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.     

含HIV-1 gag、gagV3基因的重组腺病毒伴随病毒的构建及其免疫原性的研究

将HIV－1中国株42（B亚型）gag基因及gag与gp120 V3区的嵌合基因gag V3插入腺病毒伴随病毒（AAV）表达载体（pSNAV）质粒中,构建重组质粒pSNAV-gag及pSNAV-gagV3;采用脂质体转染的方法分别将重组质粒转入BHK细胞,G418筛选得到转入重组质粒并能表达外源基因的细胞系,命名为BHK－gag及BHK－gagV3。用具有重组腺病毒伴随病毒（rAAV）包装功能的一种重组单纯疱疹病毒（rHSV）分析感染这两株细胞系,纯化后得到rAAV,电镜观察可见到大量实心病毒颗粒,核酸杂交检测重组病毒滴度达到10^12病毒颗粒／ml,重组病毒感染293细胞,ELISA检测有gag及gagV3基因的表达。用重组病毒免疫Balb/C小鼠,检测抗体及细胞免疫水平,证明重组病毒可以在小鼠体内诱导产生细胞及体液免疫。     

重组腺相关病毒2型/人肿瘤坏死因子受体:Fc(rAAV2/hTNFR:Fc)的构建和生物学活性研究

为研究腺相关病毒2型载体应用于类风湿性关节炎进行基因治疗的可行性,首先构建携带人肿瘤坏死因子Ⅱ型受体胞外区和人免疫球蛋白IgG1Fc段融合基因的重组2型腺相关病毒(rAAV2／hTNFR：Fc),并对其生物学活性进行研究。以RT—PCR分别从U937和人淋巴细胞总RNA中扩增人肿瘤坏死因子Ⅱ型受体胞外区和人免疫球蛋白IgG1Fc段基因,并以重叠延伸PCR的方法将二者融合后克隆入pSNAV1载体质粒进行重组病毒生产,在进行重组病毒理化分析后,以TNFa细胞毒中和试验来研究该重组病毒的生物学活性。结果显示：所构建的重组病毒rAAV2／hTNFR：Fc基因结构与预期一致;病毒在体外感染BHK-21细胞后,含TNFR：Fc融合蛋白的表达上清可以有效中和人、大鼠、小鼠TNFα对L929的细胞毒性。所研究构建的重组腺相关病毒可以用来作为阻断TNFα的手段,进行类风湿性关节炎的基因治疗研究。     

Therapeutic level of functional human alpha 1 antitrypsin (hAAT) secreted from murine muscle transduced by adeno-associated virus (rAAV1) vector

Alpha 1 antitrypsin (AAT) is a serine proteinase inhibitor (serpin). One well-known function of this protein is to inactivate neutrophil elastase and other neutrophil-derived proteinases, and prevent the destruction of pulmonary extracellular matrix. Deficiency of AAT can cause emphysema due to degradation of interstitial elastin by elastase. The majority of circulating AAT is secreted from the liver. Muscle-directed gene therapy using recombinant adeno-associated virus 2 (rAAV2) vectors has been tested to increase the serum levels of AAT. However, inefficient transduction of rAAV2 vector makes it difficult to reach therapeutic levels of AAT in clinical trials and it remains unclear as to whether muscle-secreted AAT is functional. In the present study, we evaluated five serotypes (1, 2, 3, 4, and 5) of rAAV vectors for transduction efficiency in mouse muscle. Results from these studies showed that rAAV1 is the most efficient vector among these serotypes and mediated at least 100-fold higher levels of AAT secretion than the rAAV2 vector. Western blot analysis showed that this murine muscle-secreted human AAT (hAAT) formed a complex with human neutrophil elastase in a dose-dependent manner. An anti-elastase activity assay showed that murine muscle-secreted hAAT inhibited elastase with equal capacity as hAAT purified from plasma. These results provide strong support for the functionality of AAT in ongoing clinical studies of muscle-directed AAT gene therapy.     

HIV-1B亚型gp120基因密码子优化前后免疫原性的比较

对HIV-1B亚型gp120基因按照哺乳动物优势密码子的使用原则进行优化,以Westem blot方法比较其体外表达量.将优化前的野生型gp120基因和改造后的modgp120基因插入重组腺伴随病毒载体,构建了重组病毒rAAV-wtgp120和rAAV-modgp120,比较两者免疫Balb/C小鼠后的抗体和CTL应答.Western blot检测结果显示:优化后基因的体外表达量明显高于野生型基因,rAAV-modgp120与rAAV-wtgp120相比可更好地诱导Balb/C小鼠的CTL应答,但检测不到明显的抗体反应.由此得出结论,优化后gp120基因的体外表达量明显高于野生型基因,并且可以诱导更强的特异性CTL应答,但检测不到gp120抗体.     

基于TCID50检测AAV9载体制品感染性滴度的方法

目的:建立一种基于半数组织培养感染剂量(median tissue culture infective dose,TCID₅₀)检测9型腺相关病毒(adeno-associated virus type 9,AAV9)载体制品感染性滴度的方法。方法:利用含AAV2 rep和cap基因的1型单纯疱疹病毒(herpes simplex virus type1,HSV1)做为辅助病毒与梯度稀释的AAV9载体制品共同感染HEK-293细胞,培养48 h后用实时荧光定量PCR(quantitative real-time PCR,qPCR)扩增AAV特异性反向末端重复序列(inverted terminal repeats,ITR),根据阳性及阴性感染孔数,利用Kärber法计算样品的TCID₅₀。结果:采用携带增强绿色荧光蛋白报告基因的AAV9载体制品确定辅助病毒HSV1-rc最佳感染复数(multiplicity of infection,MOI)为5,AAV9-101的感染性滴度为1.6×10⁹ TCID₅₀/mL。结论:对AAV9载体制品进行感染性滴度检测,且具有可重复性。     

一种包装AAV2/5的重组单纯疱疹病毒的构建

为了得到一种可以包装AAV2/5和表达绿色荧光蛋白的重组单纯疱疹病毒,设计并构建了一个由AAV2rep基因和AAV5cap基因嵌合而成的rep2cap5基因,然后,利用一套携带HSV1基因组的粘粒系统(cos6、cos28、cos14、cos56、cos48),将rep2cap5基因插入cos6粘粒上HSV1基因组片段的UL2基因中,而将EGFP的表达单位插入cos56粘粒上HSV1基因组片段的UL44基因中,用这2个重组粘粒与其它3个粘粒(cos14、cos28、cos48)共转染BHK-21细胞获得了重组病毒HSV1-r2c5-EGFP并进行了空斑纯化。HSV1-r2c5-EGFP病毒能够在BHK-21细胞连续传代,并且可以观察到几乎所有的感染细胞都能产生绿色荧光。用PCR方法以及Southern杂交方法表明所获得的HSV1-r2c5-EGFP中携带有rep2cap5基因,用HSV1-r2c5-EGFP感染携带报告基因LacZ的AAV载体细胞株,获得了具有感染性的重组AAV2/5-LacZ。结果表明,所获得的重组单纯疱疹病毒HSV1-r2c5-EGFP可提供AAV2/5载体包装所需的全部辅助功能,是一种能简便、高效制备重组AAV2/5病毒的通用性辅助病毒。     

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.     

Development and characterization of an antisense-mediated prepackaging cell line for adeno-associated virus vector production

One of the limitations of recombinant adeno-associated virus (rAAV) vector systems for gene therapy applications has been the difficulty in producing the vector in sufficient quantity for adequate evaluation. Since the AAV Rep proteins are cytotoxic, it is not easy to establish stable cell lines that express them constitutively. We describe a novel 293-derived prepackaging cell line which constitutively expresses the antisense rep/cap driven by a loxP-flanked CMV promoter. This cell line was converted into a packaging cell line expressing Rep/Cap for rAAV vector production through adenovirus-mediated introduction of a Cre recombinase gene. Without the introduction of the Cre recombinase gene, the cell line was shown to produce neither Rep nor Cap. rAAV vector was produced (1 x 10(9) genome copies/3.5-cm dish) 4 days after the transduction with Cre-expression adenovirus vector together with transfection of AAV vector plasmid. We further showed that the addition of Cap-expression adenovirus vector caused a 10-fold increase in the yield of rAAV vector. This system is also capable of producing rAAV as a transfection-free system by using a small amount of rAAV instead of vector plasmid.     

Design space determination to optimize DNA complexation and full capsid formation in transient rAAV manufacturing

Recombinant adeno-associated virus (rAAV) vectors are a promising platform for in vivo gene therapies. However, cost-effective, well-characterized processes necessary to manufacture rAAV therapeutics are challenging to develop without an understanding of how process parameters (PPs) affect rAAV product quality attributes (PQAs). In this work, a central composite orthogonal experimental design was employed to examine the influence of four PPs for transient transfection complex formation (polyethylenimine:DNA [PEI:DNA] ratio, total DNA/cell, cocktail volume, and incubation time) on three rAAV PQAs related to capsid content (vector genome titer, vector genome:capsid particle ratio, and two-dimensional vector genome titer ratio). A regression model was established for each PQA using partial least squares, and a design space (DS) was defined in which Monte Carlo simulations predicted < 1% probability of failure (POF) to meet predetermined PQA specifications. Of the three PQAs, viral genome titer was most strongly correlated with changes in complexation PPs. The DS and acceptable PP ranges were largest when incubation time and cocktail volume were kept at mid-high setpoints, and PEI:DNA ratio and total DNA/cell were at low-mid setpoints. Verification experiments confirmed model predictive capability, and this work establishes a framework for studying other rAAV PPs and their relationship to PQAs.     

Recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap.

Recombinant adeno-associated virus (AAV) type 2 (rAAV) vectors have recently been shown to have great utility as gene transfer agents both in vitro and in vivo. One of the problems associated with the use of rAAV vectors has been the difficulty of large-scale vector production. Low-efficiency plasmid transfection of the rAAV vector and complementing AAV type 2 (AAV-2) functions (rep and cap) followed by superinfection with adenovirus has been the standard approach to rAAV production. The objectives of this study were to demonstrate the ability of a recombinant herpes simplex virus type 1 (HSV-1) amplicon expressing AAV-2 Rep and Cap to support replication and packaging of rAAV vectors. HSV-1 amplicon vectors were constructed which contain the AAV-2 rep and cap genes under control of their native promoters (p5, p19, and p40). An HSV-1 amplicon vector, HSV-RC/KOS or HSV-RC/d27, was generated by supplying helper functions with either wild-type HSV-1 (KOS strain) or the ICP27-deleted mutant of HSV-1, d27-1, respectively. Replication of the amplicon stocks is not inhibited by the presence of AAV-2 Rep proteins, which highlights important differences between HSV-1 and adenovirus replication and the mechanism of providing helper function for productive AAV infection. Coinfection of rAAV and HSV-RC/KOS resulted in the replication and amplification of rAAV genomes. Similarly, rescue and replication of rAAV genomes occurred when rAAV vector plasmids were transfected into cells followed by HSV-RC/KOS infection and when two rAAV proviral cell lines were infected with HSV-RC/KOS or HSV-RC/d27. Production of infectious rAAV by rescue from two rAAV proviral cell lines has also been achieved with HSV-RC/KOS and HSV-RC/d27. The particle titer of rAAV produced with HSV-RC/d27 is equal to that achieved by supplying rep and cap by transfection followed by adenovirus superinfection. Importantly, no detectable wild-type AAV-2 is generated with this approach. These results demonstrate that an HSV-1 amplicon expressing the AAV-2 genes rep and cap along with HSV-1 helper functions supports the replication and packaging of rAAV vectors in a scaleable process.     

腺相关病毒介导的RNA干扰抑制HeLa细胞中CTCF的表达

目的:利用RNA干扰(RNAi)稳定抑制HeLa细胞中CTCF的表达。方法:构建能够抑制转录因子CTCF表达的短发夹RNA(shRNA)的腺相关病毒载体,重组病毒感染HeLa细胞后挑取单克隆,用Western印迹法和实时荧光定量PCR检测CTCF的表达水平。结果:获得3株CTCF被显著抑制的HeLa细胞株,Western印迹法和实时荧光定量PCR结果均显示CTCF的表达水平被抑制,其中最明显的被下调76%。结论:shRNA病毒表达载体构建成功,HeLa细胞中CTCF的表达可被长期稳定地抑制。     

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。     

Tet-On调控HSVtk表达的重组腺相关病毒载体的构建和感染活性的检测

构建含有Tet基因调节系统及自杀基因HSVtk的重组腺相关病毒载体pAAV TRE HSVtk Tet_On ,并使用PCR技术和限制性内切酶消化进行鉴定。用构建好的重组质粒分别与辅助质粒pAAV_RC、pHelper以磷酸钙共沉淀法转染HEK2 93细胞,进行病毒包装后得到了AAV TRE HSVtk Tet_On重组腺相关病毒,以氯化铯密度梯度离心对包装好的病毒进行纯化。用纯化后的重组腺相关病毒感染乳腺癌细胞株MCF_7后,斑点杂交检测结果显示,HSVtk基因整合进入MCF_7细胞基因组中。有感染活性的重组腺相关病毒能将目的基因转移到宿主细胞中,在Dox诱导下,GCV对AAV感染的MCF_7细胞具有明显的杀伤作用。     

Scalable production of adeno-associated virus type 2 vectors via suspension transfection

Vectors derived from adeno-associated virus type 2 (AAV2) are promising gene delivery vehicles, but it is still challenging to get the large number of recombinant adeno-associated virus (rAAV) particles required for large animal and clinical studies. Current transfection technology requires adherent cultures of HEK 293 cells that can only be expanded by preparing multiple culture plates. A single large-scale suspension culture could replace these multiple culture preparations, but there is currently no effective co-transfection scheme for generating rAAV from cells in suspension culture. Here, we weaned HEK 293 cells to suspension culture using hydrogel-coated six-well culture plates and established an efficient transfection strategy suitable for these cells. Then the cultures were gradually scaled up. We used linear polyethylenimine (PEI) to mediate transfection and obtained high transfection efficiencies ranging from 54% to 99%, thereby allowing efficient generation of rAAV vectors. Up to 10(13) rAAV particles and, more importantly, up to 10(11) infectious particles were generated from a 2-L bioreactor culture. The suspension-transfection strategy of this study facilitates the homogeneous preparation of rAAV at a large scale, and holds further potential as the basis for establishing a manufacturing process in a larger bioreactor.     

Development and characterization of a cell line for large-scale, serum-free production of recombinant adeno-associated viral vectors

BACKGROUND: One of the major limitations to the use of adeno-associated virus (AAV) vectors for gene therapy has been the difficulty in producing enough vector to supply a clinical trial. More than 20 000 roller bottles may be required to generate AAV by the traditional transient transfection process to treat 50 patients. A scalable AAV producer cell line grown in serum-free media will meet the needs for the manufacture of AAV gene therapeutics. METHODS: A packaging cell line was generated by introducing the AAV rep and cap genes into A549 cells. From this packaging cell line, a number of producer cell lines were generated by infecting the packaging cell with the appropriate AAV vector. Producer cell lines were then adapted to serum-free suspension conditions for growth in bioreactors. RESULTS: We report here the development of six AAV producer cell lines that generate > 10(4) particles/cell. The rAAV vector preparations from these cell lines have physical and functional characteristics similar to rAAV vectors prepared by transient transfection. To enable large-scale production, producer cell lines were adapted to serum-free suspension and we demonstrate production of AAV at the 15 L scale. In addition, vector preparations from these cell lines were shown to be free of wild-type AAV. CONCLUSIONS: AAV producer cell lines can be readily scaled to meet the needs of clinical trials. One 500 L bioreactor of these producer cells can produce the equivalent of 2500 high capacity roller bottles or 25 000 T-175 tissue culture flasks.     

A concept of eliminating nonhomologous recombination for scalable and safe AAV vector generation for human gene therapy

Scalable and efficient production of high-quality recombinant adeno-associated virus (rAAV) for gene therapy remains a challenge despite recent clinical successes. We developed a new strategy for scalable and efficient rAAV production by sequestering the AAV helper genes and the rAAV vector DNA in two different subcellular compartments, made possible by using cytoplasmic vaccinia virus as a carrier for the AAV helper genes. For the first time, the contamination of replication-competent AAV particles (rcAAV) can be completely eliminated in theory by avoiding ubiquitous nonhomologous recombination. Vector DNA can be integrated into the host genomes or delivered by a nuclear targeting vector such as adenovirus. In suspension HeLa cells, the achieved vector yield per cell is similar to that from traditional triple-plasmid transfection method. The rcAAV contamination was undetectable at the limit of our assay. Furthermore, this new concept can be used not only for production of rAAV, but also for other DNA vectors.     

Long-term modifications of blood pressure in normotensive and spontaneously hypertensive rats by gene delivery of rAAV-mediated cytochrome P450 arachidonic acid hydroxylase

INTRODUCTION It is well known that arachidonic acid (AA) exists ex- tensively in eukaryotic cells and it is metabolized by cycloxygenases and lipoxygenases to produce prostag- landins (PGs) and hydroperoxyeicosatetraenoic acid(HPETE) [1-3]. In the 1980’s…     

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.