , we found an obvious reduction in GFP expression. This was determined using fluorescence microscopy and quantitative reverse transcription PCR to assess the mRNA levels of the auxin-sensitive reporter DR5-EGFP Arabidopsis thaliana . Our data revealed that applying target gene-specific exogenous dsRNAs can induce suppression of target genes of interest whether the dipping or spray method is used. This study therefore provides a foundation for understanding how to apply and deliver dsRNAs in plants.

RNA Interference-Mediated Simultaneous Silencing of Four Genes Using Cross-Shaped RNA

이태연,이동기,장찬일,이두영,홍선우,신찬석,Chiang J. Li,김소연,Dirk Haussecker 한국분자세포생물학회 2013 Molecules and cells Vol.35 No.4

The structural flexibility of RNA interference (RNAi)-trig-gering nucleic acids suggests that the design of uncon-ventional RNAi trigger structures with novel features is possible. Here, we report a cross-shaped RNA duplex structure, termed quadruple interfering RNA (qiRNA), with multiple target gene silencing activity. qiRNA trig-gers the simultaneous down-regulation of four cellular target genes via an RNAi mechanism. In addition, qiRNA shows enhanced intracellular delivery and target gene silencing over conventional siRNA when complexed with jetPEI, a linear polyethyleneimine (PEI). We also show that the long antisense strand of qiRNA is incorporated intact into an RNA-induced silencing complex (RISC). This novel RNA scaffold further expands the repertoire of RNAi-triggering molecular structures and could be used in the development of therapeutics for various diseases including viral infections and cancer.

Note : Note : Simultaneous and Systemic Knock-down of Big Defensin 1 and 2 gene Expression in the Pacific Oyster Crassostrea gigas using Long Double-stranded RNA-mediated RNA Interference

( Bo Young Jee ),( Min Sun Kim ),( Mi Young Cho ),( Soon Jeong Lee ),( Myung Ae Park ),( Jin Woo Kim ),( Seung Hyuk Choi ),( Hyun Do Jeong ),( Ki Hong Kim ) 한국수산과학회(구 한국수산학회) 2014 Fisheries and Aquatic Sciences Vol.17 No.3

RNA interference (RNAi)-mediated transcriptional knock-down of Crassostrea gigas big defensin 1 and 2 genes (Cg-BigDef1 and Cg-BigDef2) was investigated. The cDNA sequences of Cg-BigDef1 and Cg-BigDef2 were identical, excluding an additional fragment of 20 nucleotides in Cg-BigDef1; thus, a long double-stranded RNA (dsRNA) targeting the mRNA of Cg-BigDef2 effectivelydownregulated both Cg-BigDef2 and Cg-BigDef1. In addition, long dsRNA targeting green fluorescent protein (GFP) did not affect transcription of the two big defensin genes. These results suggest that the transcriptional downregulation of Cg-BigDef1 and Cg-BigDef2 was mediated by sequence-specific RNA interference (RNAi). Despite injection of long dsRNA targeting Cg-BigDef2 into only the adductor muscle, knock-down of Cg-BigDef1 and Cg-BigDef2 was observed in the adductor muscle, hemocytes, mantle, and gills, suggestive of systemic spread of RNAi in C. gigas. Furthermore, the inhibitory effect of dsRNA persisted until 72 h post-injection, indicative of a long-lasting RNAi-mediated knock-down of target genes.

RNA Therapy: Current Status and Future Potential

김영국 전남대학교 의과학연구소 2020 전남의대학술지 Vol.56 No.2

Recent studies identified diverse RNAs including noncoding RNAs and their various action mechanisms in the cells. These RNAs regulate a variety of cellular pathways and are therefore expected to be important targets for the treatment of human diseases. Along with their extensive functional studies, RNA-based therapeutic techniques have developed considerably in recent years. After years of research and various trial and error, antisense RNAs and small interfering RNAs-based drugs have been developed and are now being used in the clinic. In addition, active research is ongoing to develop drugs based on RNA aptamer and messenger RNA. Along with the development of these RNA-based drugs, diverse strategies have been developed to transport RNA drugs into the cells efficiently. RNA therapy has many advantages over existing small molecule or monoclonal antibody-based therapies, including its potential to target all genes in the cells. This review will introduce the history of RNA therapy, and explain the basic concepts of RNA therapy and RNA-based drugs on the market or clinical trials. In addition, the future potential of RNA therapy will be discussed.

자연적으로 유발된 고혈압 백서에서 안지오텐신 전환효소를 억제하는 Small Hairpin RNA 투여 효과

홍영미,이혜련,김관창 대한고혈압학회 2012 Clinical Hypertension Vol.18 No.3

Background: Interfering RNA (iRNA) represents a recent breakthrough in effective blocking of the target genes in mammalian cells. Angiotensin-converting enzyme (ACE) has been shown to play an important role in the pathogenesis of hypertension. The purposes of this study were to investigate the effects on blood pressure, myocardial hypertrophy and gene expressions of iRNA targeting ACE. Methods: Twelve week old male Wistar-Kyoto rats were grouped as follows: control group (C group), spontaneously hypertensive rat (SHR) group (H group), and ACE-iRNA group (A group) in which SHR was treated with recombinant lentiviral vectors carrying small hairpin RNA targeting ACE. Reverse transcriptionpolymerase chain reaction and western blot analysis of ACE, endothelin (ET)-1, angiotensin (AT) II receptor type 1A,neutrophil cytosolic factor, caspase 3, Bax, and Bcl-2 were performed in the heart tissues. Serum AT, ACE, and high sensitive-C reactive protein were estimated. Results: Systolic blood pressure was significantly decreased in the A group compared with the H group in weeks 3 and 5. Serum AT level was significantly lower on day 1, weeks 3 and 5 after ACE-iRNA treatment. ACE protein contents were significantly lower after ACE-iRNA treatment in week 5. ET-1 and Bcl-2protein contents were significantly lower after ACE-iRNA treatment in weeks 3 and 5. Bax protein contents were significantly lower after ACE-iRNA treatment in week 3. Conclusions: Recombinant lentiviral vectors carrying shRNA targeting ACE prevented hypertension. Serum AT and gene expressions such as ACE, ET-1, Bax, and Bcl-2 were significantly decreased after ACE-iRNA treatment. 연구배경: RNA interference는 이중 가닥의 RNA (double-stranded RNA)가 dicer에 의해 절단되어 생성되는 21-25 뉴클레오타이드 크기의 작은 RNA조각으로 상보적인 서열을 갖는 mRNA에 특이적으로 결합하여 단백질 발현을 억제하는 것으로 밝혀졌다. 본 연구에서는 자연적으로 유발된 고혈압 쥐(spontaneously hypertensive rat, SHR) 모델에서 안지오텐신 전환효소(angiotensinconverting enzyme, ACE)에 대한 small hairpin RNA (shRNA)가 주입된 렌티바이러스 벡터를 투여하여 혈역학적, 해부학적 변화를 알아보고, 이와 같은 조절 효과가조직학적 변화 또는 유전자 수준에서 어느 시기에 어느정도 일어나는지를 규명하고자 하였다. 방법: 12주의 자성 성숙 Wistar-Kyoto 백서를 대조군으로 SHR을 고혈압군(H군)으로 shRNA가 주입된 렌티바이러스 벡터를 투여한 치료군을 ACE-interfering RNA (iRNA) (A군)으로 하였다. shRNA 투여는 렌티바이러스에 형질주입된 상태로 1 mL를 실험 시작일에 SHR의 꼬리에 정맥투여하였다. 대조군 및 실험군 모두 21일, 35일에 도살하였다. 혈청 알도스테론, ACE, 안지오텐신치를측정하였고, reverse transcription-polymerase chain reaction과Western blot분석을 이용하여 endothelin (ET)-1,ACE, angiotensin II receptor (AT II R) 1A, neutrophil cytosolic factor (NCF), caspase-3, Bax, Bcl-2 유전자 발현을 측정하였다. 결과: 혈압은 H군에서 유의하게 증가하였고 A군에서유의하게 감소하였다. 혈청 안지오텐신치는 H군에서 유의하게 증가하였고, A군에서 유의하게 감소하였다. ACE 농도는 세 군 간에 유의한 차이가 없었다. ACE 농도는 5주에, ET-1와 Bcl-2 단백질 농도는 ACE-iRNA 치료 후 3주, 5주째 유의하게 감소하였디. Bax 단백질 농도는ACE-iRNA 치료 후 3주에 유의하게 감소하였다. AT II R, NCF, caspase-3 단백질 농도는 ACE-iRNA 치료 후 유의한 감소가 없었다. 결론: ACE에 대한 shRNA가 주입된 렌티바이러스 벡터는 항고혈압 효과가 뚜렷하였고, ACE, ET-1, Bax,Bcl-2 유전자 발현의 감소를 초래하였다. 향후 shRNA 주입 양을 증량시킨 연구가 추후에 이루어져야 할 것으로생각한다.

Animal Biotechnology : Changes in Apoptosis-related Gene Expression Induced by Repression of FGFR1 by RNA Interference in Embryonic Fibroblasts and Cancerous Cells from Chicken

( Sang In Lee ),( Bo Ram Lee ),( Young Sun Hwang ),( Jae Yong Han ),( Deivendran Rengaraj ) 한국동물자원과학회 ( 구 한국축산학회 ) 2010 한국축산학회지 Vol.52 No.6

Fibroblast growth factor receptor 1 (FGFR1) plays roles in angiogenesis, wound healing, and embryonic development via the regulation of cell proliferation, differentiation, and survival. It is well known that ectopic expression of FGFR1 is associated with cancer development. To characterize the function of FGFR1 in the normal and cancer cell lines DF-1 and DT40, respectively, we performed FGFR1 knockdown by RNA interference. In the DT40 cells, FGFR1 knockdown induced upregulation of FGFR2 and FGFR3 expression, downregulation of pro-apoptosis-related genes, and upregulation of anti-apoptosis-related genes. However, in DF-1 cells, FGFR1 knockdown induced upregulation of pro-apoptosis-related genes and downregulation of anti-apoptosis-related genes. Our data suggest that repression of FGFR1 induced upregulation of other FGF receptors and anti-apoptosis-related genes in cancer cells and pro-apoptosis-related genes in normal cells.