Skip to main content
SpringerLink
Log in

Construction of Capsid-Modified Adenoviruses by Recombination in Yeast and Purification by Iodixanol-Gradient

  • Protocol
  • First Online:
Oncolytic Viruses

Part of the book series: Methods in Molecular Biology ((MIMB,volume 797))

Abstract

Adenovirus represents a valuable tool for the treatment of cancer, but tumor targeting remains a pending issue. Most common procedures to modify adenovirus genome are time-consuming due to the requirement of multiple cloning steps, and the low efficacy of the recombination process. Here, we present a new method for homologous recombination in yeast to fast construct recombinant adenoviruses. Also, an alternative procedure to purify viral stocks, based on iodixanol gradient is described. Compared to classical methods, iodixanol is nontoxic to cells, which avoids desalting to use in vitro and in vivo. Moreover, viral stocks are more viable and it can be used for large-scale purifications. Finally, a protocol for analyzing blood persistence of modified vector in in vivo biodistribution is presented.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Alemany, R., and Curiel, D. T. (2001) CAR-binding ablation does not change biodistribution and toxicity of adenoviral vectors, Gene therapy8, 1347–1353.

    Article  CAS  Google Scholar 

  2. Bayo-Puxan, N., Cascallo, M., Gros, A., Huch, M., Fillat, C., and Alemany, R. (2006) Role of the putative heparan sulfate glycosaminoglycan-binding site of the adenovirus type 5 fiber shaft on liver detargeting and knob-mediated retargeting, The Journal of general virology87, 2487–2495.

    Article  CAS  Google Scholar 

  3. Einfeld, D. A., Schroeder, R., Roelvink, P. W., Lizonova, A., King, C. R., Kovesdi, I., and Wickham, T. J. (2001) Reducing the native tropism of adenovirus vectors requires removal of both CAR and integrin interactions, Journal of virology75, 11284–11291.

    Article  CAS  Google Scholar 

  4. Smith, T. A., Idamakanti, N., Rollence, M. L., Marshall-Neff, J., Kim, J., Mulgrew, K., Nemerow, G. R., Kaleko, M., and Stevenson, S. C. (2003) Adenovirus serotype 5 fiber shaft influences in vivo gene transfer in mice, Human gene therapy14, 777–787.

    Article  CAS  Google Scholar 

  5. Kritz, A. B., Nicol, C. G., Dishart, K. L., Nelson, R., Holbeck, S., Von Seggern, D. J., Work, L. M., McVey, J. H., Nicklin, S. A., and Baker, A. H. (2007) Adenovirus 5 fibers mutated at the putative HSPG-binding site show restricted retargeting with targeting peptides in the HI loop, Mol Ther15, 741–749.

    Google Scholar 

  6. Shayakhmetov, D. M., Gaggar, A., Ni, S., Li, Z. Y., and Lieber, A. (2005) Adenovirus binding to blood factors results in liver cell infection and hepatotoxicity, Journal of virology79, 7478–7491.

    Article  CAS  Google Scholar 

  7. Kalyuzhniy, O., Di Paolo, N. C., Silvestry, M., Hofherr, S. E., Barry, M. A., Stewart, P. L., and Shayakhmetov, D. M. (2008) Adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo, Proceedings of the National Academy of Sciences of the United States of America105, 5483–5488.

    Google Scholar 

  8. Waddington SN, M. J., Bhella D, Parker AL, Barker K, Atoda H, Pink R, Buckley SM, Greig JA, Denby L, Custers J, Morita T, Francischetti IM, Monteiro RQ, Barouch DH, van Rooijen N, Napoli C, Havenga MJ, Nicklin SA, Baker AH. (2008) Adenovirus serotype 5 hexon mediates liver gene transfer., Cell132, 397–409.

    Google Scholar 

  9. Alba, R., Bradshaw, A. C., Parker, A. L., Bhella, D., Waddington, S. N., Nicklin, S. A., van Rooijen, N., Custers, J., Goudsmit, J., Barouch, D. H., McVey, J. H., and Baker, A. H. (2009) Identification of coagulation factor (F)X binding sites on the adenovirus serotype 5 hexon: effect of mutagenesis on FX interactions and gene transfer, Blood114, 965–971.

    Article  CAS  Google Scholar 

  10. Carlisle, R. C., Di, Y., Cerny, A. M., Sonnen, A. F., Sim, R. B., Green, N. K., Subr, V., Ulbrich, K., Gilbert, R. J., Fisher, K. D., Finberg, R. W., and Seymour, L. W. (2009) Human erythrocytes bind and inactivate type 5 adenovirus by presenting Coxsackie virus-adenovirus receptor and complement receptor 1, Blood113, 1909–1918.

    Google Scholar 

  11. Stone, D., Liu, Y., Shayakhmetov, D., Li, Z. Y., Ni, S., and Lieber, A. (2007) Adenovirus-platelet interaction in blood causes virus sequestration to the reticuloendothelial system of the liver, Journal of virology81, 4866–4871.

    Article  CAS  Google Scholar 

  12. Morrison, J., Briggs, S. S., Green, N., Fisher, K., Subr, V., Ulbrich, K., Kehoe, S., and Seymour, L. W. (2008) Virotherapy of ovarian cancer with polymer-cloaked adenovirus retargeted to the epidermal growth factor receptor, Mol Ther16, 244–251.

    Article  CAS  Google Scholar 

  13. Korn, T., Nettelbeck, D. M., Volkel, T., Muller, R., and Kontermann, R. E. (2004) Recombinant bispecific antibodies for the targeting of adenoviruses to CEA-expressing tumour cells: a comparative analysis of bacterially expressed single-chain diabody and tandem scFv, The journal of gene medicine6, 642–651.

    Article  CAS  Google Scholar 

  14. Dmitriev, I., Kashentseva, E., Rogers, B. E., Krasnykh, V., and Curiel, D. T. (2000) Ectodomain of coxsackievirus and adenovirus receptor genetically fused to epidermal growth factor mediates adenovirus targeting to epidermal growth factor receptor-positive cells, Journal of virology74, 6875–6884.

    Article  CAS  Google Scholar 

  15. Bayo-Puxan, N., Gimenez-Alejandre, M., Lavilla-Alonso, S., Gros, A., Cascallo, M., Hemminki, A., and Alemany, R. (2009) Replacement of adenovirus type 5 fiber shaft heparan sulfate proteoglycan-binding domain with RGD for improved tumor infectivity and targeting, Human gene therapy20, 1214–1221.

    Article  CAS  Google Scholar 

  16. Magnusson, M. K., Henning, P., Myhre, S., Wikman, M., Uil, T. G., Friedman, M., Andersson, K. M., Hong, S. S., Hoeben, R. C., Habib, N. A., Stahl, S., Boulanger, P., and Lindholm, L. (2007) Adenovirus 5 vector genetically re-targeted by an Affibody molecule with specificity for tumor antigen HER2/neu, Cancer gene therapy14, 468–479.

    Article  CAS  Google Scholar 

  17. Coughlan, L., Vallath, S., Saha, A., Flak, M., McNeish, I. A., Vassaux, G., Marshall, J. F., Hart, I. R., and Thomas, G. J. (2009) In vivo retargeting of adenovirus type 5 to alphavbeta6 integrin results in reduced hepatotoxicity and improved tumor uptake following systemic delivery, Journal of virology83, 6416–6428.

    Article  CAS  Google Scholar 

  18. He, T. C., Zhou, S., da Costa, L. T., Yu, J., Kinzler, K. W., and Vogelstein, B. (1998) A simplified system for generating recombinant adenoviruses, Proceedings of the National Academy of Sciences of the United States of America95, 2509–2514.

    Google Scholar 

  19. Hokanson, C. A., Dora, E., Donahue, B. A., Rivkin, M., Finer, M., and Mendez, M. J. (2003) Hybrid yeast-bacteria cloning system used to capture and modify adenoviral and nonviral genomes, Hum Gene Ther14, 329–339.

    Article  CAS  Google Scholar 

  20. Gietz, R. D., and Woods, R. A. (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method, Methods in enzymology350, 87–96.

    Article  CAS  Google Scholar 

  21. Gietz, R. D., and Woods, R. A. (2006) Yeast transformation by the LiAc/SS Carrier DNA/PEG method, Methods in molecular biology (Clifton, N.J) 313, 107–120.

    CAS  Google Scholar 

  22. Peng, H. H., Wu, S., Davis, J. J., Wang, L., Roth, J. A., Marini, F. C., 3rd, and Fang, B. (2006) A rapid and efficient method for purification of recombinant adenovirus with arginine-glycine-aspartic acid-modified fibers, Analytical biochemistry354, 140–147.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank to Manel Cascalló and Lynda Coughlan for critical ­reading of the manuscript.

Author information

Authors and Affiliations

  1. Institut Català d’Oncologia – IDIBELL, L’Hospitalet de Llobregat, Barcelona, Spain

    Marta Giménez-Alejandre

  2. National Cancer Institute, US National Institutes of Health, Bethesda, MD, USA

    Alena Gros

  3. Laboratori de Recerca Translacional, Institut Català d’Oncologia – IDIBELL, L’Hospitalet de Llobregat, Barcelona, Spain

    Ramón Alemany

Authors
  1. Marta Giménez-Alejandre
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alena Gros
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ramón Alemany
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ramón Alemany .

Editor information

Editors and Affiliations

  1. Jennerex Biotherapeutics, Inc., Market Street 1, San Francisco, 94105, California, USA

    David H. Kirn

  2. School of Medicine, Dept. Pathology & Immunology, Washington University, S. Euclid Ave. 660, St.Louis, 63110, Missouri, USA

    Ta-Chiang Liu

  3. Hillman Cancer Center, University of Pittsburgh, Centre Avenue 5117, Pittsburgh, 15213, Pennsylvania, USA

    Stephen H. Thorne

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Giménez-Alejandre, M., Gros, A., Alemany, R. (2012). Construction of Capsid-Modified Adenoviruses by Recombination in Yeast and Purification by Iodixanol-Gradient. In: Kirn, D., Liu, TC., Thorne, S. (eds) Oncolytic Viruses. Methods in Molecular Biology, vol 797. Humana, Totowa, NJ. https://doi.org/10.1007/978-1-61779-340-0_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-340-0_2

  • Published:

  • Publisher Name: Humana, Totowa, NJ

  • Print ISBN: 978-1-61779-339-4

  • Online ISBN: 978-1-61779-340-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation