重组腺相关病毒包装容量研究进展

在以病毒载体介导的基因治疗研究中,重组腺相关病毒(rAAV)因其疗效和安全性方面的优势,是最有临床应用前景的载体。但其转基因包装容量一般不能超过5.0kb,给需要转导大片段基因的应用带来了困难,限制了rAAV在基因治疗研究中的应用。随着对rAAV细胞转导生物学过程研究的不断深入,发现了一些可以突破rAAV包装容量限制的技术,如反式剪接和同源重组策略,为拓展该载体应用范围提供了可能性。另外,rAAV包装容量限制的特点还可以被用来减少生产过程中具有可复制能力的类病毒杂质的污染,为rAAV的临床安全性提供了保障。     

重组腺相关病毒生产方法研究进展

重组腺相关病毒(rAAV)作为基因治疗的载体,具有感染范围广、能在宿主细胞中长久稳定表达外源基因和非致病性等优点,因此在基因治疗领域倍受青睐。rAAV的大规模制备技术一直是限制其临床广泛应用的瓶颈。近年来,有许多改进rAAV制备工艺的尝试。我们在介绍rAAV载体制备所需各种元件的基础上,对这些方法进行了简要综述。     

携带Bclxl基因的重组腺相关病毒的制备和鉴定

目的:制备携带Bclxl基因的重组腺相关病毒,并鉴定其感染细胞有效性。方法:抽提BALB/c小鼠脾脏总RNA,通过RT-PCR获得cDNA,然后设计引物通过PCR获得Bclxl基因的编码序列。将得到的Bclxl编码序列插入腺相关病毒质粒pAAV-MCS中的多克隆位点,从而构建出重组腺相关病毒质粒pAAV-Bclxl。将质粒转染细胞后使用Western Blot和流式分析检测Bclxl蛋白的表达。进一步包装制备了携带Bclxl基因的重组腺相关病毒颗粒,用其感染HT1080细胞后用流式细胞仪分析了感染前后细胞中Bclxl基因的表达情况。结果:质粒转染后细胞中Bclxl蛋白的表达水平显著高于转染前,病毒感染后细胞中Bclxl蛋白的表达水平显著高于感染前。结论:携带Bclxl基因的重组腺相关病毒颗粒包装成功,能高效感染细胞,为后续的遗传改造树突细胞打下了基础。     

重组腺相关病毒：很有潜力的基因治疗载体

腺相关病毒(AAV)是细小病毒家族的一员,为无包膜的线性单链DNA病毒.由于AAV具有长期潜伏于人体而不具有任何明显致病性等优点,人们对AAV作为一种理想的基因治疗载体给予了很大期望.但是,近来发现,这类载体在应用上有许多明显的缺陷,包括某些细胞膜上病毒受体数量极少,重组AAV载体位点特异性整合不足,AAV衣壳成分和转基因产物引起宿主的免疫反应等等.这些缺陷促使人们加大对AAV生物学特性和转染过程的研究,从而更好地对AAV载体进行改进,使新一代重组AAV载体具备基因治疗所必需的安全性、高效性和靶向性,以期更广泛地应用于临床.     

重组腺相关病毒载体的研究进展

重组腺相关病毒(rAAV)载体作为基因治疗的工具,具有无致病性、长期表达、低免疫原性和感染多种细胞等特点.因此,rAAV载体越来越受到重视.就rAAV载体在生物学特性、构建、安全性和应用方面作一介绍.     

重组腺相关病毒载体在肝疾病基因治疗中的应用及局限性

基因治疗(gene therapy)是近十年来随着现代分子生物学技术的发展而诞生的新的生物医学治疗技术.重组腺病毒伴随病毒是一种具有开发潜质的病毒载体,因此近年来对它的研究很是受人关注.肝炎、肝硬化、肝癌在我国乃至全世界是严重损害人们的健康,据此,国内外的研究人员对rAAV在肝疾病基因治疗方面做了大量的工作,本文对rAAV在肝疾病基因治疗中的应用和局限性研究进行综述.     

化学修饰的重组腺相关病毒载体

重组腺相关病毒(Recombinant adenovirus-associated virus,rAAV)载体源于腺相关病毒(Adeno-associated virus,AAV),以其无致病性、低免疫原性、可定点整合及在宿主细胞内长期表达等优势,广泛应用于肿瘤和遗传疾病等基因治疗研究,被视为最有前途的基因治疗载体之一。但是人体内广泛存在AAV中和抗体,以及rAAV对体内特定组织或细胞的靶向性欠理想,限制了其在临床上的应用。经化学修饰的rAAV可以抵御血清中和抗体,提高转导靶向性,还可实现rAAV体内动态监测,这为解决当前rAAV载体的问题提供了新的思路和方法。本文对化学修饰的rAAV展开系统阐述,并对其未来发展趋势作出展望。     

重组腺相关病毒载体诱导的天然免疫反应及机制

重组腺相关病毒(rAAV)已成为基因治疗领域应用最广泛的载体之一。临床前研究显示其具有很高的安全性,但人体免疫毒性仍是制约其临床疗效的关键,因此有关rAAV免疫机制的研究成为近期热点。尽管天然免疫在获得性免疫反应中发挥重要作用,但与rAAV有关的天然免疫研究过去一直未被重视。直到最近,才确认有至少3种人体细胞(树突状细胞、巨噬细胞和内皮细胞)参与了rAAV的天然免疫,作用机制为可识别载体基因组的TLR9或病毒衣壳TLR2所介导,NF-κB或干扰素调节因子(IRFs)信号通路被激活,导致各种炎性因子及I型干扰素的大量表达。自身互补型rAAV诱导的TLR9依赖性天然免疫较单链rAAV更为强烈。本文重点对近期发现的激活天然免疫反应的宿主与rAAV的相互作用、涉及的信号通路、天然免疫对获得性免疫以及转基因表达的影响进行综述。     

重组腺相关病毒2型转导人外周血单核细胞来源树突状细胞的研究

To find out the infection efficiency of recombinant adeno-as sociated virus 2-mediated exogenou s genes in human peripheral blood monocyte-derived dendritic cells(D Cs),the process of transfection wa s investigated by FITC-labeled rAA V2,and observed under confocal mic roscope.Newly separated dendritic cells were tranfected by rAAV2-luc and rAAV2-GFP at different MOI,and transfection efficiency were detec ted by luminometer for rAAV2-luc a nd flow cytometry for rAAV2-GFP.Th e results were elucidated in four different assay systems:(1)60min w as needed for rAAV2 to bind on den dritic cells,and got into cells in the following 10min;(2)the express ion of luc could be detected at th e MOI as low as 1×10~5v.g/cell,and the expression plateau was reached by the MOI of 10~6～10~7v.g/cell,furt her increase of MOI had no functio n on expression level;(3)transgene expression was detected after 48h, and maintained a higher expression level from 96h to 240h after infec tion;(4)7days postinfection of rAA V2-GFP,5%～18% dendritic cells were GFP positive. These data suggest th at rAAV2 vector can efficiently in fect monocyte-derived dendritic ce lls and mediate exogenous gene exp ression,and that the application o f rAAV2 as vector may be useful fo r gene transfer to dendritic cells in ex vivo immunotherapy.     

利用BmNPV-家蚕表达系统包装重组腺相关病毒2型（rAAV2）研究

腺相关病毒（adeno-associated virus,AAV）是基因治疗中最常用的病毒载体之一,目前用于基因治疗的AAV多利用苜蓿银纹夜蛾核型多角体病毒表达系统（AcMNPV-sf9）包装,但较高的包装成本限制了AAV在基因治疗中的广泛应用。家蚕杆状病毒表达系统与AcMNPV-sf9系统相比,具有包装量更高、成本更低的优势,因此更适用于包装重组腺相关病毒（recombinant adeno-associated virus,rAAV）。首先,将AAV2功能基因cap和rep进行序列优化后合成,克隆到杆状病毒转移载体pVL1393上,将增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）基因和荧光素酶（luciferase,Luc）基因分别作为报告基因克隆到含有巨噬细胞病毒IE（cytomegalovirus-IE,CMV-IE）启动子的病毒转移载体pVL1393-ITRs-MCS上。随后,将构建好的转移载体分别与缺陷型家蚕杆状病毒reBmBac共转染BmN细胞系,获得分别重组有cap、rep和报告基因的家蚕杆状病毒（Bombyx mori nucleopolyhedrovirus,BmNPV）。再将纯化的重组病毒（reBm-Cap2、reBm-Rep2）与reBm-EGFP、reBm-Luc分别混合后感染家蚕,收获其表达产物,纯化得到含有目的基因的rAAV病毒。利用rAAV病毒感染哺乳动物细胞后,通过检测EGFP、Luc的表达状态来验证rAAV包装成功与否。结果显示,利用家蚕杆状病毒系统成功包装了rAAV2,并且在哺乳动物细胞中实现了报告基因的表达。     

Molecular design for recombinant adeno-associated virus (rAAV) vector production

Applied Microbiology and Biotechnology - Recombinant adeno-associated virus (rAAV) vectors are increasingly popular tools for gene therapy applications. Their non-pathogenic status, low...     

Critical challenges and advances in recombinant adeno-associated virus (rAAV) biomanufacturing

Gene therapy is a promising therapeutic approach for genetic and acquired diseases nowadays. Among DNA delivery vectors, recombinant adeno-associated virus (rAAV) is one of the most effective and safest vectors used in commercial drugs and clinical trials. However, the current yield of rAAV biomanufacturing lags behind the necessary dosages for clinical and commercial use, which embodies a concentrated reflection of low productivity of rAAV from host cells, difficult scalability of the rAAV-producing bioprocess, and high levels of impurities materialized during production. Those issues directly impact the price of gene therapy medicine in the market, limiting most patients’ access to gene therapy. In this context, the current practices and several critical challenges associated with rAAV gene therapy bioprocesses are reviewed, followed by a discussion of recent advances in rAAV-mediated gene therapy and other therapeutic biological fields that could improve biomanufacturing if these advances are integrated effectively into the current systems. This review aims to provide the current state-of-the-art technology and perspectives to enhance the productivity of rAAV while reducing impurities during production of rAAV.     

A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.     

A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus (rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1 (rHSV-1) designated HSV1-rc/AUL2, which expressed adeno-associated virus type2 (AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein (GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/AUL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit (TU) or 4.28×104 particles per cell. Therefore, compared     

Identification of a replication-defective herpes simplex virus for recombinant adeno-associated virus type 2 (rAAV2) particle assembly using stable producer cell lines

BACKGROUND: The development of stable producer cell lines for recombinant adeno-associated virus (rAAV) assembly is a strategy followed by many groups to develop scalable production methods suitable for good manufacturing practice (GMP) requirements. The major drawback of this method lies in the requirement for replicating adenovirus (Ad) for rAAV assembly. In the present study, we analyzed the ability of several replication-defective herpes simplex type 1 (HSV-1) helper viruses to induce rAAV2 particle production from stable producer cell lines. METHODS: Several stable rAAV producer cell clones were infected with wild-type and replication-defective HSV strains and analyzed for rep-cap gene amplification, viral protein synthesis and rAAV titers achieved. In vivo analysis following rAAV injection in the murine brain was also conducted to evaluate the toxicity and biopotency of the rAAV stocks. RESULTS: We demonstrated that an HSV strain mutated in the UL30 polymerase gene could efficiently be used in this context, resulting in rAAV titers similar to those measured with wild-type HSV or Ad. Importantly, with respect to clinical developments, the use of this mutant resulted in rAAV stocks which were consistently devoid of contaminating HSV particles and fully active in vivo in the murine central nervous system with no detectable toxicity. CONCLUSIONS: This study, together with our previous report describing a rAAV chromatography-based purification process, contributes to the definition of an entirely scalable process for the generation of rAAV particles.     

Enabling PAT in insect cell bioprocesses: In situ monitoring of recombinant adeno-associated virus production by fluorescence spectroscopy

The process analytical technology (PAT) initiative shifted the bioprocess development mindset towards real-time monitoring and control tools to measure relevant process variables online, and acting accordingly when undesirable deviations occur. Online monitoring is especially important in lytic production systems in which released proteases and changes in cell physiology are likely to affect product quality attributes, as is the case of the insect cell-baculovirus expression vector system (IC-BEVS), a well-established system for production of viral vectors and vaccines. Here, we applied fluorescence spectroscopy as a real-time monitoring tool for recombinant adeno-associated virus (rAAV) production in the IC-BEVS. Fluorescence spectroscopy is simple, yet sensitive and informative. To overcome the strong fluorescence background of the culture medium and improve predictive ability, we combined artificial neural network models with a genetic algorithm-based approach to optimize spectra preprocessing. We obtained predictive models for rAAV titer, cell viability and cell concentration with normalized root mean squared errors of 7%, 4%, and 7%, respectively, for leave-one-batch-out cross-validation. Our approach shows fluorescence spectroscopy allows real-time determination of the best time of harvest to maintain rAAV infectivity, an important quality attribute, and detection of deviations from the golden batch profile. This methodology can be applied to other biopharmaceuticals produced in the IC-BEVS, supporting the use of fluorescence spectroscopy as a versatile PAT tool.     

Cation-exchange high-performance liquid chromatography of recombinant adeno-associated virus type 2

There has been much interest recently in the development of recombinant viruses as vectors for gene therapy applications. We have constructed a recombinant adeno-associated viral (AAV) vector containing the gene encoding CFTR (cystic fibrosis transmembrane chloride regulator). This vector is currently being used in clinical trials as a treatment for cystic fibrosis. In the course of scale-up and process optimization efforts, a variety of analyses have been developed to characterize yield and quality. Although these methods produce quantitative and highly reproducible results, most are very time intensive. For example, a standard bioassay requires a 72-h incubation period followed by an additional day of analysis. Other tests such as UV spectrophotometry are fast, but unable to distinguish between whole virus, free protein, and DNA. Here, we describe an analytical cation-exchange high-performance liquid chromatographic method utilizing a TSKgel SP-NPR strong cation-exchange column. Unlike the bioassay which requires a 96-h wait for information, this method yields data in less than 20 min. In addition to the quick assay turn-around, the material eluting in the single peak was found to be intact, infectious, nuclease resistant AAV particles. This offers a significant advantage over the limited information one gains from UV spectrophotometry. This demonstrates the utility of chromatography for analysis and purification of viral vectors.     

The DNA minor groove binding agents Hoechst 33258 and 33342 enhance recombinant adeno-associated virus (rAAV) transgene expression

BACKGROUND: Recombinant adeno-associated viruses (rAAV) are commonly used in pre-clinical and clinical gene transfer studies. However, the relatively slow kinetics of rAAV transgene expression complicates in vitro and in vivo experiments. METHODS: 293 and COS-1 cells were transduced with rAAV2-EGFP, rAAV1-EGFP, or rAAV5-EGFP. The rAAV-EGFP expression was analyzed in the presence of Hoechst 33 258 or 33 342 as a function of time and concentration by flow cytometry and fluorescent microscope. Effects of Hoechst on cell cycle populations were determined by flow cytometry. Enhanced green fluorescent protein (EGFP) expression plasmids with or without AAV inverted terminal repeats (ITR) were constructed and gene expression by transient transfection was compared in the presence of Hoechst. RESULTS: We found that Hoechst 33 258 and 33 342 increase both the level and the population of EGFP gene expressing cells, transduced by several different serotypes of rAAV-EGFP. The augmentation of rAAV-EGFP expression occurs in different cell types in a concentration-dependent manner. In addition, the Hoechst 33 258 or 33 342 mediated enhancement of rAAV gene expression correlated with an increase of cells in S phase and G2/M phases of the cell cycle. Finally, gene expression from transfected ITR-containing plasmid DNA was also enhanced by Hoechst dyes. CONCLUSIONS: Our results revealed that two different, although related, DNA-binding drugs, Hoechst 33 258 and 33 342, accelerate the kinetics of rAAV transgene expression. These findings may provide the basis for more sensitive assessment of rAAV biological activity and also extend the applications of rAAV for in vivo gene transfer.