重组腺相关病毒载体相关性杂质

可以稳定表达治疗基因而无明显不良反应的重组腺相关病毒 (rAAV) 载体被认为是最有发展前景的基因治疗载体。但如何建立可以有效去除rAAV载体内具有潜在致病危害的杂质、产品质量符合临床使用要求的纯化工艺是研究人员面临的巨大挑战。其中针对载体相关性杂质的纯化工艺尤为关键,因为该类杂质的性质与真正的rAAV载体极其相似,一旦存在便难以去除,且会引起严重不良反应。以下总结了该类杂质形成的过程及有别于rAAV载体的特点,并对可以防止其生成或将其与rAAV载体有效分离的技术手段进行了评价。     

一种简单有效的基于阳离子交换层析的重组腺相关病毒纯化方法

本研究旨在探索出一套简单有效的重组腺相关病毒(rAAV)纯化方法,为基因治疗提供有力载体,推进rAAV的基因治疗走进临床.我们选用国际通用的无辅助病毒包装体系,将pAAV-cTnT-eGFP,pAAV-R2C9和pHelper三种质粒共转染HEK293T细胞系进行病毒包装,并通过荧光显微镜观察包装效率;应用阳离子交换层析法对收获处理后的病毒液进行纯化,包括第Ⅰ轮高pH下的除杂纯化和第Ⅱ轮以严格密集梯度洗脱为特点的进一步纯化.通过SDS-PAGE和Western Blot(WB)对纯化产物进行检测,使用Q-PCR法测定病毒滴度,最后再通过透射电镜对病毒颗粒进一步验证和分析.荧光显微镜结果显示,病毒包装效率较高,可达70％～80％;SDS-PAGE结果表明病毒纯化效果显著,第Ⅰ轮纯化除去了大量的蛋白杂质,第Ⅱ轮纯化也明显除去部分杂质;WB和电镜结果证明,纯化产物实为rAAV病毒,经Q-PCR测得病毒的滴度可达2.15X1012GC/mL,而且电镜进一步观察到我们纯化的终成品病毒中空颗粒含量极少,包含基因的实心颗粒占比可达99％.本研究成功开发出了一套的简单方便、切实有效的rAAV纯化方法,并且该方法能有效去除空壳病毒,提高包含基因的实心病毒占比.     

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。     

重组腺伴随病毒载体介导的hEPO转移及表达

为实现人促红细胞生成素 (humanerythropoietin ,hEPO)基因在体内的持续表达 ,构建了携带hEPO的重组腺伴随病毒 (recombinantadeno associatedvirus,rAAV)载体质粒 ,建立了稳定携带hEPO表达盒的rAAV载体细胞株 ,采用“一种辅助病毒感染一个载体细胞株”的rAAV生产策略 ,制备并纯化了携带hEPO的rAAV(rAAV hEPO)。结果表明 ,rAAV介导的hEPO转移能够使hEPO在培养的BHK 2 1细胞中得到有效表达 ;用rAAV hEPO对Balb/c小鼠进行一次肌肉注射 ,可使hEPO在小鼠体内持续表达 10周以上 ,并可明显升高小鼠的红细胞比容     

重组腺相关病毒载体基因表达效率与辐射的关系

重组腺相关病毒(rAAV)载体被认为是最有希望的临床应用基因治疗载体之一。rAAV载体基因传递的有效性与病毒衣壳蛋白的免疫原性之间的矛盾是制约该类基因药物开发的关键难题,提高其表达效率是rAAV载体研发的主攻方向之一。研究发现,紫外线、γ射线等辐射可以提高rAAV载体的基因表达效率,其作用效果与辐射种类和剂量、细胞种类和状态、载体感染指数和感染时间等因素有关。其机制可能与DNA损伤反应,特别是DNA双链断裂修复有关。对辐射与rAAV载体的表达效率之间关系的深入了解,为两者的联合应用打开方便之门。本文将对UV、γ射线等辐射提高rAAV载体表达效率的机制、影响因素及其在基因治疗领域的应用进展进行综述。     

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。     

茶氨酸提取纯化工艺研究

系统研究了从茶多酚工业废液中提取纯化茶氨酸的工艺。采用絮凝、吸附、阳离子树脂交换、重结晶工艺来分离纯化茶氨酸。结果表明,絮凝能有效的去除茶多酚工业废液中的蛋白质等杂质,杂质的去除率为50％;吸附能进一步去除色素、多酚类物质及大分子有机物;阳离子交换树脂能较专-吸附氨基酸。茶多酚工业废液经絮凝→吸附→阳离子树脂交换工艺可得纯度50%的茶氨酸,得率为1．8％;通过重结晶可得到纯度90％的茶氨酸,得率为0．8%。     

表达HCV包膜蛋白的两种重组腺相关病毒疫苗的制备及免疫原性分析

重组腺相关病毒载体(rAAV)可在动物体内高水平地持久表达外源基因,本研究采用两种rAAV载体(rAAV1与rAAV2)构建了表达丙型肝炎病毒中国分离株包膜糖蛋白(E1E2)的载体疫苗并以之免疫小鼠,分别采用免疫荧光证实其表达与总抗体,用HCV假病毒系统检测其中和抗体水平,用ELISpot分析其细胞免疫应答,结果表明:rAAV1-E1E2重组载体疫苗单针免疫激发的体液应答明显高于rAAV2-E1E2,rAAV1-E1E2单针注射后3个月可在肌肉组织中检出E2蛋白表达及特异性T细胞应答。上述结果提示HCV重组腺相关病毒载体疫苗单针免疫可引起明显持久的体液与细胞免疫应答。     

添加SNS能显著提高CHT填料使用寿命的研究

陶瓷羟基磷灰石广泛用于各种生物分子的分离和纯化,但在层析应用上,纯化洗脱过程中填料表面氢原子的释放容易导致其性能和寿命降低。研究了一种可以延长CHT填料使用寿命的方法,通过在洗脱步骤前加入一种称为表面中和体系(SNS)的溶液,结合释放的氢原子,使得整个体系处于一个接近中性pH的状态。先筛选出有效调整pH的SNS条件,通过钙离子流失的监测证实该条件的有效性。再通过在WLB304单克隆抗体纯化工艺中加入SNS之后,对羟基磷灰石填料载量、pH和压力的变化、杂质的去除能力进行考察。结果表明,加入SNS可以有效减少钙离子流失,同时并不会影响CHT层析工艺的效果。     

制备液相色谱分离纯化埃博霉素B

基于制备液相色谱法开发与优化埃博霉素B的分离纯化工艺，制备得高纯度埃博霉素B样品。实验首先对埃博霉素B粗品进行了液相色谱（HPLC）分析，定位目标峰和杂质情况。其次，对两款正相色谱填料进行筛选和模拟制备，考查不同填料对埃博霉素B相关杂质的去除效果和回收率。结果表明，上样量为0.2%时，埃博霉素B在NPLC-2分析柱（250 mm×4.6 mm, 10μm）上保留得较好，可与杂质有效分离。最后，将优化好的纯化方法在制备水平上进行放大，以正庚烷和乙酸丁酯为洗脱剂，使用NPLC-2制备柱（260 mm×50 mm, 10μm）对埃博霉素B粗品进行分离纯化，样品的色谱纯度可达99.63%，回收率为90%，各项有关杂质均符合限量规定。该方法对埃博霉素B的相关杂质去除效果好，纯化效率和回收率均很高，为埃博霉素B分离纯化生产工艺的开发提供了新方法。     

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.     

Towards a scalable bioprocess for rAAV production using a HeLa stable cell line

The majority of recombinant adeno-associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale-up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa-based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3-based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3-4 months, and presented rAAV2 volumetric production (bulk) > 3 × 10¹¹ vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity-based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time-efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 10¹¹ vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines.     

Unique biologic properties of recombinant AAV1 transduction in polarized human airway epithelia

The choice of adeno-associated virus serotypes for clinical applications is influenced by the animal model and model system used to evaluate various serotypes. In the present study, we sought to compare the biologic properties of rAAV2/1, rAAV2/2, and rAAV2/5 transduction in polarized human airway epithelia using viruses purified by a newly developed common column chromatography method. Results demonstrated that apical transduction of human airway epithelia with rAAV2/1 was 100-fold more efficient than rAAV2/2 and rAAV2/5. This transduction profile in human airway epithelia (rAAV2/1 > rAAV2/2 = rAAV2/5) was significantly different from that seen following nasal administration of these vectors to mouse lung (rAAV2/5 > rAAV2/1 > rAAV2/2), emphasizing differences in transduction of these serotypes between these two species. In stark contrast to rAAV2/2 and rAAV2/5, rAAV2/1 transduced both the apical and basolateral membrane of human airway epithelia with similar efficiency. However, the overall level of transduction across serotypes did not correlate with vector internalization. We hypothesized that differences in post-entry processing of these serotypes might influence the efficiency of apical transduction. To this end, we tested the effectiveness of proteasome inhibitors to augment nuclear translocation and gene expression from the three serotypes. Augmentation of rAAV2/1 apical transduction of human polarized airway epithelia was 10-fold lower than that for rAAV2/2 and rAAV2/5. Cellular fractionation studies demonstrated that proteasome inhibitors more significantly enhanced rAAV2/2 and rAAV2/5 translocation to the nucleus than rAAV2/1. These results demonstrate that AAV1 transduction biology in human airway epithelia differs from that of AAV2 and AAV5 by virtue of altered ubiquitin/proteasome sensitivities that influence nuclear translocation.     

Novel strategy for generation and titration of recombinant adeno-associated virus vectors

Recombinant adeno-associated virus (rAAV) vectors have many advantages for gene therapeutic applications compared with other vector systems. Several methods that use plasmids or helper viruses have been reported for the generation of rAAV vectors. Unfortunately, the preparation of large-scale rAAV stocks is labor-intensive. Moreover, the biological titration of rAAV is still difficult, which may limit its preclinical and clinical applications. For this study, we developed a novel strategy to generate and biologically titrate rAAV vectors. A recombinant pseudorabies virus (PrV) with defects in its gD, gE, and thymidine kinase genes was engineered to express the AAV rep and cap genes, yielding PS virus, which served as a packaging and helper virus for the generation of rAAV vectors. PS virus was useful not only for generating high-titer rAAV vectors by cotransfection with an rAAV vector plasmid, but also for amplifying rAAV stocks. Notably, the biological titration of rAAV vectors was also feasible when cells were coinfected with rAAV and PS virus. Based on this strategy, we produced an rAAV that expresses prothymosin alpha (ProT). Expression of the ProT protein in vitro and in vivo mediated by rAAV/ProT gene transfer was detected by immunohistochemistry and a bioassay. Taken together, our results demonstrate that the PrV vector-based system is useful for generating rAAV vectors carrying various transgenes.     

Unrestricted hepatocyte transduction with adeno-associated virus serotype 8 vectors in mice

Recombinant adeno-associated virus (rAAV) vectors can mediate long-term stable transduction in various target tissues. However, with rAAV serotype 2 (rAAV2) vectors, liver transduction is confined to only a small portion of hepatocytes even after administration of extremely high vector doses. In order to investigate whether rAAV vectors of other serotypes exhibit similar restricted liver transduction, we performed a dose-response study by injecting mice with beta-galactosidase-expressing rAAV1 and rAAV8 vectors via the portal vein. The rAAV1 vector showed a blunted dose-response similar to that of rAAV2 at high doses, while the rAAV8 vector dose-response remained unchanged at any dose and ultimately could transduce all the hepatocytes at a dose of 7.2 x 10(12) vector genomes/mouse without toxicity. This indicates that all hepatocytes have the ability to process incoming single-stranded vector genomes into duplex DNA. A single tail vein injection of the rAAV8 vector was as efficient as portal vein injection at any dose. In addition, intravascular administration of the rAAV8 vector at a high dose transduced all the skeletal muscles throughout the body, including the diaphragm, the entire cardiac muscle, and substantial numbers of cells in the pancreas, smooth muscles, and brain. Thus, rAAV8 is a robust vector for gene transfer to the liver and provides a promising research tool for delivering genes to various target organs. In addition, the rAAV8 vector may offer a potential therapeutic agent for various diseases affecting nonhepatic tissues, but great caution is required for vector spillover and tight control of tissue-specific gene expression.     

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.     

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。     

重组腺相关病毒载体的整合性研究进展

重组腺相关病毒(rAAV)载体是一种具有高靶向性和良好安全性的病毒载体,在基因治疗中得到了较为广泛的应用。目前全球范围内已有70余项以rAAV为基因药物的临床研究已经完成或正在进行中。与野生型AAV(wtAAV)定点整合不同,不表达Rep蛋白的rAAV载体与宿主染色体发生的是随机整合,而这给临床应用带来了可能的潜在的安全隐患。该文在综述wtAAV和rAAV整合机理的基础上对rAAV的因随机整合而可能导致的致癌性及其他后果进行探讨,并总结了相应应对策略,特别是目前利用Rep蛋白所开展的定点整合研究。     

Superior neovascularization and muscle regeneration in ischemic skeletal muscles following VEGF gene transfer by rAAV1 pseudotyped vectors

Recombinant adeno-associated virus serotype 2 (rAAV2) vector has been widely employed for gene therapy. Recent progress suggests that the new serotypes of AAV showed a better performance than did AAV2 in normal tissues. Here, we evaluate the potential role of human vascular endothelial growth factor (VEGF) gene transfer using rAAV vector pseudotyped with serotype 1 capsid proteins (rAAV1) in the treatment of muscle ischemia. In ischemic skeletal muscles, the rAAV1-LacZ vector allowed higher level, broader distribution, and long-lasting gene expression compared with the rAAV2-LacZ vector. Muscle VEGF165 production following the rAAV1-VEGF165 vector injection was 5-10 times higher than that following the rAAV2-VEGF165 vector injection. VEGF165 production mediated by the rAAV1-VEGF165 vector stimulated a large set of neovascularization with relatively mature vascular structures and enhanced muscle regeneration in the ischemic skeletal muscles. Thus, the rAAV1-VEGF165 vector mediated gene transfer may be a therapeutic approach to peripheral vascular diseases.