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.     

Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography

Adeno-associated virus (AAV) empty capsids typically co-purify with genome containing AAV2 vectors purified by column chromatography. This study describes a method to remove empty capsids from genome containing vector particles by anion exchange chromatography. The separation is based on the slightly less anionic character of empty particles compared to vectors. Detailed methods to achieve AAV2 vector purification and particle separation using cation exchange resin POROS 50HS followed by anion exchange resin Q-Sepharose^xl are described. Chromatographic separation of AAV2 particles was achieved using gradients based on sodium acetate and ammonium acetate, and was optimal at pH 8.5. Efficient removal of particle surface nucleic acid impurities was found to be important to achieve good particle separation. In a large scale experiment performed using partially purified vector containing a mixture of 1.56 × 10¹⁴ vg and 2.52 × 10¹⁵ empty capsids as a starting material, the optimized anion exchange chromatography method resulted in a vector peak of 1.15 × 10¹⁴ vg containing 0.25 × 10¹⁴ empty capsids, corresponding to 74% vector yield and 86-fold reduction in empty capsids in the vector product.     

Transduction of human adipose-derived mesenchymal stem cells by recombinant adeno-associated virus vectors

Human adipose-derived stem cells (ASCs) are attractive targets for genetic manipulation and cellular therapies. However, current methods of gene transfer are limited by lack of efficiency, toxicity, or safety concerns. Recombinant adeno-associated virus (rAAV) has been extensively assessed as a gene therapy vector and has an excellent safety profile. This study reports the efficient transduction of well-characterized, homogeneous cultures of human ASCs by rAAV serotypes 2, 5, and 6. Transduction with rAAV2 at high multiplicity of infection was associated with reduced cell viability; however, no adverse effect was seen with serotypes 5 and 6. A further increase in transduction efficiency was observed using a rAAV6 Y731F tyrosine capsid mutant. rAAV-transduced ASCs retained their adipogenic potential. Therefore, rAAV serotypes 2, 5, and 6 should be considered the vectors of choice for genetic manipulation of ASCs.     

Purification of a recombinant baculovirus of Autographa californica M nucleopolyhedrovirus by ion exchange membrane chromatography

Significant progress in the application of viral vectors for gene delivery into mammalian cells and the use of viruses as biopesticides requires downstream processing that can satisfy application-specific demands on performance. In the present work the stability and ion exchange membrane chromatography of a recombinant of Autographa californica M nucleopolyhedrovirus is studied. To adjust the degree of purification the effect of ionic conductivity or pH on the viral infectivity was assessed (0.77-78.00mS/cm, pH 3-8). Infectivity decreased rapidly by several orders of magnitude at below 5mS/cm (i.e., 0.49MPa osmotic pressure change) or at below pH 5.5 (rationalized with particle aggregation). The virus was concentrated and purified via adsorption (0.2-1.1×10(16)pfu/m(3) chromatographic bed volume, 0.6-1.1×10(12)pfu/m(2) membrane area facing the incident fluid flow) and elution at pH 6.1 and 6.35mS/cm from three strong anion exchange membranes. Virus recovery and concentration in accord with the volume reduction were obtained using a polyether sulfone-based membrane with quaternary ammonium ligands. The level of host cell protein (down to below the detection limit) and suspended DNA (below 93pg DNA per 10(6)pfu) are reported for each membrane employed, for the purpose of comparability, under equal adsorption or elution conditions respectively.     

Purification of norovirus-like particles (VLPs) by ion exchange chromatography

Recombinant expression of the norovirus capsid protein VP1 leads to self-assembly of non-infectious virus-like particles (VLPs), which are recognized as promising vaccine candidates against norovirus infections. To overcome the scalability issues connected to the ultracentrifugation-based purification strategies used in previous studies, an anion exchange-based purification method for norovirus VLPs was developed in this study. The method consists of precipitation by polyethylene glycol (PEG) and a single anion exchange chromatography step for purifying baculovirus-expressed GII.4 norovirus VLPs, which can be performed within one day. High product purity was obtained using chromatography. The purified material also contained fully assembled monodispersed VLPs, which were recognized by human sera containing polyclonal antibodies against norovirus GII.4.     

PEG-modulated column chromatography for purification of recombinant adeno-associated virus serotype 9

Column chromatography has been described for purification of recombinant adeno-associated viral vectors (rAAV) serotypes 1, 2, 5, 6 and 8. Some of these purification processes have been used in manufacturing pre-clinical grade and clinical grade rAAV vectors. Recently, recombinant AAV9 has been reported to be highly efficient in transducing cardiac muscle in animal models. Systemic or cardiac gene delivery and other applications may require large quantities of rAAV9 vectors, thus a scalable method supporting large scale purification of rAAV9 is needed for clinical development. However, column chromatography-based purification has not been reported to date for rAAV9. This study reports a polyethylene glycol (PEG) modulated chromatography process for purification of AAV9 vectors. Inclusion of PEG in chromatography buffers modulated rAAV9 elution profiles in a manner that resulted in significantly improved resin binding capacity, vector purity and yield. PEG-modulated methods were developed and optimized for hydroxyapatite and ion exchange chromatography, and shown to result in vectors of high purity and functional activity.     

Delivery of recombinant adeno-associated virus by jet injection

The jet-injection technology was used for delivery of recombinant adeno-associated virus (rAAV). Although AAV-based vectors are an attractive tool in gene therapy, some methodological and technical problems of their targeted delivery remain to be solved. We tried to address some of these cell-targeting problems by using a new low-volume needleless injection device the Swiss Injector. First we tested, by electron microscopy, whether jet-injection would have any detrimental effect on rAAV particle integrity. Second, we compared transgene expression after infection of 293T cells with fired or control (non-fired) rAAV that expressed the green fluorescent protein (GFP), beta-galactosidase (beta-gal), the B7.1 molecule, and interleukin 2 (IL2). Third, an rAAV carrying the genes coding for beta-gal was jet-injected into mouse subcutaneous (s.c.) tumours. The staining of tumour cryosections revealed beta-gal expression 72 h after the delivery. Our study demonstrated the applicability of the Swiss Injector for the delivery of rAAV into tumour tissue without either vector particle integrity or the level of expression of the transgenes, as tested in vitro, being affected. The jet-injection technology could improve the distribution of vector particles in the tumour mass without leakage of liquid from the injection site.     

Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by approximately 25-fold in WT MEFs, but only by approximately 4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency approximately 23-fold in WT MEFs, but only approximately 4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, approximately 59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only approximately 28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.     

Scalable serum-free production of recombinant adeno-associated virus type 2 by transfection of 293 suspension cells

Recombinant adeno-associated virus (rAAV) has emerged in recent years as a promising gene therapy vector that may be used in the treatment of diverse human diseases. The major obstacle to broadening the usage of rAAV vectors remains the limited capacity of available production systems to provide sufficient rAAV quantities for preclinical and clinical trials. The impracticality of expanding commonly used adherent cell lines represents a limitation to large-scale production. This paper describes successful productions of rAAV type 2 using suspension-growing human embryonic kidney (HEK293) cells in serum-free medium. The developed process, based on triple transfection employing polyethylenimine (PEI) as DNA transporter, allowed for a serum-free production of AAV, yielding viral vector titer up to 4.5x10(11) infectious viral particles (IVP) in a 3.5-L bioreactor. A maximum ratio of VG:IVP in the order of 200:1 was obtained, indicating the efficient encapsidation of viral vectors in HEK293 cells. The effect of varying the ratio of three plasmids and the influence of cell density at transfection were studied. The conditioned medium did not limit or inhibit the rAAV production; therefore, the elimination of the medium exchange step before or after transfection greatly simplified the scale-up of rAAV production. The cell-specific viral titers obtained in bioreactor suspension cultures were similar or higher than those obtained with control adherent cell cultures which further supported the scalability of the process. From multiple aspects including process simplicity, scalability, and low operating costs, this transfection method appears to be the most promising technology for large-scale production of rAAV.     

Optimization of stealth adeno-associated virus vectors by randomization of immunogenic epitopes

Therapeutic gene transfer by adeno-associated virus of serotype 2 (AAV-2) vectors is hampered in patients with pre-existing immunity. Molecular engineering was recently used to identify key immunogenic amino acid residues of the viral capsid and generate mutants with decreased antibody recognition. Here we explored the importance of finely tuning amino acid identity at immunogenic sites to optimize vector phenotype. A capsid library was generated by codon randomization at five positions where substitutions were shown to yield antibody evading phenotypes. Screening this library to isolate immune-escaping mutants allowed an exhaustive scan of combinations of the 20 natural amino acids at each position and yielded variants that remained infectious when incubated with serum or IVIG concentrations that completely neutralize AAV-2. Clones obtained replacing different residues at the same positions displayed strikingly different phenotypes, demonstrating that a precise choice of amino acid substitutions is fundamental to optimize immune-escaping, packaging ability, infectivity and tropism.     

含HPV16L1基因重组腺病毒和1型重组AAV载体联合免疫效果的研究

目的 对表达HPV16L1抗原的重组腺病毒及1型重组AAV载体联合免疫效果进行研究.方法 分别构建含密码子优化型HPVl6LI基因重组腺病毒rAd-mod.HPV16L1及1型重组从V载体rAAV1-mod-HPV 16L1,将纯化的重组AAV病毒载体以肌注及滴鼻途径单独及联合免疫C57BL/6小鼠,使用体外中和实验检测各组小鼠血清中特异性中和抗体.结果 rAAV1-med-HPV16L1单独及与rAd-mod-HPV16L1联合肌注可诱导高滴度的血清中和抗体,在初免后第16周抗体滴度显著高于其他免疫组,联合肌注组诱导的抗体滴度高于单独肌注组;重组病毒联合滴鼻虽能产生一定的免疫加强作用,但抗体滴度仍显著低于rAAV1-mod-HPV16L1单独及联合肌注组.结论 型重组从V载体联合重组腺病毒以初免.加强模式肌注可诱导更高滴度的血清中和抗体.     

Purification of hemoglobin by ion exchange chromatography in flow-through mode with PEG as an escort

Development of hemoglobin-based blood substitutes requires production of highly purified hemoglobin. Process of hemoglobin purification by ion exchange chromatography in flow-through mode was researched and optimized. Three kinds of media including, QMA Spherosil LS (Biosepra, France) and Q Sepharose Big Beads (Amersham Bioscience, Sweden), and an anion exchange membrane column, Mustang Q (PALL, USA) were investigated and compared. Adding polyethylene glycol (PEG) as an escort in ion exchange chromatography improved the purity and recovery, and the recovery in the chromatography was increased from 75 to 95%. The mechanism of PEG effects on chromatography was discussed. The optimal chromatography step, in combination with hypotonic dilution hemolyzing and membrane separation, formed an integrated hemoglobin purification process. The total recovery in the process was 87.6%. The activity of hemoglobin was well preserved: P50 23.2 mmHg, and Hill coefficient 2.31. The product appeared as a single band in SDS-PAGE, and GF-HPLC showed only one peak. The purity of the prepared hemoglobin was more than 99.9%. The optimized process is time saving and suitable for large-scale preparation of hemoglobin to provide materials for further preparation of blood substitutes.     

Pim-1基因重组腺相关病毒2载体的构建及感染大鼠视网膜

目的构建携带大鼠原癌基因Pim-1的重组腺相关病毒2载体(rAAV2-Pim-1),检测其体内感染大鼠视网膜的细胞类型及目的基因Pim-1在视网膜中的表达。方法 p AOV-CAGMINI-EGFP-2A-MCS-3FLAG载体及Pim-1基因PCR产物用Nhel酶切,琼脂糖凝胶电泳鉴定后回收载体及目的基因DNA并连接转化,鉴定质粒阳性克隆及测序。rAAV2-Pim-1表达质粒p AOV-CAGMINI-EGFP-2A-Pim-1-3FLAG及包装质粒p AAV-RC和辅助质粒p Helper,通过Lipofectamine 2000共转染293细胞,纯化获得高滴度的rAAV2-Pim-1。大鼠玻璃体注射rAAV2-Pim-1,用免疫荧光组织化学检测其感染视网膜的细胞类型;用Real-time PCR和Western blotting检测Pim-1在视网膜中的表达。结果rAAV2-Pim-1质粒构建成功并且核苷酸序列比对正确;质粒转染293细胞后出现绿色荧光;包装出的病毒浓缩滴度为5.7×1015vg/L。rAAV2-Pim-1组体内感染视网膜神经节细胞(RGCs)达71%,并感染少量无长突细胞,几乎不感染星形胶质细胞;Pim-1 mRNA和蛋白在视网膜中的表达约为rAAV2-EGFP组的6.61倍和2.29倍。结论成功构建rAAV2-Pim-1病毒载体,并在感染后的大鼠视网膜RGCs中过表达Pim-1。     

Inhibition of NF-kappaB mediated inflammation by siRNA expressed by recombinant adeno-associated virus

NF-kappaB mediated inflammation is a key process to many diseases. RNA interference (RNAi) is the specific suppression of genes by short double-stranded RNA. It was the aim of the present study to modify NF-kappaBdependent inflammation by small interfering RNA (siRNA) expressed by recombinant adeno-associated virus (rAAV). To study the kinetics of rAAV mediated expression of siRNA, the expression of the luciferase gene was targeted and resulted in a significant decrease of luciferase activity as compared to a control vector in the human 293 cell line. The effect was dose dependent and was detectable 24 h after infection. rAAV coding for siRNA against the p65 subunit of NF-kappaBsignificantly reduced the p65 protein. In a cellular model of TNF-alpha induced inflammation, expression of siRNA against p65 significantly suppressed the secretion of IL-8 from BEAS-2B cells. In conclusion, rAAV vectors coding for siRNA are an useful tool for efficient gene silencing in mammalian cells and can be used to modify NF-kappaB mediated inflammation.     

NT4-Apoptin-HA2-TAT融合基因重组腺相关病毒的构建及鉴定

目的:构建编码融合基因NT4-Apoptin-HA2-TAT的重组腺相关病毒表达载体.方法:利用限制性内切酶切相应载体后将Apoptin和HA2-TAT连入pUC19/NT4质粒, 再将融合基因NT4-Apoptin-HA2-TAT亚克隆至腺相关病毒的穿梭质粒内, 与辅助质粒pAAV/Ad、 腺病毒质粒pFG140共同转染HEK-293细胞, 通过同源重组获得NT4-Apoptin-HA2-TAT重组腺相关病毒载体, 收集病毒上清, Dot blot法测定其滴度.MTT比色法观察NT4-Apoptin-HA2-TAT重组腺相关病毒表达载体, 对HepG2细胞存活率的影响.结果:经酶切及测序证实克隆出NT4-Apoptin-HA2-TAT融合基因; 得到高滴度的(3.14×1015 pfu/L)重组腺相关病毒表达载体.NT4-Apoptin-HA2-TAT重组腺相关病毒表达载体, 对HepG2细胞有强烈的诱导凋亡作用, 与对照组比较, 处理组细胞的存活率明显降低.结论:通过分子克隆体外重组技术成功制备了NT4-Apoptin-HA2-TAT重组腺相关病毒载体, 为下一步的Apoptin应用于基因治疗奠定了基础.     

晚期糖化终产物受体反义 RNA 腺相关病毒载体的构建及在肾脏系膜细胞中表达

目的 构建晚期糖化终产物受体(RAGE)反义RNA腺相关病毒载体,并在大鼠肾脏系膜细胞中表达. 方法 构建腺相关病毒介导的RAGE反义RNA载体,3个质粒共转染293细胞,获得病毒原液,感染大鼠肾脏系膜细胞,流式细胞术、RT-PCR、ELISA检测重组病毒感染的细胞RAGE的表达和分泌细胞外基质的情况.结果 经酶切鉴定、序列分析显示RAGE基因片段正确完整反向插入pAAV-MCS.利用293细胞包装获得病毒原液的滴度为8.7×107VP/mL.感染重组病毒的细胞与正常细胞比较RAGE表达被抑制(48.2±6.1)%,分泌Ⅳ型胶原(ColⅣ)水平明显下降(P＜0.05).结论 成功构建具有抑制功能的RAGE反义RNA腺相关病毒载体,为进一步研究RAGE的作用机制,以及基因治疗RAGE相关疾病提供一个重要工具.     

Stable transduction of large DNA by high-capacity adeno-associated virus/adenovirus hybrid vectors

Viral vectors with high cloning capacity and host chromosomal integration ability are in demand for the efficient and permanent genetic modification of target cells with large DNA molecules. We have generated a hybrid gene transfer vehicle consisting of recombinant adeno-associated virus (AAV) replicative intermediates packaged in adenovirus (Ad) capsids. This arrangement allows cell cycle-independent nuclear delivery of recombinant AAV genomes with lengths considerably above the maximum size (i.e., 4.7 kb) that can be accommodated within AAV capsids. Here we show that high-capacity AAV/Ad hybrid vector gene transfer mediates cellular genomic integration of large fragments of foreign DNA and accomplishes stable long-term transgene expression in rapidly proliferating cells. Southern blot and polymerase chain reaction analyses of chromosomal DNA extracted from clones of stably transduced cells revealed that most of them contained a single copy of the full-length hybrid vector genome with AAV inverted terminal repeat (ITR) sequences at both ends. The high-capacity AAV/Ad hybrid vector system can thus be used for the transfer and expression of transgenes that cannot be delivered by conventional integrating viral vectors.