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A rapid protocol for construction and production of high-capacity adenoviral vectors

Nature Protocols volume 4, pages 547–564 (2009)Cite this article

Abstract

High-capacity adenoviral vectors (HC-AdVs) lacking all viral coding sequences were shown to result in long-term transgene expression and phenotypic correction in small and large animal models. It has been established that HC-AdVs show significantly reduced toxicity profiles compared with early-generation adenoviral vectors. Furthermore, with capsid-modified HC-AdV becoming available, we are just starting to understand the full potential of this vector system. However, for many researchers, the wide-scale use of HC-AdV is hampered by labor-intensive and complex production procedures. Herein, we provide a feasible and detailed protocol for efficient generation of HC-AdV. We introduce an efficient cloning strategy for the generation of recombinant HC-AdV vector genomes. Infection and amplification of the HC-AdV are performed in a spinner culture system. For purification, we routinely apply cesium chloride gradients. Finally, we describe various methods for establishing vector titers. Generation of high-titer HC-AdV can be achieved in 3 weeks.

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Figure 1: Schematic diagram for high-capacity adenoviral vector (HC-AdV) production.
Figure 2: Schematic maps of pHM5 and pAdFTC.
Figure 3: Flowchart of cloning the transgene expression cassette into pAdFTC.
Figure 4: Amplification of high-capacity adenoviral vectors (HC-AdV) in spinner flasks.
Figure 5: Monitoring adenoviral amplification steps during large-scale production.
Figure 6: Flowchart of CsCl purification of HC-AdV.
Figure 7: Transducing units in final vector preparations as determined by Southern blot analysis.

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Acknowledgements

This work was supported by DFG grants SFB455 and SPP1230 and the Wilhelm Sander Stiftung to A.E. We thank Philip Ng for helpful discussions and advice.

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Author notes

  1. Lorenz Jager, Martin A Hausl and Christina Rauschhuber: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Virology, Max von Pettenkofer-Institute, Ludwig-Maximilians University Munich, Munich, 80336, Germany

    Lorenz Jager, Martin A Hausl, Christina Rauschhuber, Nicola M Wolf & Anja Ehrhardt

  2. Department of Pediatrics, School of Medicine, Stanford University, 269 Campus Drive, CCSR Building, Room 2105, Stanford, 94305-5164, CA, USA

    Mark A Kay

  3. Department of Genetics, School of Medicine, Stanford University, 269 Campus Drive, CCSR Building, Room 2105, Stanford, 94305-5164, CA, USA

    Mark A Kay

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  1. Lorenz Jager
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Correspondence to Anja Ehrhardt.

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Jager, L., Hausl, M., Rauschhuber, C. et al. A rapid protocol for construction and production of high-capacity adenoviral vectors. Nat Protoc 4, 547–564 (2009). https://doi.org/10.1038/nprot.2009.4

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