Bovine Vira1 Diarrhea Virus를 이용한 포유동물세포 발현벡터의 개발

이영민 한국미생물학회 2002 미생물학회지 Vol.38 No.2

최근 인간을 비롯한 다양한 생명체의 genome project연구결과 밝혀진 유전자들의 염기서열을 토대로, 생명체 구성성분의 실질적 인 역할을 하는 단배질의 기능을 밝히는 proteomics에 관한 연구의 필요성 이 대두되고 있다. 따라서, 이 연구는 post-genomics시대에 다양한 종류의 단백질 기능과 상호작용의 기초연구에 필수적 인 새로운 포유동물세포 유전자 발현벡터를 RNA 바이러스인 소설사성 바이러스(Bovine Viral Diarrhea Virus)의 infectious CDNA molecular clone을 이용하여 개발하였다. 먼저 BVDV의 infectious CDNA molecular clone (pNADLclns-)을 이용하여 puromycin 항생제에 저항성을 나타내는 puromycin acetyltransferase (pac) 유전자를 삽입하여 recombinant full-length infectious CDNA clone을 합성하였다. 합성된 recombinant CDNA clone을 주형으로 T7 RNA polymerase를 사용하여 in vitro transcribed full-length viral RNA를 합성하였다. 합성된 viral RNA의 자가복제 여부는 MDBK세포에 transfection시킨 후, $^{32}P$ 로 metabolically label함으로써 확인하였다. 또한, transfection된 세포에서의 바이러스 단백질 발현여부는 바이러스에 특이적으로 반응하는 anti-NS3 단클론항체를 사용하여 분석하였다. 또한, infectious CDNA clone을 응용하여 새로운 포유동물세포유전자 발현벡터의 개발을 위해서, 먼저 바이러스의 구조단백질이 바이러스의 자가복제에 필수적인 지를 평가하였다. 실험결과, 각각의 구조단백질 유전자를 deletion한 recombinant cDNA clone으로부터 합성된 viral RMA의 자가복제여부는pac유전자의 발현여부로 recombinant cDNA clone으로부터 합성된 recombinant viral RMA를 MDBK 세포에 transfection시킨 후, puromycin으로 selection함으로써 할 수 있었다. Deletion실험결과, 각각의 구조단백질 capsid및 E0, El, E2는 바이러스의 자가복제에 영향을 기치지 않음을 알 수 있었다. 이와 더불어, 바이러스의 모든 구조단백질을 함께deletion하였을 경우에도 자가복제에는 영향을 기치지 않는 것을 합성된 viral replicon을 이용한 실험에서 알 수 있었다. 이렇게 합성된 BVDV의 replicon을 사용하여 포유동물의 발현벡터로써 사용할수 있는 지의 여부를 분석하기 위해서 pac유전자 이외에 luciferase유전자를 사용하여 MDBK및 HeLa, BHK세포에서의 단백질 발현정도를 시간 별로 분석한 결과, BVDV의 replicon을 다양한 종류의 유전자 발현벡터로사용할 수 있음을 알 수 있었다. 그러므로, RNA바이러스의 하나인 BVDV의 viral replicon을 이용하여 다양한 종류의 포유동물 세포에 유전자 발현벡터로써 사용할 수 있음으로 post-genomics시대에 다양한 종류의 단백질 기능연구에 맡은 도움이 되리라 기대한다. As a result of genome projects, the research to elucidate the function of a protein of interest has recently been well-recognized. In order to facilitate functional genomics, a useful mammalian gene expression vector is required. Using an infectious CDNA clone of BVDV pNADLclns-, we have developed a mammalian gene expression vector. In this study, a replication-competent full-length infectious CDNA clone containing puremycin acetyltransferase (pac) gene (pNADLclns-/pac) was successfully generated. The viral RNA replication and viral protein NS3 synthesis were examined by detecting metabollically $^{32}P$-labelled genomic viral RNA and immunoblotting with a mouse anti-NS3 antibody. To generate viral replicon as an expression vector, we examine if the viral structural genes (C, E0, El, E2) are required for viral replication by deletion analysis. As a result, all of the structural proteins are dispensable for viral replication per se, but essential for infectious viral particle formation. Based on our deletion analysis, we have generated a replication-competent BVDV viral replicon (pNADLclns-/pac/${\Delta}S$), whose structural genes are all deleted. In addition to NADLclns- /pac/${\Delta}S$, NADLclns-/ luc/${\Delta}S$ viral replicon containing luciferase gene as a reporter was constructed and fecund to be replication-compotent in HeLa and BHK cells as well as MDBK cells. Therefore, BVDV viral replicon developed in our study will be a useful tool to express a protein of interest in various mammalian cells.

유전자치료를 위한 벡터 개발의 연구 동향 : A Current Research Insight

손은화,손은수,표석능 한국약제학회 2004 Journal of Pharmaceutical Investigation Vol.34 No.5

The basic concept underlying gene therapy is that human diseases may be treated by the transfer of genetics material into specific cells of a patient in order to correct or supplement defective genes responsible for disease development. There are several systems that can be used to transfer foreign genetic material into the human body. Both viral and non-viral vectors are developed and evaluated for delivering therapeutic genes. Viral vectors are biological systems derived from naturally evolved viruses capable of transferring their genetics materials into host cells. However, the limitations associated with viral vectors, in terms of their safety, particularly immunogenecity, and their limited capacity of transgenic materials, have encouraged researchers to increasingly focus on non-viral vectors as an alternative to viral vectors. Although non-viral vectors are less efficient than viral ones, they have the advantages of safety, simplicity of preparation and high gene encapsulation capability. This article reviews the most recent studies highlighting the advantages and the limitation of gene delivery systems focused on non-viral systems compared to viral systems.

Utilizing carbon dots as non-viral vectors in gene delivery

박소연,한지은,나건 한국공업화학회 2020 한국공업화학회 연구논문 초록집 Vol.2020 No.-

Vectors are the most important component for gene therapy. The viral vectors are widely used because of their higher transfection efficiency. However, viral vectors have limitation of genetic material size, oncogenic potential and immunogenicity. Thus, the non-viral vectors have been developing to substitute viral vectors. In this study we utilized carbon dots (c-dots) as a non-viral vector and c-dots have unique properties such as great water solubility and low toxicity. The c-dots were generated using cationic polymer for gene delivery. The therapeutic gene complexed with c-dots (gene/c-dots) were transfected into human mesenchymal stem cells (hMSCs). C-dots have been found to be safer because c-dots are less toxic than cationic polymer. In addition, c-dots enable bioimaging after transfection of gene/c-dots into hMSCs (gene/c-dots@hMSCs). Even, the amount of secreted therapeutic protein from gene/c-dots@hMSCs was 25-fold higher than that from gene/cationic polymer@hMSCs.

Establishment of a Dual-Vector System for Gene Delivery Utilizing Prototype Foamy Virus

( Soo-yeon Cho ),( Yoon Jae Lee ),( Seong-mook Jung ),( Young Min Son ),( Cha-gyun Shin ),( Eui Tae Kim ),( Kyoung-dong Kim ) 한국미생물생명공학회 2024 Journal of microbiology and biotechnology Vol.34 No.4

Foamy viruses (FVs) are generally recognized as non-pathogenic, often causing asymptomatic or mild symptoms in infections. Leveraging these unique characteristics, FV vectors hold significant promise for applications in gene therapy. This study introduces a novel platform technology using a pseudo-virus with single-round infectivity. In contrast to previous vector approaches, we developed a technique employing only two vectors, pcHFV lacking Env and pCMV-Env, to introduce the desired genes into target cells. Our investigation demonstrated the efficacy of the prototype foamy virus (PFV) dual-vector system in producing viruses and delivering transgenes into host cells. To optimize viral production, we incorporated the codon-optimized Env (optEnv) gene in pCMV-Env and the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) at the 3’ end of the transgene in the transfer vector. Consequently, the use of optEnv led to a significant enhancement in transgene expression in host cells. Additionally, the WPRE exhibited an enhancing effect. Furthermore, the introduced EGFP transgene was present in host cells for a month. In an effort to expand transgene capacity, we further streamlined the viral vector, anticipating the delivery of approximately 4.3 kbp of genes through our PFV dual-vector system. This study underscores the potential of PFVs as an alternative to lentiviruses or other retroviruses in the realm of gene therapy.

Recent advances in the development of gene delivery systems

YK sung,SW KIM 한국생체재료학회 2019 생체재료학회지 Vol.23 No.2

Background: Gene delivery systems are essentially necessary for the gene therapy of human genetic diseases. Gene therapy is the unique way that is able to use the adjustable gene to cure any disease. The gene therapy is one of promising therapies for a number of diseases such as inherited disorders, viral infection and cancers. The useful results of gene delivery systems depend open the adjustable targeting gene delivery systems. Some of successful gene delivery systems have recently reported for the practical application of gene therapy. Main body: The recent developments of viral gene delivery systems and non-viral gene delivery systems for gene therapy have briefly reviewed. The viral gene delivery systems have discussed for the viral vectors based on DNA, RNA and oncolytic viral vectors. The non-viral gene delivery systems have also treated for the physicochemical approaches such as physical methods and chemical methods. Several kinds of successful gene delivery systems have briefly discussed on the bases of the gene delivery systems such as cationic polymers, poly(L-lysine), polysaccharides, and poly(ethylenimine)s. Conclusion: The goal of the research for gene delivery system is to develop the clinically relevant vectors such as viral and non-viral vectors that use to combat elusive diseases such as AIDS, cancer, Alzheimer, etc. Next step research will focus on advancing DNA and RNA molecular technologies to become the standard treatment options in the clinical area of biomedical application.

Foamy Virus Integrase in Development of Viral Vector for Gene Therapy

김지선,이가은,신차균 한국미생물·생명공학회 2020 Journal of microbiology and biotechnology Vol.30 No.9

Due to the broad host suitability of viral vectors and their high gene delivery capacity, many researchers are focusing on viral vector-mediated gene therapy. Among the retroviruses, foamy viruses have been considered potential gene therapy vectors because of their non-pathogenicity. To date, the prototype foamy virus is the only retrovirus that has a high-resolution structure of intasomes, nucleoprotein complexes formed by integrase, and viral DNA. The integration of viral DNA into the host chromosome is an essential step for viral vector development. This process is mediated by virally encoded integrase, which catalyzes unique chemical reactions. Additionally, recent studies on foamy virus integrase elucidated the catalytic functions of its three distinct domains and their effect on viral pathogenicity. This review focuses on recent advancements in biochemical, structural, and functional studies of foamy virus integrase for gene therapy vector research.

Current Trends in Viral Gene Therapy for Human Orthopaedic Regenerative Medicine

Jagadeesh Kumar Venkatesan,Ana Rey-Rico,Magali Cucchiarini 한국조직공학과 재생의학회 2019 조직공학과 재생의학 Vol.16 No.4

Background: Viral vector-based therapeutic gene therapy is a potent strategy to enhance the intrinsic reparative abilities of human orthopaedic tissues. However, clinical application of viral gene transfer remains hindered by detrimental responses in the host against such vectors (immunogenic responses, vector dissemination to nontarget locations). Combining viral gene therapy techniques with tissue engineering procedures may offer strong tools to improve the current systems for applications in vivo. Methods: The goal of this work is to provide an overview of the most recent systems exploiting biomaterial technologies and therapeutic viral gene transfer in human orthopaedic regenerative medicine. Results: Integration of tissue engineering platforms with viral gene vectors is an active area of research in orthopaedics as a means to overcome the obstacles precluding effective viral gene therapy. Conclusions: In light of promising preclinical data that may rapidly expand in a close future, biomaterial-guided viral gene therapy has a strong potential for translation in the field of human orthopaedic regenerative medicine.

Current trends in large‐scale viral surveillance methods in mosquitoes

Park Ki Beom,Patnaik Hongray Howrelia,Kim Tae‐Yun,Jo Yong Hun,Kim Nam‐Yeon,Yang Sung‐Chan,Lee Wook‐Gyo,Lee Hee‐Il,Cho Shin‐Hyeong,Han Yeon Soo 한국곤충학회 2020 Entomological Research Vol.50 No.6

Vector-borne and zoonotic infectious diseases are serious public health concerns that affect approximately half of the world’s population. In particular, arthropod-borne viruses (arboviruses) have contributed to more mortality and morbidity worldwide with the emergence of dengue, chikungunya, yellow fever, and Zika virus diseases. The infections have scaled up due to urbanization, globalization, and international mobility. Traditionally, the spread of mosquito-borne viral diseases to humans was considered a low health priority concern. However, their categorization as emerging infectious diseases and public health emergencies of international concern has heightened the attention given by the government, academia, research, and industry for the development of timely, cost-efficient, and sustainable solutions. The urgency has increased in the wake of global climate change. The focus on effective interventions includes epidemiological monitoring, vector control measures, molecular diagnostics, vaccines, and environmental determinants. In this review, we discuss the etiology and predisposition of mosquito-borne viruses that are detrimental to public health and economically damaging when disseminated as epidemics. We focus on the large-scale virus surveillance methods with special reference to innovations and interventions in molecular detection science and technologies that include viral nucleic acid isolation, polymerase chain reaction (PCR)-based diagnostics, and high-throughput sequencing technologies. In addition, we discuss the development of a viral RNA extraction and PCR-based diagnostic kit (Invirustech) that can extract viral RNA from mosquitoes with verified applications in PCR-based molecular diagnostics of Pan-flavivirus.

의료용 단백질 생산을 위한 바이러스 운반체 시스템

김순희 ( Sun Hee Kim ),강한철 ( Han Chul Kang ),노경희 ( Kyung Hee Roh ),김현욱 ( Hyun Uk Kim ),이경렬 ( Kyeong Ryeol Lee ),김원용 ( Won Yong Kim ),박종화 ( Jong Hwa Park ),정인식 ( In Sig Chung ),김종범 ( Jong Bum Kim ) 한국국제농업개발학회 2015 韓國國際農業開發學會誌 Vol.27 No.2

Most of pharmaceutical proteins are usually produced by the animal, insect cell and yeast culture. The proteins must be produced in strictly controlled-facilities, which is one of major causes for increasing production cost. So, in spite of increasing demand on the proteins in worldwide, their generalization has been very restrictive. To decrease the production cost, many scientists have been interested in production of pharmaceutical proteins by using the plant system for a long time. The plant system must be alternative production means, for it is easily scalable, more production cost effective, and safer than animal system. For this purpose, several techniques have been developed: stable expression by transformation of nuclear and chloroplast genome and transient expression by viral vectors. Among them, the deconstructed viral vector system has made it possible to produce recombinant proteins massively, rapidly, and transiently. So, the plant viral expression systems might be a suitable platform for production of pharmaceutical proteins in plants. In this review, we will describe the plant viral vector systems and their application.

Transient expression of hemagglutinin antigen from canine influenza virus H3N2 in Nicotiana benthamiana and Lactuca sativa

Puna Maya Maharjan,최성화 대한백신학회 2019 Clinical and Experimental Vaccine Research Vol.8 No.2

Purpose: Canine influenza virus (CIV), H3N2, carries potentiality for zoonotic transmission and genetic assortment which raises a concern on possible epidemics, and human threats in future. To manage possible threats, the development of rapid and effective methods of CIV vaccine production is required. The plant provides economical, safe, and robust production platform. We investigated whether hemagglutinin (HA) antigen from Korea-originated CIV could be produced in Nicotiana benthamiana and lettuce, Lactuca sativa by a DNA viral vector system. Materials and Methods: We used DNA sequences of the HA gene from Korean CIV strain influenza A/canine/Korea/S3001/2015 (H3N2) for cloning into a geminiviral expression vectors to express recombinant HA (rHA) antigen in the plant. Agrobacterium-mediated infiltration was performed to introduce HA-carrying vector into host plants cells. Laboratory-grown N. benthamiana, and grocery-purchased or hydroponically-grown lettuce plant leaves were used as host plants. Results: CIV rHA antigen was successfully expressed in host plant species both N. benthamiana and L. sativa by geminiviral vector. Both complex-glycosylated and basal-glycosylated form of rHA were produced in lettuce, depending on presence of endoplasmic reticulum (ER) retention signal. In terms of rHA expression level, canine HA (H3N2) showed preference to the native signal peptide than ER retention signal peptide in the tested geminiviral vector system. Conclusion: Grocery-purchased lettuce leaves could serve as an instant host system for the transient expression of influenza antigen at the time of emergency. The geminiviral vector was able to induce expression of complex-glycosylated and basal-glycosylated rHA in lettuce and tobacco.