基于人类腺病毒5型高容量腺病毒载体的克隆及规模化生产
Summary
A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.
Abstract
High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.
Introduction
用于基因治疗应用是非常重要的，以避免细胞毒性和免疫原性的副作用引起的病毒蛋白质的表达，转基因本身或者由传入的病毒蛋白。腺病毒载体（腺病毒）被广泛用于将外源DNA成多种细胞的研究转基因表达1,2的影响。进阶最先进的版本是由高容量腺病毒载体（HCAdV）缺少所有的病毒编码序列3,4，从而提供包装容量高达35 KB加上低免疫原性和低毒性5-8来表示。由于其高的包装容量它们允许输送的使用单一载体剂量大或多个转基因。因此，它们代表了研究界的宝贵工具。
在对比第一或第二代腺病毒缺乏早期基因的E1和/或E3，可以使用商业试剂盒可容易地制造，vec的器的基因组结构和病毒产生HCAdV的更为复杂。该系统为HCAdV基因组的结构是基于质粒pAdFTC携带HCAdV基因组缺乏所有病毒编码序列和穿梭质粒pHM5 9-12中 。的高达14千碱基（kb的）任何感兴趣的基因可以被克隆到穿梭载体pHM5其中多克隆位点被识别侧翼/裂解归巢内切核酸酶位点PI- SCE我和I- CEU I.因此，感兴趣的克隆的基因可以通过连续的PI-SCE我和I- CEU我被释放消化用于随后定向插入到存在于包含于质粒pAdFTC的HCAdV基因组中的相同限制性位点。在pAdFTC位于PI- SCE I和I-CEU I切割位点之间的转基因插入位点是由填塞DNA和所需的基因组的包装，如5'和3'末端反向重复的非编码腺病毒序列（ITRS）侧翼在端部和5'ITR下游的包装信号两者。额外填充的DNA提供了最后的HCAdV基因组范围从27到36 KB的最优规模，以确保病毒在生产过程中高效的包装。因为pAdFTC是一个大质粒高达45千字节（依赖于插入的转基因的大小）和归巢核酸内切酶具有同等的长DNA识别位点的使用具有强DNA结合，从pHM5传递转基因的过程中几个净化步骤是必要的到pAdFTC。小心处理，避免剪切力的建议。
所述HCAdV基因组的的ITR由直接位于5'ITR的上游和3'ITR 12的下游Not I限制性内切酶识别位点侧接。因此，HCAdV可以通过的Not I消化用于随后转染的病毒基因组进入HCAdV生产者细胞系被释放。需要注意的是限制性内切酶的使用不余为相对便于从质粒pAdFTC病毒基因组，则表示插入转基因是缺乏的Not I DNA识别位点。在HEK293细胞根据生产细胞（116细胞）稳定表达Cre重组酶。对于病毒扩增116细胞被共感染辅助病毒（HV）提供所需要的复制和包装的反式 3,4-所有的AdV基因。高压是第一代腺病毒与被在病毒扩增通过Cre重组酶表达116细胞4移除的两侧装接loxP的包装信号。这确保了含有完整包装信号主要HCAdV基因组包被。
预放大的HCAdV的通过进行在116细胞连续传代步骤上生长在组织培养皿的表面进行的。每次传代后的病毒颗粒通过导电三个连续的冻融步骤释放从感染细胞。随着每一个通道增加细胞数量被感染三分之一从前面的传代细胞裂解物。最后溶解物从最后预扩增步骤用于感染在转瓶中大规模扩增中悬浮生长生产细胞。病毒颗粒从悬浮液中的细胞通过在氯化铯密度梯度4,12进行超速离心纯化。与此过程空粒子和完全组装颗粒分离成两个不同的频带。进一步浓缩HCAdV颗粒进行第二非渐进超速离心步骤。接着含有HCAdV所得频带被收集和透析生理缓冲液。最终载体制剂的特征在于相对于绝对病毒颗粒，感染颗粒和高压污染水平的数字。绝对的病毒颗粒可以通过裂解病毒颗粒，并测量260nm处的吸光度或通过进行定量实时PCR（qPCR的）12来确定。在PUR的感染性指明分数病毒颗粒可通过感染细胞3小时后感染内本的qPCR测量HCAdV基因组来确定。
Protocol
Representative Results
Discussion
这里介绍的协议允许根据基于先前描述的方法4,12人腺病毒5型HCAdV矢量纯化。在pAdFTC质粒内的基因组HCAdV是缺乏所有腺病毒基因和仅携带5'-和3'-的ITR和包装信号。在这一战略的HV AdNG163R-2 4提供了所有必要的基因的高效病毒生产反式 。这提供了一个包装容量高达35 KB，这显然outcompetes第一代和第二代副词或广泛使用的慢病毒（LV） – 或腺相关病毒（AAV）为基础的载体。 HCAdV…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This work was supported by DFG grant EH 192/5-1 (A.E.), the EU (E-rare-2) project Transposmart (A.E.), the UWH Forschungsförderung (E.S. and W.Z), and the PhD programme of the University Witten/Herdecke (P.B.). J.L. was supported by a stipend of the Chinese Scholarship council and T.B. and M.G by the Else Kröner-Fresenius foundation (EKFS).
Materials
| I-CeuI | New England Biolabs | R0699S | restriction digest |
| PI-SceI | New England Biolabs | R0696S | restriction digest |
| T4 Ligase | New Engand Biolabs | M0202S | ligation |
| SwaI | New England Biolabs | R0604S | restriction digest |
| NotI | New England Biolabs | R0189S | restriction digest |
| Calf Intestinal Alkaline Phosphatase (CIP) | New England Biolabs | M0290S | dephosphorylation of digested plasmids |
| Hygromycin B | PAN Biotech | P02-015 | selection of CRE expressing 116 cells |
| DMEM | PAN Biotech | P04-03590 | Hek293T cell culture medium |
| Minimal Essential Medium (MEM) Eagle | PAN Biotech | P04-08500 | 116 cell culture medium |
| Dulbecco’s phosphate buffer saline (DPBS) | PAN Biotech | P04-36500 | washing of cells, resuspension of cells |
| 250 ml storage bottle | Sigma | CLS430281-24EA | infection of 116 cells grown in suspension |
| 500mL PP CentrifugeTubes | Sigma | CLS431123-36EA | sedimentation of cells from suspension culture |
| Spinner flask | Bellco | 1965-61030 | growth of 116 cells in suspension |
| Ultra Clear Ultracentrifuge tubes | Beckmann Coulter | 344059 | density gradient centrifugation |
| Ultracentrifuge | Beckmann Coulter | – | density gradient centrifugation |
| SW-41 rotor | Beckmann Coulter | – | density gradient centrifugation |
| Spectrum Laboratories Spectrapor Membrane | VWR | 132129 | dialysis tubing |
| ready-to-use dialysis cassettes | Thermo | 66383 | dialysis |
| one shot DH10B electrocompetent E. coli | invitrogen | C4040-52 | transformation of ligation reactions |
| PureYield™ Plasmid Midiprep System | Promega | A2495 | midiprep |
| peqGOLD Tissue DNA Mini Kit | Peqlab | 12-3396-02 | isolation of genomic DNA |
| SuperFect Transfection Reagent | Qiagen | 301305 | tranfection of 116 cells |
| opti MEM (10 % FBS) | Gibco | 31985-062 | transfection of 116 cells |
| iQ SYBR Green Supermix | BioRad | 170-8882 | q-PCR |
| CFX 96 C1000 touch | Biorad | qPCR machine | |
| Phenol/Chloroform/Isoamyl alcohol | Carl Roth | A156.1 | purification of DNA |
| Cesium chloride | Carl Roth | 8627.1 | density gradient centrifugation |
| sodium acetate 99% | Carl Roth | 6773.2 | DNA precipitation |
| LB medium | Carl Roth | X968.3 | bakterial growth medium |
| ethanol 99,8% pure | Carl Roth | 9065.5 | DNA precipitation and washing |
| SDS 99,5% | Carl Roth | 2326.2 | lysis buffer |
| EDTA | Carl Roth | 8043.2 | lysis buffer |
| Tris-HCl 99% | Carl Roth | 9090.3 | dialysis buffer/ lysis buffer |
| glycerol 99,5% | Carl Roth | 3783.1 | dialysis buffer |
| MgCl2 98,5% | Carl Roth | KK36.2 | dialysis buffer |
| NaCl | Carl Roth | 3957.1 | optional dialysis buffer |
| KH2PO4 | Carl Roth | 3904.2 | optional dialysis buffer |
| sucrose | Carl Roth | 9286.1 | optional dialysis buffer |
| Na2HPO4x2H2O | Carl Roth | 4984.2 | optional dialysis buffer |
| 1,5ml tubes | sarstedt | 72,730,005 | storage of virus preparations at -80°C |
Referencias
- Benihoud, K., Yeh, P., Perricaudet, M. Adenovirus vectors for gene delivery. Curr Opin Biotechnol. 10 (5), 440-447 (1999).
- Crystal, R. G. Adenovirus: the first effective in vivo gene delivery vector. Hum Gene Ther. 25 (1), 3-11 (2014).
- Parks, R. J., et al. A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sci U S A. 93 (24), 13565-13570 (1996).
- Palmer, D., Ng, P. Improved system for helper-dependent adenoviral vector production. Mol Ther. 8 (5), 846-852 (2003).
- Morral, N., et al. High doses of a helper-dependent adenoviral vector yield supraphysiological levels of alpha1-antitrypsin with negligible toxicity. Hum Gene Ther. 9 (18), 2709-2716 (1998).
- Muruve, D. A., et al. Helper-dependent adenovirus vectors elicit intact innate but attenuated adaptive host immune responses in vivo. J Virol. 78 (11), 5966-5972 (2004).
- Ehrhardt, A., et al. A gene-deleted adenoviral vector results in phenotypic correction of canine hemophilia B without liver toxicity or thrombocytopenia. Blood. 102 (7), 2403-2411 (2003).
- Cots, D., Bosch, A., Chillon, M. Helper dependent adenovirus vectors: progress and future prospects. Curr Gene Ther. 13 (5), 370-381 (2013).
- Mizuguchi, H., Kay, M. A. Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method. Hum Gene Ther. 9 (17), 2577-2583 (1998).
- Mizuguchi, H., Kay, M. A. A simple method for constructing E1- and E1/E4-deleted recombinant adenoviral vectors. Hum Gene Ther. 10 (12), 2013-2017 (1999).
- Ehrhardt, A., Kay, M. A. A new adenoviral helper-dependent vector results in long-term therapeutic levels of human coagulation factor IX at low doses in vivo. Blood. 99 (11), 3923-3930 (2002).
- Jager, L., et al. A rapid protocol for construction and production of high-capacity adenoviral vectors. Nat Protoc. 4 (4), 547-564 (2009).
- Sambrook, J., Russell, D. W. . Molecular Cloning: A Laboratory Manual. 1-3, (2001).
